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LSU Historical Dissertations and Theses Graduate School
1994
The Production and Characterization of Alginate Produced by

Pseudomonas Syringae.
Richard David Ashby
Louisiana State University and Agricultural & Mechanical College
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Ashby, Richard David, "The Production and Characterization of Alginate Produced by Pseudomonas
Syringae." (1994). LSU Historical Dissertations and Theses. 5775.
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T he p rod u ction and characterization o f algin ate produced by

Pseudomonas syringae
Ashby, Richard David, Ph.D.

The Louisiana State University and Agricultural ana Mechanical Col., 1994
UMI
300 N. ZeebRd.
Ann Arbor, MI 48106
THE PRODUCTION AND CHARACTERIZATION
OF ALGINATE PRODUCED
BY PSEUDOMONAS SYRINGAE
A D issertation
Subm itted to the G raduate Faculty of the

Louisiana State U niversity an d
A gricultural an d M echanical College
in p artial fulfillm ent of the
req u irem en ts for the degree of
Doctor of Philosophy
in
The D epartm ent of Microbiology
by
Richard David Ashby
B.S., Brigham Young University, 1987
August 1994
ACKNOWLEDGMENTS
I w o u ld like to ex p ress m y d e e p e st a p p re c ia tio n to m y

ad visor, Dr. D onal. F. Day, fo r h is advise, en c o u ra g em e n t, a n d
valuable discussions th ro u g h o u t this work, an d also fo r his h elp in
the p rep aratio n of this dissertation.
S in cere a p p re c ia tio n is also e x te n d e d to m y co m m ittee
m em bers, Drs. E. A chberger, A. Biel, D. C hurch, R. Laine, a n d J.
Larkin fo r valuable discussions, a n d suggestions on each aspect of
th is w ork, a n d th e ir h elp fu l in sig h ts a n d co n stru ctiv e criticism
pertain in g to the p re p a ra tio n of th is d issertatio n . I w ould like to
th an k Ms. C. Henk fo r h e r helpful assistance in p h o to developm ent,
Dr. B. G unn (C h em istry , LSU) fo r h e r h e lp in c o m p u te riz e d
m a cro m o lec u la r m odeling, a n d Ms. D. S ark ar (A u d u b o n S ugar
In stitu te, LSU) fo r h e r v ario u s h elp a n d suggestions d u rin g th is
study. I w ould also like to th an k m y lab collegues, D. Kim, J. Lee,
a n d M. Ott fo r th e ir v alu able discussion, a n d especially th a n k the
A udubon Sugar In stitu te fo r providing the facilities a n d fu n d s to
com plete this research.
Finally, I dedicate this w ork to m y wife, Shelley, m y d aughter,
Kailee, an d to b o th sides of m y extended family. T heir love, patience
an d u n d erstan d in g m ade this w ork possible.
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS.......................................................................... ii
LIST OF TABLES...................................................................................... vi
LIST OF FIGURES..................................................................................... vii
ABSTRACT............................................................................................... xi
INTRODUCTION........................................................... 1
REVIEW OF LITERATURE....................................................................... 3
I. Bacterial Exopolysaccharides: G eneral C h aracteristics.. 3
II. Alginate: O verview ................................................................ 5
1. Compositional Differences,
Seaweed vs Bacterial A lg in ate................................ 5
2. Properties...................................................................... 10
3. A pplications.................... 14
4. Commercial M anufacture.......................................... 15
III. A lginate Biosynthesis........................................................... 21
1. P ath w ay . .................................................................. 21
2. Enzymology................................................................... 25
3. Regulation...................................................... 30
IV. Pseudom onas syringae Alginates.................................... 32
V. Polysaccharide Analysis....................................................... 32
1. D epolym erization........................................................ 32
2. Acid Sensitivity............................................................ 36
VI. Goals of this S tudy................................................................ 38
MATERIALS AND METHODS............................................................... 39
I. Organisms, Growth Conditions, an d M aintenance 39
II. Analytical M ethods................................................................ 40
1. Cell Mass D eterm in atio n ........................................... 40
2. Total C arbohydrate Q uantitation............................. 40
3. Alginate Q uantitation....................... 41
4. Acetyl Q uantitation..................................................... 43
iii
III. O ptim ization of Bacterial A lginate Production............. 45
1. M edia C om position...................................................... 45
A. C arbon Source.................................................... 45
B. N itrogen Source................................................ 45
2. pH a n d T em p eratu re ...................................... 46
3. Agar vs Broth C ulture................................................. 46
IV. Batch Ferm entations............................................................ 48
V. Purification of Bacterial A lginate......................................... 48
VI. D eacetylation of Bacterial Alginate.................................. 49
VII. Alginate Size a n d Q uantity D eterm inations................. 50
VIII. Sugar Sensitivity to Acid................................................... 50
1. Thin Layer C hrom atography.................................... 50
2. Ion C hrom atography.................................................. 51
IX. Identification o f the Acid Hydrolysis Product of
L-Gulose................................................................................... 52
1. Thin Layer C hrom atography ........................... 52
2. Stability of 1,6 A nhydro p-L-Gulopyranose 53
X. Alginate Reductions.................. 53
XI. Alginate Com positions......................................................... 54
XII. P roperties of Bacterial Alginates a n d Effects of
A cetylation............................................................................. 55
1. Viscosity.......................................................................... 55
2. W ater Holding Capacity.............................................. 56
3. Surface T ension.................................................. 56
4. Precipitation by Metal Ions....................................... 57
RESULTS................................................................................................... 58
I. Production o f Bacterial A lginate...................................... 58
1. M edia Compositions an d C onditions ........ 58
2. Agar vs Broth C ulture................................................. 62
II. C haracterization of Bacterial A lginate.............................. 69

1. Recovery................................................................. 69
2. M olecular W eight......................................................... 69
3. Com position.......................... 71
iv
III. Functional P roperties......................................................... 76
1. Viscosity.......................................................................... 76
2. Physical Effects.............................................................. 85
3. Cation Precipitation by Alginates.............................. 94
DISCUSSION............................................................................................ Ill
REFERENCES............................................................................................ 125
VITA........................................................................................................... 141
v
LIST OF TABLES
Page
Table 1. The stru ctu res o f some bacterial exopolysaccharides. 6
Table 2. Some food applications of alginates................................ 16

Table 3. Some in d u strial applications of alginates...................... 17
Table 4. Estimates of U. S. consum ption an d price of
polysaccharides........................................... 20
Table 5. Effects of carb o n source o n alginate yield from

P. syringae ATCC 19304..................................................... 60
Table 6 . A lginate yield b y P. syringae ATCC 19304 on
d ifferen t m ed ia.................................................................... 67
Table 7. M olecular w eights of alginates......................................... 70
Table 8 . Thin lay er chro m ato g rap h y (Rf values)........................ 74
Table 9. The energetics an d stability of 1,6 an h y d ro
p-L-gulopyranose................................................................ 79
Table 10. Effects of calcium concentration on th e w ater
holding capacities of alginate gels.................................. 86
Table 11. Effects of acetylation o n b ead surface ten sio n 91

Table 12. The p recip itatio n of M acrocystis alginate an d
acetylated an d d eacetylated P. syringae alginate
by m etal ions........................................................................ 1 1 0
vi
LIST OF FIGURES
Page
Figure 1. Structures of the uronic acids of alginates................... 8
Figure 2. The block structures of alginate..................................... 11

Figure 3. The "egg box" m odel for Ca4f in duced gelation of
poly a-L -guluronate..................... 13
Figure 4. Flow diagram fo r the extraction of sodium alginate
from seaw eed.................................................................... 19
Figure 5. The proposed biosynthetic pathw ay of b acterial
alginate in A zotobacter vinlandii an d
Pseudom onas aeruginosa............................................... 22
Figure 6 . A com parison of the overall ro u tes of
in corporation of fructose a n d glucose in alginate
p ro d u ced by Pseudom onas aeruginosa....................... 24
Figure 7. The relative location of the alginate (alg) genes
on the chrom osom e linkage m ap of
Pseudom onas aeruginosa................................................. 26
Figure 8 . The m echanism of acid hydrolysis of glycosides 34
Figure 9. The relatio n sh ip betw een cell m ass an d
alginate accum ulation by P. syringae ATCC
19304 w ith tim e in shake flask cu ltu re...................... 59
Figure 10. The effect of the initial am m onium co ncentration

on cell mass, an d total alginate accum ulation
by P. syringae ATCC 19304 a t 48 h o u rs...................... 61
Figure 11. The effect of the initial am m onium co ncentration

on specific yields of alginate by P. syringae ATCC
19304 a t 48 h o u rs............................................................ 63
Figure 12. The effect of initial pH on cell mass, an d

alginate accum ulation by P. syringae ATCC 19304
a t 48 h o u rs........................................................................ 64
vii
Figure 13. The effect of tem p eratu re on cell mass, a n d
alginate accum ulation by P. syringae ATCC 19304
at 48 h o u rs....................................................... 65
Figure 14. The effect of initial ferric ion (Fe^+) concentration

on th e specific yields o f alginate by P. syringae
ATCC 19304 a t 48 h o u rs............................... 6 8
Figure 15. Thin layer chrom atograph of HC1 hydrolyzed
D -m annose....................................................... 72
Figure 16. Thin layer chrom atograph of HC1 hydrolyzed
L-gulose............................................................. 73
Figure 17. Stability of m onom eric D-m annose a n d L-gulose
in HC1 an d H 2 SO4 u n d er hydrolysis conditions
at 100°C, as d eterm in ed by ion c h ro m a to g ra p h y ... 75
Figure 18. Thin lay er chrom atograph of the acid hydrolyzed
p ro d u ct of L-gulose com pared to 1,6a n h y d rid e s .. . 77
Figure 19. C om puter d erived im age of 1,6 an h y d ro
p-L-gulopyranose............................................ 78
Figure 20. Gulose recovered, expressed as a p ercen t of

the total sugar p resen t in the HC1 hydrolyzed
red u ced alginates, from M acrocystis pyrifera,
an d P. syringae ATCC 19304, as d eterm in ed
by ion ch ro m ato g rap h y.................................................. 80
Figure 21. Com parative viscosity, (N/No), of M acrocystis

alginate, acetylated P. syringae alginate, an d
d eacetylated P. syringae alginate as a function
of alginate co n cen tratio n................................................ 82
Figure 22. The effects of acetylation on th e com parative

viscosity, (N/No), of M acrocystis alginate, an d
P. syringae alginate........................................ 83
Figure 23. The effects of tem p eratu re on the com parative
viscosity, (N/N 0), of M acrocystis alginate,
acetylated P. syringae alginate, an d
d eacetylated P. syringae alginate................................ 84
viii
Figure 24. Percent difference in w ater holding capacity of
M acrocystis alginate gels m ade w ith ferric iron,
an d lead, as com pared to calcium alginate g e ls .. . . 87
acetylated P. syringae alginate gels m ad e w ith
ferric iron, an d lead, as com pared to calcium
alginate gels....................... 89
d eacetylated P. syringae alginate gels m ad e w ith
ferric iron, a n d lead, as com pared to calcium
alginate gels...................................................................... 90
Figure 27. Rate of w ater loss of calcium alginate gels m ade

from M acrocystis alginate, acetylated P. syringae
alginate, an d deacetylated P. syringae alginate
as a function of incubation tim e a t 50°C................... 93
Figure 28. C om parison of th e w ater loss of ferric iro n
alginate gels of M acrocystis alginate,
acetylated P. syringae alginate, an d deacety lated
P. syringae alginate com pared to calcium alginate
gels as a function of incubation tim e a t 50°C.......... 95
Figure 29. Precipitation of M acrocystis alginate, acetylated

P. syringae alginate, a n d d eacetylated P. syringae
alginate by u ran iu m ( U ^ ) io n s.................................. 97

P. syringae alginate, an d d eacetylated P. syringae
alginate by copper (Cu2+) io n s.................................... 98

P. syringae alginate, an d d eacetylated P. syringae
alginate by lead (Pb2+) io n s........................................ 99

P. syringae alginate, an d deacetylated P. syringae
alginate by ferric (Fe^+) io n s...................................... 100
ix
alginate b y calcium (Ca2+) io n s............................. 101

alginate b y stro n tiu m (Sr^+) io n s.............................. 103
Figure 35. Precipitation o f M acrocystis alginate, acetylated

alginate by zinc (Zn^+) io n s........................................ 104
Figure 36. Precipitation o f M acrocystis alginate, acetylated

alginate b y m anganese (M n^+ ) io n s......................... 105

alginate b y cobalt (Co2+) io n s...................................... 106

P. syringae alginate, a n d deacetylated P. syringae
alginate b y cesium (Csl+) io n s.................................... 107

alginate by m agnesium (M g 2+) io n s......................... 108
x
ABSTRACT
A lginate h a s m a n y in d u s tria l uses b ecau se o f its u n iq u e

colloidal b eh a v io r, a n d its ab ility to thick en , stabilize, em ulsify,
suspend, form films, a n d p ro d u ce gels. C urrently, all com m ercial
alg in ate com es fro m b ro w n algae, w h ere it exists n a tu ra lly as a
stru ctu ra l m aterial. M any d iffe re n t types of brow n algae p ro d u ce
alg in a te b u t it ca n o n ly be o b ta in e d in su fficien t q u a n tity a n d
quality fro m a lim ited n u m b e r of species. On th e basis of location,
a t least h alf of th e w orld's resources of this polym er are p o ten tially
a t risk d u e e ith e r to p o litical in sta b ility o r in d u stria l pollution.
B acteria p ro v id e a p o te n tia lly u n lim ite d a lte r n a te so u rce fo r
alg in ate. Pseudom onas syringae p v phaseolicola ATCC 19304,
p ro d u ced a n acety lated alginate-like p o ly sacch arid e w ith a w eight
a v e ra g e m o le cu lar w eight (Mw ) o f 1.2 x 1 0 $. T his b a c te ria l
p olym er was com posed o f 82% m an n u ro n ic acid a n d 18% guluronic
acid. C om positional an a ly sis o f th e re d u c e d a lg in a te p o ly m e r
show ed th a t L-gulose was m ore sensitive to acid d eg rad a tio n th a n
D -m an n o se. T he p e rc e n ta g e o f gulose re c o v e re d a t v a rio u s
h y d ro ly sis tim es w as e x tra p o la te d to ze ro h y d ro ly sis tim e to
account for the loss of gulose. A cetylation affected th e solution an d
gelling p ro p e rtie s o f th e p olym er. A cety lated b a c teria l alg in ate
show ed in creased viscosity, an d w ater holding capacity, a n d altered
cation p recip itab ility o v er u n ac ety late d alginates. By co n tro llin g
th e degree of acetylation on the bacterial alginate, th e solution a n d
gelling p ro p e rtie s of th e p o ly m e r ca n be m a n ip u la te d a n d th e
polym er targ eted to specific applications.
xi
INTRODUCTION
A lginate is a w ater-soluble gum used in the food an d chem ical

in d u s tr y p rim a rily as a n em u lsifier, stab ilizer, o r th ic k en in g
(gelling) agent (117, 144). The com m ercial sources of alginate are
th e b ro w n m a r in e se a w e e d s o f th e fa m ily Phaeopliyceae,
specifically, th o se of th e g en e ra Ascophyllum , Ecklonia, Fusarium,
Laminaria, a n d Macrocystis. These alginates form viscous solutions
a t low concentrations, show polyelectrolytic behavior, an d form gels
a n d film s (97). T ypically, th ese alginates are block copolym ers,
lin k ed 1-4, o f p-D -m annuronic acid (M) a n d its C-5 ep im er a-L-
guluronic acid (G). The ratio of M/G varies from one algal species to
th e n e x t a n d d ic ta te s th e gelling p ro p e rtie s of th e p o ly m e r.
A lginates w ith h ig h M /G ratio s are m o re ex ten d ed a n d p ro d u c e
elastic, p liab le gels d u e to th e sm aller regions o f poly-G blocks.
Alginates w ith low M/G ratio s p ro d u ce strong, b rittle gels, because
of th e h ig h er affinity of poly-G for C a ^ , an d the g reater com paction
of the m olecule.
A lginate is o n e of th e few eu k ary o tic p o ly sacch arid es th a t
h av e a p o te n tia l p ro k ary o tic source. M any species of b a c te ria
p ro d u ce "alginate-like" polysaccharides. This was first discovered
in studies o n th e "slime" p ro d u ced by Pseudom onas aeruginosa (13,
75, 76) in in fe c tio n s o f cystic fib ro sis p a tie n ts . A zotobacter
vinlandii (51, 99) was also stu d ied as a po ten tial source of alginate
because of its n o n p ath o g en ic n atu re. At high resp iratio n ra te s the
m ajority of th e carb o n used by A. vinlandii goes to resp iratio n an d
the form ation of poly p h y d ro x y b u ty rate (PHB, 23). This b acteriu m
1
2
p ro d u ces alg inate only d u rin g en cy stm en t a n d does n o t p ro d u ce

sufficient polym er for in d u strial production.
O ther nonpathogenic pseudom onads, including th e fluorescent
pseudom onads (58, 120), an d the phytopathogenic pseudom onads,
specifically P seudom onas syringae (37, 38, 91), p ro d u ce alginates.
P. syringae produces an alginate conspicuously d ifferen t from th a t
o f th e b ro w n seaw eeds. As w ith o th e r b a c te ria l alg in ates, P.
syringae alginates are generally acetylated a t position C-2 a n d /o r C-
3 of th e m a n n u ro n ic acid resid u es. U nlike seaw eed alg in ate, P.
syringae p o ly m ers do n o t co n tain an extensive block stru c tu re .
The bacterial alginate pro d u ces bulky, elastic gels w ith h igh w ater
re te n tio n b e c a u se o f its lack o f ex ten siv e poly-G blocks, O-
acetylation, an d high M/G ratio (47).
T he goals of th is stu d y w ere to d eterm in e th e co n d itio n s
w hich in flu en c e alg in a te p ro d u c tio n fro m P. sy rin g a e a n d to
ch aracterize the p ro p erties o f the polym er in o rd e r to d eterm in e if
th e use of this org an ism is feasible fo r large scale p ro d u c tio n o f
alginate.
REVIEW OF LITERATURE
I. Bacterial Exopolysaccharides: General Characteristics.

Most species of b acteria pro d u ce exopolysaccharides (EPS), i.e.,
polysaccharides fo u n d o u tsid e th e cell wall, e ith e r a tta c h e d in th e
form of a capsule o r secreted into the extracellular en v iro n m e n t as
a slime (135). T here have been m any theories p ro p o sed to explain
th e role o f bacterial EPS a n d all rela te to the survival ad v an tag es it
o ffers to th e organism s. O riginally, th ese p o ly sacch arid es w ere
stu d ied because of th e ir link to pathogenicity. This was especially
tru e o f th e capsules of strains of Streptococcus pneum oniae, Bacillus
anthracis, a n d Klebsiella pneum oniae (2). The v iru len ce o f th ese
organism s is due, in p art, to the protection p rovided b y th e capsular
polysaccharide su rro u n d in g the cell. W ithin anim al hosts, capsules
in terfere w ith b o th phagocytosis an d an tib o d y binding (87).
Som e b a c te ria se c re te EPS as a n aid to co lo n izatio n by
a s sistin g in su rfa c e a tta c h m e n t (16, 5 0 ). The a b ility of
Streptococcus m u ta n s a n d Streptococcus salivarius cells to a d h e re to
th e surface o f te e th is p artially a fu n ctio n of the EPS p ro d u c e d by
th ese o ral b a c te ria (87). O th er ro les of EPS in clu d e p re v e n tin g
desiccation, en erg y storage, co n c en tratio n a n d u p tak e of c h a rg e d
m olecules, p artic u larly m etal ions (11, 17, 46), p ro te c tio n ag ain st
th e effects o f u ltrav io let ra d ia tio n (17), a n d m ed iatio n of biofilm
a n d m icrocolony form ation (18).
B a cte rial EPS is sp ecies sp ecific. S u g ars a re common
co m pon en ts in m ost b acterial EPS. D-glucose, D -m annose, a n d D-
galactose o ccur freq u en tly , a n d L-rham nose a n d L-fucose a re less
3
4
com m on. The p resen ce of n eg ativ e ch arg es is a fe a tu re o f some

b a c te ria l EPS. U su ally th e se c h a rg e s a r e th e r e s u lt o f th e
in co rp o ratio n of uronic acids into th e polym er. The m ost com m on
negatively ch arg ed sugar p re se n t in b a c te ria l EPS is D -glucuronic
acid w hich is a co m p o n en t of h y alu ro n a n , x an th an gum, a n d m ost
teichuronic acids. D -m annuronic acid, D -galacturonic acid a n d L-
guluronic acid are also com ponents of som e b acterial polym ers. D-
m annuro n ic acid an d L-guluronic acid are associated p rim arily w ith
b a c te ria l a lg in a te s. D -g a lac tu ro n ic a c id is fo u n d in som e
lip o p o ly sa c c h a rid e s p ro d u c e d b y P roteus m ira b ilis, (77) a n d
Xanthom onas cam pestris (142).
B io sy n th esis o f EPS n o rm a lly o c c u rs b y e ith e r o f tw o
m echanism s. EPS m ay be p ro d u c e d at th e cytoplasm ic m em b ran e
using p re c u rso rs fo rm ed in trac ellu larly , o r th e y m ay be fo rm e d
fro m sp ecific p r e c u r s o r s in th e e x tra c e llu la r e n v iro n m e n t.
G enerally, th e first ty p e is characteristic of h etero p o ly sacch arid e
p r o d u c tio n , w h ile th e s e c o n d is c h a r a c te r is tic o f c e r ta in
hom opolysaccharides including levans a n d d e x tra n s (133). Both
p a th w a y s r e q u ir e th e p r o d u c tio n o f a c tiv a te d n u c le o tid e
d ip h o sp h a te form s of th e m onosaccharides a n d th e p recu rso rs for
sub stitu en t groups, specifically acetate, p yruvate, an d succinate (25,
37). Acetyl CoA was recen tly confirm ed as th e source of acetate in
x a n th a n gum b io sy n th esis b y Xanthom onas cam pestris ( 6 6 ). The
p re c u rs o r fo r p y ru v a te is p h o sp h o e n o lp y ru v a te (7 1 ). T h ese
substituents in tro d u ce negative charges in to th e polysaccharides. A
high n u m b e r of n eg ativ e charges, co u p led w ith a h igh m o lecu lar
w eight, p ro d u ce polym ers th a t form h ig h ly h y d ra te d gels. W hile
5
th e a b ility to p ro d u c e EPS is w id e s p re a d , u n d e r la b o ra to ry
conditions this ability is often un stab le an d lost on re p e a te d cu ltu re
(17).
Bacterial EPS varies enorm ously in stru ctu re a n d com position,
ra n g in g fro m sim p le h o m o p o ly sacc h arid es to com plex, h ig h ly
su b stitu te d a n d b ran ch e d h etero p o ly sacch arid es (Table 1). These
polysaccharides can be divided in to five distinct groups: 1 ) d ex tran s
a n d levans, o r h o m o p o ly sacch arid es p ro d u c e d b y b a c te ria using
sucrose as a specific su b strate, 2 ) hom opolysaccharides o th e r th a n
d e x tr a n a n d le v a n , i.e., cellu lo se, 3) h e te ro p o ly s a c c h a rid e s
containing m ore th an one type of m onosaccharide synthesized from
a specific ca rb o n su b strate, 4) h etero p o ly sacch arid es fo rm ed from
re p e a tin g u n it s tr u c tu r e s , i.e., x a n th a n gum , and 5)
heterop o ly sacch arid es com posed of two types o f m o n o m er w ith no
rep eatin g unit, i.e., alginate (134).
II. Alginate: Overview

1. Com positional Differences, Seaweed vs. Bacterial Alginate
A lginate, th e m ajo r stru c tu ra l p o ly sacch arid e of th e brow n
algae (Phaeophyceae), was first disco v ered b y E. C. C. S tan fo rd in
1881 (19). It was n o t u n til 30 y ears la te r th a t u ro n ic acids w ere
identified as the m ajor com ponents (118). Initially, it was assu m ed
th a t p -D -m an n u ro n ate was th e o n ly m o n o sa cc h arid e p re s e n t in
alginate. S ubsequent rese arch estab lish ed th e copolym eric n a tu re
of alginate an d the p resence of v ariable am ounts of a second uronic
acid, a-L -guluronate (41).
Table 1. The stru ctu res of some b acterial exopolysaccharides.
Species Polysaccharide S tructure
Leuconostoc m esenteroides D extran ( lit 6 glucose)n

A cetobacter xylin u m Cellulose (16.4 glucose)n
Alcaligenes faecalis var. niyxogcnes C urdlan (163 glucose)n
Streptococcus m utans M utan (1“ 3 glucose)n
Streptococcus salivarius Levan ( 26.6 fructose)n
A zotobacter vinlandii Alginate 1-4 linked ManA an d GulA
Pseudom onas aeruginosa A lginate 1-4 linked ManA an d GulA
Streptococci H yaluronan (-3GlcNAcl6.4GlcAl6.)n
(Haemolytic group A)
X anthom onas cam pestris X anthan (-4G lclh4G lclh4G lclli)n
I
1
Aa
A bbreviations: Gal, galactose; Glc, glucose; GlcA, glucuronic acid; GulA, guluronic acid; Man,
m annose; ManA, m annuronic acid; GlcNAc, N-acetylglucosamine; OAc, O-acetyl; Pyr, pyruvate.
a A= (-3«lM an(O A c)2hlG lcA 4lilM an4-Pyr)
7
W ithin the last 30 years, alginate was fo u n d to be p ro d u c e d

b y so m e p r o k a r y o te s . T he o b s e rv a tio n th a t Pseudom onas
aeruginosa was able to synthesize an "alginate-like" polysaccharide
w as f irs t re p o rte d b y Linker a n d Jo n es (75), a lth o u g h m u co id
s tra in s o f th is b a c te riu m w ere iso la te d m u ch e a rlie r. T h ey
d em o n strate d th a t this polym er h ad com ponents id en tical to algal
alginate. In a su b seq u en t study, th ey show ed th a t an O -acetylated
a lg in a te w as th e m ajo r co m p o n en t o f P. aeruginosa slim e (76).
Carlson an d M atthews (13) confirm ed th e com position of b acterial
alg in ates in a stu d y of 13 d iffe re n t m u co id isolates fro m cystic
fib ro sis p a tie n ts. M ore rece n tly , th e re la te d p se u d o m o n a d s P.
fluorescens, P. m endocina, P. p u tid a (53), an d m any p ath o v ars of P.
syringae (37) have b een identified as alginate pro d u cers. The sam e
y ea r th a t P. aeruginosa was rep o rted to produce alginate, Gorin an d
Spencer (51) show ed th a t the polysaccharide p ro d u ced b y strain s of
A zotobacter vinlandii w as th e sam e as th e ac ety late d alg inate
p ro d u ced b y pseudom onads. Later it was d eterm in ed th a t alginate
b io sy n th e sis b y A. vinlandii was clo sely a s so c ia te d w ith th e
form atio n of vegetative cysts (93).
Alginates are u n b ran ch ed copolym ers of p-D -m annuronic acid
a n d its C-5 ep im er cx-L-guluronic acid, lin k ed 1-4 (Fig. 1). The
actual com position an d sequence of the polym er is d e p e n d e n t u p o n
th e source (73, 96, 130). Alginates fro m seaw eed a n d A. vinlandii
g e n e ra lly co n tain extensive regions o f h o m opolym eric blocks of
m an n u ro n ic acid an d guluronic acid to g eth er w ith some ran d o m o r
a lte r n a tin g se q u e n c e s. A lg in ates d e riv e d fro m s tr a in s of
Pseudom onas co n tain few block hom opolym eric regions. Instead,
p-D -m annuronate
C-(i
ooc
a-L -g u lu ro n ate
Figure 1 . Structures of the uronic acids of alginates.

9
th e y a re co m p o sed p re d o m in a n tly of ra n d o m se q u e n c e s. In
addition, the m an n u ro n ic acid residues are highly O -acetylated ( 2 1 ,
125). T he stru ctu re, as well as th e position of acety latio n in these
alginates, has b een extensively stu d ied . The O-acetyl g ro u p s are
associated exclusively w ith th e C-2 a n d /o r C-3 p o sitions of th e D-
m a n n u r o n a te r e s id u e s . n u c le a r m a g n e tic reso n a n ce
sp e c tro m e try (NMR) re v e a le d th a t som e of th e m a n n u ro n a te
resid u es w ere 2,3 di-O -acetylated, alth o u g h the m ono-O -acetylated
form s w ere m ore com m on (21, 125, 127). The re a so n fo r th e O-
ac e ty la tio n in b a c te ria l alg in ates is n o t e n tire ly clear. It was
suggested th a t acetylation m ay be p a rt of a control m echanism th a t
o p e ra te s d u rin g b io sy n th e sis th a t c o n tro ls th e M /G ra tio b y
p re v e n tin g ep im eriza tio n of th e c a rb o x y l g ro u p o n th e C-5
m a n n u ro n a te ca rb o n (128). A lternatively, it m ay p lay a ro le in
dictating the solution p ro p erties of the polysaccharide (129).
Seaw eed a n d b ac teria l alginates d iffer in th e ir M /G ratio s.
M ost alginates isolated fro m the brow n seaw eeds show M/G ratio s
in th e ran g e of 0.45 (Laminaria hyperborea) to 1.85 (A scophyllum
nodosum , 117). The m ost com m on in d u strially available alginate is
extracted from the brow n alga, M acrocystis pyrifera. This p olym er
contain s 60% 4-linked m an n u ro n a te a n d 40% 4-linked g u lu ro n ate
residues giving an M/G ratio of 1.5. The alginates p ro d u c e d b y P.
aeruginosa co n tain 80% 4-linked m a n n u ro n a te a n d 20% 4-lin k ed
guluron ate residues, giving an M/G ratio of 4.0 (150).
H aw orth pro jectio n s o f th e (5-D -m annuronate a n d th e a-L-
g u lu ro n a te resid u es show little d ifferen ce in th e two stru c tu re s.
Epim erization of th e carboxyl group at C-5 does, how ever p ro d u ce a
10
m a rk e d ch an g e in th e th re e d im en sio n al co n fo rm atio n of th ese

m onosaccharides. Since th e carb o x y l g ro u p is th e m o st b u lk y
s u b s titu e n t o n th e rin g , th e m o st e n e rg e tic a lly fa v o ra b le
co n fo rm atio n o rien ts th is g roup to th e eq u a to rial p o sition. As a
result, p-D -m annuronate exists p referen tially in th e ch air form,
w hereas th e a-L -guluronate resid u es a d o p t th e IC 4 conform ation
(Fig. 1). C hains of sugars co n tain in g co m b in atio n s of th e se two
fo rm s p ro d u c e p o ly m e rs w ith d if f e r e n t th r e e d im e n s io n a l
stru ctu re s. T he p-D -m annuronate linkage positions, C -l a n d C-4,
a re e q u a to rial to th e p lan e of th e sugar ring. T hese linkages are
axial fo r th e a-L -g u lu ro n ate re sid u e s. X -ray fib e r d iffra c tio n
studies o f alginates co n tain in g h ig h p ro p o rtio n s o f m a n n u ro n a te
residues in dicate a flat rib b o n -lik e co n fo rm atio n in th e solid state
(5, 109, 110). T his c o n fo rm a tio n is sim ila r to th e p 1-4
dieq u ato rially linked polym ers such as cellulose. A lginates rich in
po ly g u lu ro n ate, w hich is 1-4 diaxially linked, a d o p t a b u ckled 2-
fold chain conform ation ( 6 , Fig. 2).
2. Properties
The physical com position of alginates dictates th e solution an d
gelling p ro p e rtie s o f th e p o ly m e rs. A lg in ate is a s tr u c tu ra l
com ponent in the brow n seaw eeds w here it co n trib u tes to b o th th e
tensile stren g th a n d th e flexibility of th e algal tissue (64, 143). In
A. vin la n d ii it serves a stru c tu ra l fu n ctio n fo r th e m etab o lically
d o rm an t cysts. Evidence exists th a t it is an im p o rtan t com p o n en t of
b o th th e cyst exine an d in tin e, w hich are m icroscopically d istin ct
reg io n s o u tsid e th e c e n tra l b o d y of th e cy st (1 1 5 ). A lg in ate
11
Figure 2. T he block stru c tu re s of alg in ate. A. T he rib b o n -lik e

s tr u c tu r e o f p o ly p -D -m a n n u ro n a te. B. T he b u c k le d c h a in
conform ation of poly a-L-guluronate. C. A lternating sequences of p-
D -m annuronate an d a-L -guluronate.
12
p roductio n by P. aeruginosa was related to resistance to antibiotics,

p h ag e a n d b acterio cin s a n d p ro tec tio n ag ain st phagocytosis a n d
an tib o d y attachm ent. This suggests, th a t fo r P. aeruginosa, alginate
functions as a general protective barrier.
A lginates w ith low M/G ratios p ro d u ce strong b u t b rittle gels,
w hereas alginates w ith h igh M/G ratios form m ore elastic gels (62,
110). T his d iffe re n ce is d u e to th e a rra n g e m e n t of th e block
stru ctu res w ithin th e polym ers an d is particu larly ev id en t w hen the
polysaccharide in teracts w ith cations. Alginate, being a polyanion,
is a n a tu ra l io n -ex ch an g er w here th e selectivity a n d stre n g th of
b inding d e p e n d o n th e cation, the conform ational characteristics,
a n d th e lin e a r ch arg e d en sity of th e polym er. M ost m u ltiv alen t
cations b in d alginates a n d cross-link th e po ly sacch arid e to fo rm a
gel m atrix . Some reg io n s of th e p o ly sa cc h arid e fo rm stro n g e r
ch elatio n com plexes th a n o th e rs w ith d iv alen t cations, especially
calcium ions (Ca+h). The "egg box m odel" was p ro p o sed to explain
this interactio n (82, 83, 8 8 , 109, 110, Fig. 3,). The binding of Ca_l+ is
stro n g b ecau se in a d d itio n to th e ionic b in d in g to th e carboxyl
groups, various ring an d hydroxyl oxygen atom s are able to chelate
th e c a tio n s . P o ly g u lu r o n a te b in d s Ca4f v e r y s tr o n g ly .
Polym annuronate o r m ixed sequences do n o t b ind Ca_l+ w ith as high
a n affin ity . W hen v a rio u s block seq u en ces w ere iso lated from
in ta ct alginate, p o lyguluronate blocks show ed an en h an ced binding
o f C a ^ in p o ly m ers ab o v e 20 resid u e s (61). This in d ic a te d a
cooperative m ech an ism w h ere bin d in g sites exist in a n o rd e re d
array, an d binding of one ion facilitates binding of a second. Similar
Ca++
Ca++
S^°-V
Figure 3. The "egg box" m odel for C a ^ induced gelation of poly «- L-

guluronate.
14
effects w ere n o t seen w ith p o ly m an n u ro n ate blocks o r altern atin g

sequences ( 6 8 ).
B acterial alginates th a t contain few p o ly g u lu ro n ate blocks (a
h ig h M /G ra tio ), a n d th a t a re O -acetylated, p ro d u c e relativ ely
bulky, flexible gels w ith h igh w ater re te n tio n in th e p rese n ce of
Ca++( 109). The high n u m b e r of m an n u ro n ate residues an d O-acetyl
groups red u ce th e cooperative binding of Ca'l+ a n d w eaken th e gel
netw ork. This fact, in association w ith th e g reater positive osm otic
p r e s s u r e c a u s e d b y th e in c re a s e d n u m b e r of d is s o c ia te d
co u n terio n s, significantly en h an ces th e w ater holding cap acity of
bacterial gel m atrices (129). The resulting gel p rovides cells w ith
h ydrop h ilic capsules th a t p ro tec t th em from o th e r m icroorganism s,
chemicals, antibiotics, an d desiccation (116). In P. syringae, alginate
is th o u g h t to p lay an im p o rta n t ro le in th e p ath o g en icity o f th e
bacterium , allowing adhesion to the h o st surface (137).
3. Applications
W hile alginate p ro d u ctio n is w idespread am ong m em b ers of
th e brow n seaw eed (Phaeophyceae), only a few species of brow n
seaw eed are used for com m ercial production. The p rin cip al source
o f th e w o rld 's su p p ly o f alg in ate is th e g ian t kelp, M acrocystis
pyrifera. O ther seaweeds th a t are used in alginate m an u factu re are
A scophyllum no dosum a n d species of Laminaria an d Ecklonia.
A lg in a te is u s e d in fo o d s a n d fo r g e n e ra l in d u s tria l
applications because of its unique colloidal beh av io r a n d its ability
to thicken, stabilize, em ulsify, suspend, fo rm films, a n d p ro d u c e
gels. T he p rim a ry p ro p e rtie s o n w hich th e w id esp re ad use of
15
alg in ate is b a se d are: 1 ) th e fo rm a tio n of viscous so lu tio n s a t

relatively low co n cen tratio n s, 2 ) th e p o ly electro ly tic b eh a v io r in
solution, 3) th e ab ility to form gels b y chem ical reactio n , 4) the
form ation of films on surfaces, a n d 5) the base exchange p ro p erties
(97).
Food p ro d u c ts in w hich alg in ates are u sed in c lu d e fro zen
foods, p astry fillings, b akery products, an d syrups. Because of th e ir
gelling ab ility , a lg in a te s a re also u se d in in s ta n t a n d cooked
p u ddin g s, an d pie fillings. Alginates are excellent em ulsifiers for
salad d ressin g s a n d stabilizers fo r beverages, w h ip p ed toppings,
a n d sauces (Table 2). A lginates are also of v alue in a n u m b e r of
industrial applications, such as the m anufacture of p ap er, adhesives,
textiles, a ir fre sh en e rs, explosives, polishes, antifoam s, ceram ics,
an d cleaners (19, 97, Table 3).
Since th e in d u stria l p ro d u ctio n of seaw eed alginates began,
several com m ercially im p o rta n t derivatives of alginates have been
developed. Com m ercial derivations include th e sodium , potassium ,
am m onium , calcium, a n d m ixed am m onium -calcium salts of alginic
acid, p ro p y len e glycol alginate, a n d alginic acid. T he p ro p y len e
glycol ester is the only com m ercial, organic d eriv ativ e of alginate.
It has im p ro v ed acid stab ility a n d resistan ce to p recip itatio n by
calcium an d o th er m ultivalent m etal ions (19).
4. Commercial M anufacture
M acrocystis p yrifera grows in relativ ely calm m a rin e w aters
in large, dense beds. It is a v ery rap id ly growing p la n t th a t allows
for up to four cuttings p e r year. At the tim e of harvesting, a dense
16
Table 2. Some Food Applications of A lginatesa
P roperty Product Perform ance
W ater holding Frozen foods M aintains texture d uring

freeze-thaw cycles.
Pastry fillings Produces sm ooth, soft texture
Syrups Suspends solids, controls
pouring consistency
Bakery icings C ounteracts stickiness an d
cracking
Gelling Puddings Firms body an d texture

Pie a n d p a stry fillings Acts as a cold w ater gel base;
develops soft gel b o d y w ith
b ro ad tem p eratu re tolerance;
gives im proved flavor
release.
D essert gels Produces clear, firm , quick-
setting gels.
Emulsifying Salad dressings Emulsifies an d stabilization

Sauces Emulsifies oils a n d suspends
solids
Stabilizing Beer M aintains b ee r foam

Juices Stabilizes p ulp in
concentrates an d finished
drinks
syrups a n d toppings Suspends solids; produces
uniform body
W hipped toppings Stabilizes fat dispersion, an d
freeze-thaw break d o w n
Sauces a n d gravies Thickens a n d stabilizes for a
b ro ad range o f applications
a Table was ad ap ted from Sandford an d Baird (117).

17
Table 3. Some Industrial Applications of A lginatea
P roperty Product Perform ance
W ater holding Paper coating Controls rheology of coatings

P aper sizings Im proves surface p ro p erties,
an d ink acceptance
A dhesives Controls p en etratio n to
im prove adhesion an d
application
Textile p rinting Produces v ery fine line
p rin ts
Gelling Air fre sh en e r Firm, stable gels are

p ro d u ced from cold w ater
system s
Explosives Elastic gels p ro d u ced b y
reactio n w ith borates
Toys Nontoxic m aterials m ade for
im pressions
Hydrom ulching Holds m ulch to inclined
surfaces; prom otes seed
germ ination
Emulsifying Polishes Emulsifies oils a n d suspends

solids
A ntifoam s Emulsifies a n d stabilizes
Latexes Stabilizes latex em ulsions;
provides viscosity
Stabilizing Ceramics Suspend solids

Cleaners Suspends an d stabilizes
insoluble solids
a Table was ad a p ted fro m Sandford an d Baird (117)

18
m at of fro n d s floats on th e ocean surface. H arvesting is actually a

m assive p ru n in g of th e kelp beds. U n d erw ater b lad es m ow the
kelp approxim ately th re e feet below th e w ater surface. The kelp is
th en conveyed into the h o ld of a barge by a m oving belt.
T he p ro cess fo r alg in a te e x tra c tio n is b a s e d o n a n ion-
exchange process. In seaw eed, th e alg inate is p re se n t as a m ixed
salt of sodium a n d /o r potassium , calcium, an d m agnesium (19). The
exact com position of the polym er v aries w ith th e ty p e o f seaw eed
b u t this does n o t affect processing. The ex tractio n process begins
by grinding the seaw eed an d washing it w ith w ater. A strong alkali
is th e n a d d e d to the w ashed seaw eed a n d the m ixture is h e a te d to
e x tra c t a n d dissolve th e alginate. The cru d e alg inate so lu tio n is
th e n clarified a n d p recip itated b y th e ad d itio n of calcium chloride.
T he calcium alg in ate is acid tre a te d to p ro d u c e a n alginic acid
p recip itate. Sodium ca rb o n a te is th e n a d d e d to m ak e a so d iu m
alg in ate p aste w hich is d rie d , a n d m illed in to so d iu m alg in ate
pow der (Fig. 4).
The extraction costs of seaw eed alginate are h ig h er th a n o th er
industrially available gum s due to harvesting a n d purification costs
(117, Table 4). Because of the u n iq u e p ro p erties of alginates, an d
th e high n u m b er of p o ten tial food an d in d u strial applications, th e re
a re no good rep lacem en ts fo r this polym er. B acteria h av e b ee n
studied as po ten tial altern ate sources of alginate. A bacterial source
w ould give an unlim ited supply of alginate an d m ay red u ce costs.
Calcium
Alkali + W ater Chloride
Water * Heat Solution
Wet or Dry Dissolution C r u d e Alginate

Mi l l i ng Washing Clarification Precipitation
Seaweed of Alginates Solution
Washings Insoluble Residue
Color and O dor Sodium

Removal Acid Carbonate
Calcium Acid Alginic A cid S o d i u m Alginate Dry S odium

Drying Mi l li ng
Alginate Tr e a t m e n t Precipitate Paste Alginate P o w d e r
Water a n d
Dissolved
Impurities
Figure 4. Flow diagram for th e extraction of sodium alginate from seaw eed (117).
20
Table 4. Estimates of U. S. Consum ption an d Price of

Polysaccharides.
% U. S. ConsumDtiona
Polysaccharide Food Industrial Retail Price
(p er 1 0 0 g)b
Agar 0.4 0 .6 $ 14.25

Alginate 11.5 6.2 $ 14.55
Carrageenan 11.5 0.3 $ 15.35
Guar gum 19.3 53.8 $ 2 .2 0
Gum arabic 29.5 10.8 $ 5.25
Gum ghatti 1.3 1.6 $ 1 2 .2 0
Gum tragacanth 1.2 0.3 $ 16.35
Karaya gum 1.3 10.8 $ 2.40
Locust b ean gum 11.5 6.2 $ 2 .1 0
Pectin 9.6 0 $ 18.10
X anthan gum 2.9 9.3 $ 11.60
a P ercent U. S. co n su m p tio n is based on 1980 figures. Percentages

rep rese n t the am ount of polysaccharide used for food an d industrial
applications relative to each o th e r (117).
b Retail price is based o n 1994 figures from Sigma Chemical Co., St
Louis, Missouri.
21
III. A lginate Biosynthesis

1. Pathw ay
In 1966, th e b io sy n th etic ro u te of alg in ate p ro d u c tio n was
established for th e m a rin e brow n seaw eed Fucus gardneri (73, 74).
Later, a sim ilar p ath w ay was p ro p o sed fo r alginate p ro d u c tio n by
A zotobacter vinlandii (99). In 1981, Piggott e t al (98) p ro p o sed a
p ath w ay fo r alg inate p ro d u c tio n in P seudom onas aeruginosa (Fig.
5). This path w ay was b ased o n th e d etectio n o f all th e enzym es
n e c e ssa ry fo r b io sy n th esis of b a c te ria l alg in ate, ex cep t fo r the
epim erase a n d the O-acetyl transferase.
Fructose 6 -p h osphate is co n sid ered to be th e p re c u rso r in the
alg in ate b io sy n th etic p ath w a y . T he p rim a ry ro u te o f glucose
c a ta b o lism in p se u d o m o n a d s is th ro u g h th e E ntn er-D o u d o ro ff
p ath w a y , p ro d u cin g g ly c erald e h y d e 3-p h o sp h a te a n d p y ru v a te .
Carlson a n d M atthews (13) show ed th a t th e C-6 , b u t n o t the C -l of
glucose was in c o rp o rate d in to alginate. This im p lied th a t ca rb o n
atom s 1, 2, a n d 3 of glucose w ere co n v erted to p y ru v ate by th e 2-
keto 3-d eo x y p h o sp h o g lu co n ate ald o lase re a c tio n a n d ev e n tu ally
w ere lo st to th e alg inate b io sy n th etic p ath w ay as CO2 a n d acetyl
CoA. C arbon atom s 4, 5, a n d 6 w ere chan n eled into alginate through
g ly c e ra ld e h y d e 3 -p h o sp h a te . F ru c to se 6 - p h o s p h a te c a n b e
p ro d u c e d e ith e r th ro u g h gluconeogenesis o r b y c o n d e n sa tio n of
g ly c e ra ld e h y d e 3 -p h o s p h a te w ith d ih y d ro x y aceto n e p h o sp h ate.
S u b se q u e n t stu d ie s w ith a P. aeruginosa m u ta n t d e fic ie n t in
fru c to s e 1 ,6 d ip h o s p h a te a ld o la s e sh o w ed t h a t th e r e w as
p re fe re n tia l 14c in c o rp o ra tio n fro m C-6 in to a lg in ate co m p ared
w ith C -l labeled glucose in the w ildtype. In co rp o ratio n of C-6 an d
22
fructose 6-phosphate
Phosphomannose isorerase
Mannose 6-phosphate
Phosohomannonutase
Mannose 1-phosphate
GDP-r.annose pyrcpncspnory 1ase
GDP-mannose
GDP-nannose denvcrooenasc
GDP-nannuromc acid
Pol viiierase
Epinerase
Acetyl transferase-
ALGI NATE
F igure 5. T he p ro p o s e d b io sy n th e tic p a th w a y o f a lg in a te in
A zo to b a cter vinlandii a n d P seudom onas aeruginosa. T he firs t 4
enzym atic step s h av e b e e n id e n tifie d in b o th o rg an ism s. The
p o ly m e ra se a n d ep im erase h av e been id e n tified in A. vin la n d ii
only, an d the acetyl tran sferase has not yet been identified in eith er
organism .
23
C -l of glucose w ere sim ilar in th e m u ta n t (7, 78). A su m m ary of

th e o v erall ro u te s o f in c o rp o ra tio n of fru c to se a n d glucose in
alginate can be seen in Figure 6 .
F ru cto se 6 -p h o sp h a te can be iso m e riz e d to m a n n o se 6 -
p h o s p h a te b y p h o sp h o m a n n o se iso m erase. T ra n s f e r o f th e
p h o sp h a te g roup by p h o sp h o m an n o m u tase p ro d u ces m an n o se 1 -
p h o sp h ate fro m m annose 6 -phosphate. M annose 1-p h o sp h ate a n d
GTP a re th e n c o n v e rte d in to G D P-m annose, c a taly zed b y GDP-
m an n o se p y ro p h o sp h o ry lase. The su b se q u en t o x id atio n th ro u g h
G D P-m annose d eh y d ro g en ase re su lts in th e fo rm a tio n o f GDP-
m annu ro n ic acid. Polym erization of GDP-mannuronic acid resu lts in
p o ly m a n n u ro n a te w hich is secreted fro m th e b a c te ria l cell a n d
b ec o m es th e s u b s tra te fo r a n e x tra c e llu la r a c e ty la se a n d / o r
epim erase in the p ro d u ctio n o f the m atu re polym er (47).
The tra n sp o rt m echanism of th e b acterial alg inate across the
cytoplasm ic m e m b ra n e is believ ed to be sim ilar to th a t o f some
b acterial cell wall polym ers, w hich use iso p re n o id lip id c a rrie rs
(134). The final step in th e biosynthesis o f b acterial alginate is the
selective epim erization o f th e m a n n u ro n a te resid u es to g u lu ro n ate
b y a n ex tracellu lar epim erase. T he selectiv ity is th o u g h t to be
d ic ta te d b y th e O -a c e ty la tio n o f th e m a n n u ro n a te re sid u e s.
A cetylation is believed to in h ib it epim erization, th e re b y dictating
th e final com position of th e alginates. Acetyl CoA is th e p ro b ab le
source of th e acetyl group in b acterial alginates (136), ju st as it is
o n th e m a n n o s y l r e s id u e s o f x a n th a n g u m p r o d u c e d by
X anthom onas cam pestris ( 6 6 ). T he m e ch an ism o f b a c te ria l O
acetylation is n o t well defined.
Fructose G 1u c o s o >• G l u c o n a t e
Fructose 1-phosphate 2-Ketogluconate
Fructose 6-phosphate ■* ►Glucose 6-phosphate
6-Phosphogluconate
ALGINATE
Fructose 1,6-diphosphate
2-Keto 3-deoxy
phosphogluconate
Di h y d r o x y a c e t o n e Glyceraldehyde 3-phosphate
phosphate
Pyruvate
TCA
cycle
Figure 6 . A co m p ariso n of th e overall ro u tes of in co rp o ratio n of fructose a n d glucose in alginate

pro d u ced by Ps. aeruginosa (47).
25
2. Enzymology
O nly fo u r of th e seven p ro p o se d en zy m atic step s in th e
biosynthetic pathw ay of alginate fro m fru cto se 6 -p h o sp h ate to th e
m a tu re p o ly m er h av e b ee n positively id e n tified in Pseudom onas
aeruginosa (9 8 ). Six o f th e ste p s h a v e b e e n id e n tif ie d in
A zotobacter vinlandii ( 8 6 ). The first fo u r enzym es in th e path w ay
h av e b ee n fo u n d in b o th organism s a n d h av e b een th e subject of
m u ch in v e stig a tio n . In P. aeruginosa th e genes en co d in g these
e n z y m es a re lo c a te d a t a p p ro x im a te ly 34 m in u te s o n th e
chrom osom e linkage m ap (89, Fig. 7). W hat is know n a b o u t these
enzym es is sum m arized below.
P h o s p h o m a n n o s e is o m e ra s e : T his en zy m e cataly ses th e
re v e rsib le c o n v e rsio n o f fru c to se 6 -p h o sp h a te to m a n n o se 6 -
p h o sp h a te . The en zy m e is th e p ro d u c t of th e algA g en e in P.
aeruginosa, a n d has a m olecular w eight of approxim ately 56,000 on
th e b a s is o f s o d iu m d o d e c y l s u lf a te - p o ly a c ry la m id e gel
electrophoresis (SDS-PAGE, 82). O verexpression o f th e algA gene
p ro d u c e s in c re a s e d ac tiv ity of th e n e x t tw o en z y m e s o f th e
p a th w a y , i.e., p h o s p h o m a n n o m u ta s e and GDP m a n n o s e
p y r o p h o s p h o ry la s e (4 8 , 114). In th e o v e re x p re sse d state,
phosphom annose is o m e ra s e w as s e p a ra te d from
p h o sp h o m a n n o m u ta se b u t co uld n o t be s e p a r a te d fro m GDP
m annose p y ro p h o sp h o ry lase (114). The inability to sep arate these
two activities has led to the proposal that, in P. aeruginosa, th e algA
gene encodes a single p ro te in having p h o sp h o m an n o se isom erase
an d GDP m annose pyrophosphorylase activities.
26
Switching
G enes" v
7570' algR Modulator

I srgH Genes
a rg B
P o s it i v e
Effectors
algA
r~Z------------- \
Structural
Genes
Figure 7. The relative lo catio n of the alginate (alg) genes on the

chrom osom e linkage m ap of Pseudomonas aeruginosa. (89).
27
P h o s p h o m a n n o m u ta s e : This enzym e co n v erts m an n o se 6 -

p h o sp h a te to m annose 1-phosphate. The enzym e is th e p ro d u c t of
th e algC gene, a n d h a s a m o lecu lar w eight o f 3 8 ,0 0 0 . It was
d e te c te d in th e soluble cytoplasm ic fractio n o f b o th m u co id a n d
n o n m u co id strain s of P. aeruginosa, from clinical a n d non clin ical
sources (92). P hosphom annom utase has an ab so lu te re q u ire m e n t
fo r glucose 1,6-diphosphate for its activity (114, 151). In d u ctio n of
th e algA gene increases p h o sp h o m an n o m u tase activity b y a b o u t 10
fold.
GDP m a n n o s e p y r o p h o s p h o ry la s e : This enzym e catalyses
the form ation of GDP m annose from m annose 1-phosphate a n d GTP.
This enzym e was d etected in P. aeruginosa by Piggott e t al (98). It
was suggested th a t th e activity of this enzym e m ay re p re s e n t one
o f th e two en zy m atic activ ities o f th e algA gene p ro d u c t. This
enzym e m ay be an exam ple of a bifunctional p ro te in w here th e two
activities catalyze noncontiguous steps in a biosynthetic pathw ay.
GDP m a n n o s e d e h y d ro g e n a s e : This enzym e catalyses the
oxidation of GDP m annose to GDP m an n u ro n ic acid (98, 105). The
p ro d u c t o f th e algD gene, GDP m an n o se d eh y d ro g en a se h as b een
p u rifie d a n d fo u n d to be a h ex am er w ith a m o lecu lar w eight of
290,000 (9). Its absence in alginate negative m u tan ts suggests th a t
it is essen tial fo r alg in ate biosynthesis. The algD gene h a s b ee n
cloned a n d sequenced. It is tran scrip tio n ally activ ated in m ucoid,
b u t n o t in n o n m u co id strain s of P. aeruginosa (26, 27). T he gene
co n tain s regions th a t h av e co n sid erab le sequence hom ology w ith
two o u te r m em b ran e p ro te in genes (ompF a n d ompC) fro m E. coli
28
(9). Like ompF an d ompC, expression of the algD gene is regulated

by external osm olarity (9).
The final reactions in th e biosynthesis of b acterial alginate in
P. aeruginosa a re n o t y e t fully u n d e rsto o d . From th e stru c tu ra l
com position of alginate, it has b een p ro p o se d th a t th e fin al steps
include polym erization of th e m a n n u ro n a te residues, O -acetylation
of some of those residues, ex p o rt of th e polym er, a n d epim erization
o f th e ca rb o x y l g ro u p a t C-5. T h e a s su m p tio n is th a t th e
polym erase uses GDP m an n u ro n ic acid as its su b strate a n d th a t the
p ro d u ct of this reactio n is p o ly m an n u ro n ate. The evidence fo r this
is b ased o n th e o b serv atio n th a t in A zotobacter vinlandii a n d th e
b ro w n seaw eed s th e re is a n e p im e ra se th a t c o n v e rts c e rta in
m a n n u ro n a te resid u es to g u lu ro n a te a t th e p o ly m e r level. The
p o lym erase in P. aeruginosa h as p ro v ed ex ceptionally difficult to
m easure an d the enzym e has n o t b een p u rified . V ery low levels of
activity of this enzym e h ave been m easu red in m em b ran e fractions
p re p a re d fro m cell ex tracts of m ucoid P. aeruginosa (47), b u t th e
characterizatio n of this polym erase is still a t a p relim in ary stage.
The m e c h a n is m by w h ic h g u lu r o n a te r e s id u e s a re
in c o rp o rate d in to Pseudom onas alg in ate rem ain s a m y stery . The
a ssu m p tio n h as b e e n th a t a p o ly m an n u ro n ic acid C-5 ep im erase
acts a t th e p olym er level to co n v ert som e m a n n u ro n a te residues to
g u lu ro n a te resid u es. The ep im erase fro m A. vin la n d ii h as b e e n
p u rified to h o m o g en eity (126), a n d re c e n tly Piggott e t al (98)
is o la te d th e gene e n c o d in g a C-5 e p im e ra s e (algG) fro m P.
aeruginosa. The enzym e re q u ire d Ca44- fo r activity, m u ch like the
enzym e isolated from A. vinlandii. AlgG m u tan ts w ere fo u n d to be
29
in c ap ab le of in co rp o ratin g guluronic acid resid u es in to b acterial

alginate.
The epim erase o f P. aeruginosa ap p e ars to be d iffe re n t from
th e en zy m e iso la te d fro m A . vinlandii. T he M /G ra tio o f P.
aeruginosa alginate is n ev er less th a n 1 .0 , a n d this ratio is generally
u n altered b y changes in grow th conditions. In A. vinlandii th e M/G
ratio m ay be less th a n 1 .0 an d th e resulting block stru ctu re m ay be
a lte re d b y changes in CaH+ co n c en tratio n s in th e grow th m ed iu m
(70).
The alg in ates of P. aeruginosa a re h ig h ly O -acetylated w ith
th e O -acety l g ro u p s b e in g a s s o c ia te d e x c lu siv e ly w ith th e
m a n n u ro n a te resid u es. It is th o u g h t th a t one of th e fu n ctio n s of
acetylatio n is to p ro tec t th e m a n n u ro n a te resid u es in th e p o ly m er
fro m ep im erizatio n to g u lu ro n a te resid u e s (126). R ecently, an
alginate m odification gene, algF, was sequenced. This gene codes
fo r a 28 k d p ro tein w hich controls the ad d itio n of O-acetyl groups to
th e m a n n u ro n ic acid residues. The algF gene was re p o rte d to be
n o n e s s e n tia l fo r a lg in a te b io s y n th e s is , b u t is r e q u ir e d fo r
acetylation of the alginate polym er (44).
A m ajo r difference betw een th e alginates of A.vinlandii, an d
P. aeruginosa is th e a rra n g e m e n t of m a n n u ro n a te a n d g u lu ro n ate
resid u es w ithin each polym er. T he p o ly m er fro m A. vin la n d ii is
a r r a n g e d in to h o m o p o ly m e ric b lo ck s o f m a n n u ro n a te a n d
guluronate, w hereas in P. aeruginosa alginate p o lyguluronate blocks
are absent. This difference reflects differences in O -acetylation an d
e p im e riz a tio n a c tiv itie s b etw e en th e se two b a c te ria . T h e se
30
differences m ay in dicate alterations in th e la tte r stages of alginate

biosynthesis in P. aeruginosa from those proposed in Figure 5.
3. Regulation
In th e lungs o f cystic fibrosis p a tie n ts Alg (-) stra in s of P.
aeruginosa usually convert to the m ucoid Alg (+) form . In vitro, the
Alg (+) p h en o ty p e is u n stab le a n d Alg (-) re v e rta n ts a p p e a r o ver
tim e. Genetic m apping experim ents have show n th a t sp o n tan eo u s
alginate conversion is b ased on th e gene p ro d u cts of th e algB, algR,
algS, a n d algT genes (42, 45, 79, 90). The p rim a ry genetic ev en t
th a t regulates th e " o n /o f f switch in alginate p ro d u ctio n is handled
p rim arily by th e algS a n d algT genes located a t ap p ro x im ately 6 8
m inutes on th e chrom osom al linkage m ap of P. aeruginosa (89, Fig.
7).
Spontaneous conversion betw een th e m ucoid an d nonm ucoid
state is the resu lt of a genetic alteratio n a t th e algS gene locus. The
algS gene is a genetic sw itch th a t co n tro ls th e expression of algT
(42). Form ation of th e A lgS p ro tein resu lts in th e activation of the
algT gene. The gene p ro d u c t of algT acts as a reg u lato ry p ro te in in
th e biosynthesis of b acterial alginate in P. aeruginosa by prom oting
th e activ atio n of th e stru c tu ra l genes involved in th e bio sy n th etic
pathw ay (42, 43).
Along w ith its ro le in reg u la tio n of th e stru c tu ra l genes in
alginate pro d u ctio n , th e algT gene p ro d u c t also is involved in the
exp ressio n of th e algB gene (149). T he algB gene is lo c a te d at
approxim ately 12 m in u tes o n th e chrom osom e m ap (Fig. 7). Its
gene p ro d u ct is n o t directly involved in th e biosynthetic p ath w ay of
31
alg in ate b u t is a p p a re n tly involved in h igh level p ro d u c tio n of

alginate in P. aeruginosa (49). The algB gene p ro d u c t belongs to a
class o f p ro te in s th a t c o n tro l gene tra n s c rip tio n in resp o n se to
environ m en tal stim uli (149).
T h e la s t g en e in v o lv e d in th e r e g u la tio n o f alginate
b io sy n th e sis is th e algR gene. T his re g u la to ry g en e controls
tra n sc rip tio n o f th e algD gene, w hich en co d es fo r GDP m an n o se
dehydrogenase, an essential enzym e in the biosynthesis of b acterial
alg in ate (98). DNA seq u en ce analysis show ed a h ig h d eg ree of
h o m o lo g y b e tw e e n th e algR g en e a n d o th e r environm entally
responsive b acterial reg u lato ry genes, including ompR, phoB, ntrC,
a n d spoA (28). This in d ic a te s th a t th e p ro d u c tio n of b ac teria l
alg in ate is affected by en v iro n m en tal stim uli o r specific chem ical
com pounds p rese n t in th e environm ent.
Recently, th e m ech an ism o f alg in ate p ro d u c tio n co n tro l by
algB, algR, a n d algT has b een exam ined. C om pared w ith Alg (+)
strain s, d e letio n m u ta tio n s in th e algB a n d algT g enes show ed
h ig h ly re d u c e d tra n sc rip tio n a l activ ity in th e b io sy n th e tic gene
c lu ste r (at 34 m in u tes, 49). This in d ic a te d th a t th e p a th w a y of
alg in ate b io sy n th esis is u n d e r co n tro l n o t only by th e algR gene
p ro d u c t b u t also b y th e gene p ro d u cts of algB a n d algT. W h eth er
th e algT a n d algB gene p ro d u cts act directly on an y p ro m o ters in
th e b io sy n th etic gene clu ster o r o n o th e r re g u la to ry genes is n o t
known.
32
IV. Pseudom onas syringae Alginates

N um erous p ath o v ars of P. syringae w ere re p o rte d to produce
b acterial alginates (37). El Banoby an d R udolph (34) re p o rte d th a t
th e EPS from several p la n t pathogenic p seudom onads w ere capable
of inducing w ater soaked lesions o n com p atib le leaf tissue. This
suggested th a t alginate m ay be necessary fo r successful colonization
o f p la n t h o st tissue. In com m on w ith o th e r b a c te ria l alg in ates,
those p ro d u ced by P. syringae are highly acetylated. The M/G ratio
is v ariab le am ong strain s an d am ong d iffe re n t p re p a ra tio n s from
th e sam e strain . Fett e t al (38) re p o rte d th a t th e p erce n tag e of
g uluronic acid in P. syringae alg in ates v a rie d fro m < 1% to 28%
w hen grow n in planta, a n d th a t th e in planta sam ples h a d a h ig h er
degree of acetylation th a n the alginates p ro d u c e d in vitro. It was
also re p o rte d th a t th e P. syringae alg in ates w ere sm aller, o n the
average, th a n those fro m P. aeruginosa (37). The n u m b e r average
m o le cu lar w eights ra n g e fro m 3.8 x 10^ fo r a lg in a te s fro m P.
syringae pv glycinea produced in vitro, to 47.1 x 1 0 ^ for P. syringae
pv papulans alginate p ro d u ced in vitro, co m p ared to P. aeruginosa
alginates w hose size was re p o rte d ly in th e ran g e of 1 0 ^ (37). It
ap p e a rs th a t alg in ate b iosynthesis is a com m o n p ro p e rty o f th e
m ajority of pseudom onads in rRNA-DNA hom ology group 1 (29, 40,
94).
V. Polysaccharide Analysis
1. Depolym erization
C h a ra c te riz a tio n o f a n y p o ly s a c c h a rid e s ta r ts w ith a
com positional analysis. Acid hydrolysis is com m on to m ost m ethods
33
o f d e te rm in in g th e p h y sic a l co m p o sitio n o f a polysaccharide.

Hydrolysis is usually co n d u cted w ith dilute m in eral acids, th e m ost
com m on being sulfuric acid, hydrochloric acid, or trifluoroacetic acid
a t 100°C fo r vary in g lengths of tim e. M any facto rs influ en ce th e
r a te o f h y d ro ly sis o f a n y p o ly sa cc h arid e, in c lu d in g rin g size,
configuration, conform ation, a n d p o larity of th e co m p o n en t sugars
( 8 ).
In studying th e com position of alginic acid, m ost rese arch ers

re d u c e th e u ro n ic acids to a n e u tra l p o ly m e r to facilita te acid
h y d ro ly s is w ith o u t th e rm a l d e s tr u c tio n o f th e c o m p o n e n t
m onosaccharides (13, 37, 38, 91, 150). Acid h y d ro ly sis of n e u tra l
polysaccharides has b een studied, an d the m echanism now accepted
was first suggested b y Edward (33) an d is d ep icted in Figure 8 fo r
th e hydrolysis of m ethyl p-D glucopyranoside. The process involves
p ro to n a tio n o f th e glycosidic oxygen atom to fo rm th e conjugate
acid, follow ed by th e fo rm atio n of a cyclic carbonium -oxonium ion
w hich p ro b ab ly exists in the h alf ch air conform ation having C-2, C-
1, O, a n d C-5 in a p la n e. R eaction w ith w ate r th e n gives th e
p ro to n ate d reducing sugar an d from it the reducing sugar is form ed.
Alginic acid, being a polyuronide, contains a carboxyl g roup at
th e C-5 position of each com ponent m onosaccharide. This carboxyl
g ro u p co n fers acid resistan ce to th e glycosyluronic acid linkages
(15). M any th e o rie s h av e b ee n ad v a n ce d as to w hy. T h ere is
evidence th a t th e en h a n c e d stab ility m ay be a ttrib u te d to e ith e r
steric factors o r to inductive effects (145). Ranby a n d M archessault
(107, 108) fo rm u la te d a n in d u c tio n -sta b iliz a tio n th e o ry , w hich
p ro p o se d th a t th e glycosidic b o n d is stab ilized by th e in d u c tiv e
Figure 8 . The m echanism of acid hydrolysis of glycosides. The carbonium ion in term ed iate (3) is in
the half-chair conform ation ( 8 ). OJ
35
effect of th e p o la r carboxyl group. A ccording to this th eo ry , the

presence of an electronegative group, such as a carbonyl or carboxyl
group, can exert inductive influences on the glycosidic oxygen atom .
In a polyuronide, th e carboxyl group w ould oppose the p ro to n atio n
of th e glycosidic oxygen atom s by m aking the electro n p airs on the
glycosidic oxygen atom s less basic, resulting in a m ore stable bond.
A p o ssib le fo rm a tio n o f six m e m b e re d rin g s, w h ere h y d ro g e n
stabilizes th e negatively ch arg ed carboxyl group an d th e glycosidic
oxygen ato m h as also b ee n p o stu lated (107). A lth o u g h th e exact
ex p lan atio n fo r th e stab ility of glycosyluronic acid b o n d s has n o t
b een pin p o in ted , suffice it to say th a t these bonds are m ore difficult
to hydrolyze th a n th e n eu tra l O-glycosidic bonds.
M any f a c to r s in f lu e n c e th e ra te o f h y d ro ly s is o f
polysaccharides. The ease o f hydrolysis a t a p artic u lar te m p eratu re
a n d acid c o n c en tratio n in creases in th e o rd e r g lu co p y ran o sid e <
fru cto p y ran o sid e < fru cto fu ran o sid e (63). This in d icates th a t ring
size affects hydrolysis. T h ere is a d ire c t rela tio n sh ip betw een the
strain (or free energy) associated w ith a m olecule an d th e ra te of
hydrolysis (121). In general, aldofuranosides a n d aldoheptanosides
are h y d r o ly z e d m o re ra p id ly th a n th e c o rre s p o n d in g
aldopyran o sid es. The five an d seven m em b ered rings a re strained
because of th e distortion of the te tra h e d ra l angle of th e ring carbon
atom s. The p y ran o id rings can p ucker to elim inate strain (35).
The anom eric configuration in pyranosides also plays a role in
th e stab ilities of th ese su g ars u n d e r acid h y d ro ly sis conditions.
F e a th e r a n d H a rris (3 6 ) used a n o m e ric p a irs o f m e th y l
ald opyran o sid es to study the affects of the anom eric configuration
36
on acid stability. They fo u n d th a t an an o m er w ith an e q u a to rial

m e th o x y l g ro u p h y d ro ly z e s m o re r a p id ly th a n a n a n o m e r
containing an axially o rien ted group. Two exp lan atio n s have b een
p ro p o se d . First, e q u a to ria l b o n d s are c o n sid e re d to be m o re
accessible th a n axial bonds, th u s m aking th em m o re available for
p ro to n tra n s fe r fro m a h y d ro n iu m io n to th e glycosidic oxygen
ato m . A lte rn a tiv e ly , th e g re a te r re a c tiv ity o f th e e q u a to ria l
su b s titu e n t is d u e to its h ig h e r fre e en e rg y ca u sed b y a p o la r
in te ra c tio n betw een th e eq u a to rial m ethoxyl g ro u p a n d th e rin g
oxygen atom (33).
2. Acid Sensitivity
M onosaccharides are d eg rad ed b y acid to a g re a te r o r lesser
ex ten t d ep en d in g on the sugar a n d stren g th of th e acid (119). In
ad d itio n to d e g ra d a tio n , acids m ay co n v ert sugars in to a n h y d ro
derivatives. Spontaneous conversion to a 1,6 an h y d ro ald o p y ran o se
occurs in acidic solutions of several aldoses a n d ketoses having the
id o (1 0 0 , 102, 146), a ltro (101, 112), a n d g ulo (1 3 1 , 132)
configurations. In d ilu te acid solutions those sugars o f th e gluco,
m anno, and g a la c to c o n fig u ra tio n s a re a lm o s t com pletely
h y d ro ly z e d to th e free aldoses. Reeves (111) first suggested an
ex p lan atio n fo r this d iffe re n t b eh av io r in term s of conform ational
in teractio n s. He show ed th a t (3-D-idose, w ith all h y d ro x y l groups
eq u a to rial, h as a h ig h e r p ro p en sity to fo rm 1 ,6 a n h y d rid e s th a n
d o es p-D-glucose w hose h y d ro x y l groups a re all o rie n te d axially.
This system rep resen ts a good exam ple of conform ational control of
37
an equilibrium . The position of the equilibrium is d e p e n d e n t on the

steric arran g em en t of th e groups n o t taking p a rt in th e reaction.
The solutions of an y red u cin g sugar co n tain a n eq u ilib riu m
m ixture of the IC 4 an d 4 c i form s of the a and (3 an o m er s. Only the
IC 4 form of th e (3 an o m er can d irectly form th e an h y d rid e w ithout
change in configuration an d conform ation. 1,6 A nhydride form ation
is believed to occur in two steps: 1 ) conversion of o th e r form s of the
sugar into th e IC 4 fo rm of the |3-D anom er, an d 2) fo rm atio n of the
a n h y d rid e . P ra tt a n d R ich tm y er (104) sho w ed th a t a n axial
h y d ro x y l g roup a t C-3 is th e m ost im p o rta n t axial c o n stitu e n t in
determ in atio n of 1,6 an h y d rid e form ation. An axial hydroxyl group
a t C-3 can in te ra c t w ith a n a n h y d rid e b rid g e a n d d estab ilize th e
an h y d rid e. The fo u r hexoses having axial hydroxyl groups at C-3
(D -glucose, D -m annose, D -galactose, a n d D -talo se) fo rm less
a n h y d rid e th a n those hexoses whose C-3 hydroxyl group is o rien ted
equatorially (D-allose, D-altrose, D-gulose, an d D-idose). The overall
ability for 1,6 an h y d rid e form ation th en is:
ido > altro, gulo > talo > alio > galacto > m anno > gluco
Some d eb a te still exists a b o u t th e p lacem en t of talose co m p ared to

allose in th e above schem e. Some research ers claim talose is m ore
p ro n e to 1,6 a n h y d rid e fo rm atio n . O thers claim allose form s 1,6
an h y d rid es m ore readily (103, 141).
38
VI. Goals of This Study

T his stu d y w as d e sig n e d to o p tim ize th e c o n d itio n s fo r
p ro d u c tio n o f b ac teria l alg inate fro m P seudom onas syringae p v
phaseolicola, ATCC 19304 to d eterm in e the feasibility of large scale
p ro d u c tio n , a n d to c h a ra c te riz e th e p h y sical p ro p e rtie s o f th e
p ro d u c t, rela tin g th o se p ro p e rtie s to th e re su ltin g so lu tio n a n d
gelling p ro p erties of th e polym er.
MATERIALS AND METHODS
I. Organisms, Growth Conditions, a n d M aintenance

P seudom onas syringae subsp. phaseolicola ATCC 19304 was
o b tain ed from th e A m erican Type Culture Collection, Rockville, MD.
It was se lec ted fo r th is re se a rc h b ecau se o f its lack o f h u m a n
p a th o g en ic ity a n d its p o te n tia l fo r p ro d u cin g a h ig h ly acety lated
bacterial alginate (39).
C ultures w ere m a in ta in ed at 4°C on Dworkin Foster (DF) agar
(32) su p p le m e n te d w ith 2% (w /v) gluconic acid. T his m e d iu m
c o n ta in e d (in gram s p e r lite r of d eio n ized w ater): KH2 PO4 , 4.0;
Na 2 HTC>4 , 6.0; NaCl, 0.4; KNO3 , 9.1; (NH4 ) 2 S0 4 , 0.9; MgS0 4 • 7 H 2 O,
0.2; gluconic acid , 20; a n d ag ar, 15. T he g lu co n ic a c id was
aseptically ad d e d to th e salts m edium after sep arate sterilization in
an autoclave a t 121°C, a t 15 l b s . / i n ^ o f p re ssu re fo r 15 m in u tes.
The pH of th e ag a r was betw een 6.9 a n d 7.0 p rio r to sterilization.
Plates w ere in o c u lated w ith 0.1 m l of a sta n d a rd iz e d 48 h o u r DF
b ro th cu ltu re o f P. syringae. This cu ltu re show ed a n ab so rb a n ce
betw een 1.9 a n d 2.0 a t 660 n an o m eters. The inoculum was sp read
using a flam e sterilized, b e n t glass rod. After grow th a t 30°C fo r 48
h o u rs, th e p lates w ere sto re d a t 4°C. C ultures w ere tran sferred
every fo u rth week.
All b ro th sta rte r cultures used in this w ork w ere p re p a re d by
inoculating P. syringae ATCC 19304 from agar plates in to 100 ml of
DF b ro th in 250 m l E rlenm eyer flasks. Cultures w ere in c u b ate d at
30°C fo r 48 h o u rs a t 180 rp m on a NBS Model G25-KC ro ta ry shaker
(New B runsw ick Scientific Co. Inc., Edison, NJ). C u ltu res w ere
39
40
s ta n d a r d iz e d w ith s te rile w a te r to a n a b s o r b a n c e a t 660

n a n o m e te rs b e tw e e n 1.9 a n d 2.0 o n a G ilfo rd R e sp o n se II
spectro p h o to m eter (Gilford In stru m en t Lab., O berlin, OH) p rio r to
use.
II. A nalytical M ethods

1. Cell Mass D eterm ination
D ry cell w eight fo r calculation of specific yield of alg inate or
p e rc e n t acety latio n was m e asu red d irectly . B roth c u ltu re s w ere
cen trifu g e d at 17,000 x g fo r 15 m in u tes in a Sorval S u p ersp eed
M odel RC-5B cen trifu g e (Du Pont Co., W ilm ington, DE) to rem ove
b acterial cells. Cells fro m ag ar m ed ia w ere resu sp e n d ed in 100 m l
of deio n ized w ater. The suspension was c e n trifu g e d a t 27,000 x g
for 60 m in u tes to p ellet th e cells fro m th e highly viscous solution.
Once th e cells w ere sep arated , the pellets w ere tre a te d equally. The
p e lle ts w ere w ash ed tw ice in 10 m l o f d e io n iz e d w ate r. T he
s u p e rn a ta n t was d isc ard ed a n d th e p ellet was re s u sp e n d e d in a n
equal volum e of d eionized w ater. The cell suspension was p o u re d
into a p re-d ried , tared , alum inum w eighing d ish a n d was d rie d in a
drying oven a t 100°C to a co n stan t weight.
2. Total C arbohydrate Q uantitation

T he to ta l c a rb o h y d ra te p r e s e n t in P. sy rin g a e EPS w as
d e te rm in e d b y th e p h en o l-su lfu ric acid assay (31). The p ro to co l
was as follows:
41
1) To 2 ml of carb o h y d rate solution containing 10 to 100 |.ig of

carb o h y d rate, 0.05 ml of 80% (v/v) aqueous p h en o l was
added.
2) The solution was shaken in a Vortex m ixer fo r a co u n t of 5.
Then, 5 m l of co n cen trated sulfuric acid was ad d e d a n d th e
solution m ixed again fo r an o th er count of 5.
3) The tubes w ere in cu b ated at room te m p eratu re fo r 30
m inutes to allow the color to develop. The ab sorbance was
m easu red at 485 n an o m eters on a Gilford Response II
spectrophotom eter (Gilford Instrum ent Lab., O berlin, OH).
This value was com pared to a sta n d ard curve of sodium
alginate from M acrocystis pyrifera (Sigma Chemical Co., St
Louis, MO) fo r d eterm in atio n of the total carb o h y d rate
p rese n t in the solution.
3. Alginate Q uantitation
A lginate co n c e n tra tio n s w ere d e te rm in e d b y two d iffe re n t
m ethods. These m eth o d s inclu d ed the uronic acid assay d escrib ed
by B lum enkrantz an d A sboe-H ansen (10), a n d th e carb azo le assay
o f K nutson a n d Jean es (69). Each m e th o d h a s its b en efits. T he
uronic acid assay is rap id , b u t is only accu rate u p to 150 itig/ml of
uronic acid. The carbazole assay takes m uch longer to p erfo rm , b u t
it is accu rate up to 1 0 0 0 ng/ml.
The uronic acid assay was used p red o m in ately w ith alginates
p ro d u ced in b ro th cultures. This assay allow ed d irec t testing o f the
su p e rn a ta n t w ithout color in terferen ce from th e rem ain in g salts in
solution. The protocol was as follows:
42
1) To 0.2 m l of sam ple containing from 0.5 to 30 f.ig uronic

acid, 1.2 ml of 12.5 mM tetrab o rate in co n cen trated sulfuric
acid was added.
2) The tubes w ere chilled in an ice b ath for 10 m inutes.
3) The m ixture was shaken in a Vortex m ixer, an d th e tubes
h e a te d in a w ater b a th at 100°C for 5 m inutes.
4) A fter cooling in a water-ice bath, 20 |xl of 0.15% (w/v)
m eta-hydroxydiphenyl in 0.5% (w/v) sodium h ydroxide was
ad d e d to the above m ixture.
5) The tubes w ere shaken fo r a count of 5 an d th e ab sorbance
re a d a t 520 n m as d escrib ed previously. This value was
co m pared to a sta n d a rd curve of sodium alginate from
M acrocystis p yrifera to obtain the concentration of uronic
acid.
T he c a rb a zo le assay was u sed fo r h ig h co n cen tratio n s of
p u rified alginate. E xperim ental e rro r d im in ish ed w ith th is assay
because o f few er d ilu tio n steps. The p ro to co l fo r the carb azo le
assay was as follows:
1) 0.5 ml of a p u rified alginate solution containing 0 to 500 jxg
of alginate was eq u ilib rated in a w ater-ice b ath fo r 10
m inutes.
2) T hree ml of cold concen trated sulfuric acid was ad d e d to
this solution. The m ixture was re-eq u ilib rated in a w ater-ice
bath.
3) The solution was th e n m ixed with a Vortex m ixer fo r a
co u n t o f 4 an d h ea ted at 55°C for 20 m inutes in a w ater bath.
43
4) The solution was th en re-eq u ilib rated in a w ater-ice b ath

fo r 10 m inutes, a n d 0 .2 m l of a 0 .2 % (w/v) carbazole solution
(Eastm an Kodak, Rochester NY) in ethanol was add ed .
5) The sam ple was m ixed w ith a Vortex m ixer fo r a co u n t of
10 a n d allow ed to in cu b ate at room tem p eratu re fo r 3 h o u rs
for color developm ent.
6 ) The sam ple absorbance was re a d at 530 n m as described
previously. This value was com pared to a sta n d a rd curve of

sodium alginate from M acrocystis pyrifera to d eterm in e
bacterial alginate concentrations.
4. Acetyl Q uantitation
T h e p e rc e n t a c e ty la tio n w as d e te rm in e d b y th e m e th o d
d escrib ed b y McComb a n d M cCready (80). A s ta n d a rd cu rv e fo r
p erce n t acetylation was p re p a re d w ith glucose p e n taac eta te (Sigma
C hem ical Co., St. Louis, MO). P rio r to assay , all sam p les w ere
d esalte d b y dialysis fo r 48 h o u rs against d eio n ized w ater a t ro o m
tem p eratu re. The protocol was as follows:
1) One volum e of 9.4% (w /v) sodium hyd ro x id e was a d d e d to
one volum e of 3.75% (w/v) hydroxylam ine solution.
2) To 2 m l of th e above m ixture, 0.5 ml of the sam ple solution
was ad d e d w ith agitation.
3) A fter 5 m inutes, 0.5 m l of acid m eth an o l was ad d e d w ith
agitation, th e n 1.3 m l of th e ferric p erch lo rate solution was
added.
44
4) After 5 m inutes, the precip itated hydroxam ic acid an d

ferric com plex was rem oved by m icrocentrifugation for 3
m inutes in a Sorval M icrospin 24s M icrocentrifuge (Du Pont
Co., Wilmington, DE).
5) Color intensity was d eterm in ed b y m easuring ab sorbance
a t 520 n m as described above. This value was co m pared to
th e sta n d a rd curve of glucose p en taacetate to d eterm in e th e
p ercen t acetylation.
The reag en ts fo r acetyl q u an titatio n w ere p re p a re d as follows:
1) Acid M ethanol Solution
Chilled reag en t grade absolute m eth an o l was a d d e d to 35.2 ml
o f chilled 70% p erch lo ric acid to m ake a 500 m l so lu tio n . This
solution was used as the acidic m ethanol solution.
2) Ferric P erchlorate Solution
F erric c h lo rid e (1.93 g) was d isso lv e d in 5 m l o f 70%
p erch lo ric acid a n d e v a p o ra ted alm o st to d ry n e ss. It w as th e n
d ilu ted to 1 0 0 m l w ith w ater for use as th e stock ferric p erch lo rate.
T hen 8.3 m l of 70% p erch lo ric acid was a d d e d to 60 m l o f stock
ferric p erch lo ra te solution. This solution was cooled in an ice b a th
a n d m ad e to 500 m l with chilled reag en t grade absolute m ethanol.
3) Glucose Pentaacetate S tandard Solution
P ure cry sta llin e p-D-glucose p e n ta a c e ta te (1 0 8 .9 m g) w as
dissolved b y h eatin g w ith gentle ag itatio n in a b o u t 5 m l o f eth y l
alcohol, an d m ade to 50 m l w ith deionized w ater. 2, 4, 5, a n d 7 m l
of this solution w ere th e n taken a n d m ade to 50 m l w ith deionized
w ater. These solutions re p re se n t 120, 240, 300, a n d 4 2 0 |ng/ml of
acetyl.
45
III. O ptim ization of Bacterial Alginate Production

1. M edia Composition:
A. C arbon Source
P. syringae ATCC 19304 was tested fo r its ab ility to p ro d u c e
a n a c e ty la te d b a c teria l alg in ate w hen grow n o n d iffe re n t carb o n
sources. The carb o n sources tested w ere fructose, glucose, sucrose,
glycerol, a n d gluconic acid. Each se p arate so lu tio n o f te st ca rb o n
source was auto clav ed a n d th en aseptically a d d e d to th e DF b ro th .
The c o n c e n tra tio n of each stock so lu tio n was 20% (w /v o r v /v ).
Each carb o n source was tested a t a final co n cen tratio n of 2% (w /v or
v /v ). A fter inoculation of DF b ro th from sta rte r cultures, (3%, v /v ),
P. syringae was in c u b ate d fo r 48 h o u rs w ith shaking as d escrib ed
p rev io u sly . At 48 h o u rs, th e c u ltu re b r o th w as c e n trifu g e d to
rem ove b acterial cells, a n d th e cell m ass d e te rm in e d as d escrib ed
previously. The cell free b ro th was analyzed b y th e phenol-sulfuric
assay (to ta l c a rb o h y d ra te p ro d u c tio n ), th e u ro n ic a c id assay
(alginate p roduction), a n d the acetyl assay (degree of acetylation).
B. N itrogen Source
P. syrin g a e was te ste d fo r its ab ility to p ro d u c e acetylated
b a c te ria l a lg in ate o n d iffe re n t n itro g e n so u rces. T he n itro g e n
sources tested w ere am m onia, [(NH4 )2 9 0 4 ], n itrate, (KNO3 ), n itrite,
(KNO2 ), a n d u rea. Each DF b ro th was m ade b y in co rp o ratin g only
the test co m p o u n d as a p o ten tial n itro g en source fo r th e organism .
Each n itro g e n so u rc e w as p la c e d in to th e m e d iu m a t in itia l
co n cen tratio n s of 2 mM, 5 mM, 9 mM, 12 mM, a n d 15 mM (w /v).
Gluconic acid was used as the carb o n source a t a final co n cen tratio n
46
of 2% (w /v). After inoculation, (3%, v /v ), from stan d ard ized sta rte r
cu ltu res, P.syringae was in c u b a te d fo r 48 h o u rs w ith shaking as
d escrib ed previously. At th e a p p ro p ria te tim e th e c u ltu re b ro th
w as c e n trifu g e d to rem o v e b a c te ria l cells, a n d th e cell m ass
d e te rm in e d as d escrib ed previously. T he b ro th was an a ly z e d b y
th e uronic acid assay for alginate production.
2. pH an d T em perature
The effect of pH on bacterial cell yield a n d alginate p ro d u ctio n
was investigated in DF b ro th . The initial pH's w ere 6.0, 6.2, 6.4, 6 .6 ,
6 .8 , 7.0, an d 7.2. Cell yield an d alginate p ro d u ctio n w ere m easu red ,
as d escrib ed previously, a fte r in cu b atio n a t 30°C fo r 48 h o u rs a t

180 rp m in a NBS M odel G25-KC ro ta ry sh a k er (New Brunsw ick
Scientific Co. Inc., Edison, NJ). In each case, p h o sp h ate b u ffer (0.03
M) was u sed to m ain tain th e pH of th e cu ltu re b ro th . Gluconic acid,
a t a final co n cen tratio n o f 2 % (w /v), was the carb o n source.
The effect of te m p e ra tu re on b acterial cell yield a n d alginate
p ro d u ctio n was investigated in DF b ro th . The te m p e ra tu re s te sted
w ere 25°C, 28°C, 29°C, 30°C, 31°C, an d 32°C. Both cell y ield a n d
alginate p ro d u ctio n w ere m easured, as d escrib ed previously. Each
cultu re was grown u n d e r th e sam e conditions as the pH cultures.
3. Agar vs. Broth Culture

Cell y ie ld s a n d a lg in a te p ro d u c tio n w ere m e a s u re d in 5
d ifferen t m edia, b o th on ag ar a n d in b ro th culture. All m ed ia w ere
m a d e u p in d e io n iz e d w ater. T he m e d ia in c lu d e d DF m ed ia,
n u trie n t m ed ia (Difco Lab. D etroit, MI), m ed ia com posed o f 3 g/L
47
beef extract (Difco Lab. D etroit, MI), m edia containing 5 g/L peptone
(Difco Lab. D etroit, MI), a n d m ed ia containing a m ix tu re of 3 g/L
b eef ex tract an d 5 g/L p eptone. All m edia w ere su p p lem en ted w ith
2% gluconic acid (w /v). The gluconic acid was sterilized sep arately
by autoclaving as a stock solution of 2 0 % (w /v), a n d a d d e d to the
c u ltu re m e d ia a sep tic ally a fte r ste riliz atio n . A gar m e d ia w ere
p re p a re d w ith 15 g/L agar. The pH of each test m ed ia was betw een
6.9 a n d 7.0 p r io r to ste riliz a tio n . All in o c u la w ere fro m a
sta n d a rd iz e d 48 h o u r s ta rte r c u ltu re o f P. syrin g a e grow n in DF
b ro th a t 30°C w ith shaking as described previously. Liquid cultures
w ere in o cu lated from th e stan d ard ized 48 h o u r sta rte r cu ltu re to a
fined con cen tratio n of 2% (v/v). Solid cu ltu res w ere in o cu lated w ith
0.1 m l of th e sam e sta n d a rd iz e d s ta rte r c u ltu re p e r 100 x 15 m m
p e tri dish containing 25 m l of m edium . T he in o cu lu m was sp rea d
using a flam e sterilized , b e n t glass ro d . All te st c u ltu re s w ere
allow ed to grow fo r 48 h o u rs a t 30°C. T he liq u id cu ltu res w ere
in cu b ated w ith shaking as described previously. At th e ap p ro p riate
tim e, th e cell yield an d alg in ate p ro d u c tio n of each c u ltu re w ere
m e a s u re d as d e s c rib e d p re v io u sly . A lg in ate p ro d u c tio n was
m e asu red by th e uronic acid assay. T otal alg inate p ro d u ctio n on
solid m ed ia was m e asu red by scraping th e b ac teria l grow th fro m
h a lf of a p e tri dish (allowing 2 m easu rem en ts p e r p e tri dish) an d
resu sp en d in g th a t grow th in 10 m l of d eio n ized w ater. T he cells
w ere th e n rem oved by centrifugation a n d the cell m ass d eterm in ed ,
as d escrib ed previously. A lginate p ro d u c tio n was assayed by the
u ro n ic acid assay. The to ta l a lg in ate p ro d u c tio n fo r th e solid
cultures was rep o rted in (.ig/cm2 .
48
IV. Batch F erm entations

Batch ferm en tatio n s w ere co n d u cted in 1 lite r v olum es in a
NBS 2.5 lite r Bioflo II b a tc h /c o n tin u o u s cu ltu re fe rm e n te r (New
Brunswick Scientific Inc., Edison, NJ) in DF b ro th su p p lem en ted w ith
2% (w/v) gluconic acid. T em p eratu re was m ain tain ed a t 30°C, an d
th e pH was m a in ta in e d a t 7.0 b y titra tio n w ith 3 M so d iu m
hydroxide. Air, filtered th ro u g h a sterile W hatm an H epavent filter
(W hatm an Inc., Clifton NJ) was supplied th ro u g h a sp arg er a t a ra te
o f 1 sta n d a rd lite r p e r m in u te (SLPM), a n d ag itatio n was a t 100
rpm . Samples w ere periodically w ithdraw n aseptically, th ro u g h o u t
the bacterial grow th cycle. Growth was m easu red by ab so rb an ce at
660 n an o m eters as d escribed previously. Alginate was m easu red in
th e cell free su p e rn a tan t by the uronic acid assay.
V. Purification of Bacterial Alginate

The b acterial alginates used fo r ch aracterizatio n studies w ere
obtained fro m eith er DF ag ar o r N utrient agar plates su p p lem en ted
w ith 2% (w /v) gluconic acid. Bacterial grow th was sc ra p ed off th e
ag ar plates using a b e n t glass ro d a n d re su sp e n d e d in 150 m l of
d e io n iz e d w a te r. T h e sa m p les w ere v o rte x e d u n til evenly
su sp en d ed a n d th e n the b acterial alginate was se p a ra te d fro m the
cells by cen trifu g atio n as d escrib ed previously. T hree volum es of
isopropanol w ere ad d e d to one volum e of clarified s u p e rn a ta n t to
p re c ip ita te th e p o ly sacch arid e. This so lu tio n was m ixed fo r 15
m in u tes a n d th e p re c ip ita te d alg in ate was rem o v e d b y w inding
a ro u n d a glass ro d . T he p re c ip ita te was th e n d rie d in aceto n e
followed by a ir drying.
49
P urity was d eterm in ed by the carbazole assay using sodium

a lg in a te fro m M acrocystis p yrife ra a s th e s ta n d a r d , a n d by
w avelength scans betw een 200 n m a n d 300 n m in in c re m en ts of
0.5 n m o n a G ilfo rd R esponse II s p e c tro p h o to m e te r (Gilford
In stru m en t Lab., O berlin, OH). Absence of d etectable peaks a t 260
n m a n d 280 n m in d icated a n absence of nucleic acid a n d p ro te in
respectiv ely . A larg e p ea k a t 230 n m in d ic a te d th e p rese n ce of
large am ounts of carb o h y d rate. This carb o h y d rate was d eterm in ed
to be alginate b y th e carb azo le assay. The p u rity o f th e b acterial
alginate p ro d u ced from P. syringae ATCC 19304 was m ore th a n 98%
(w /v).
VI. D eacetylation of Bacterial Alginate

The alginates from P. syringae ATCC 19304 w ere d eacety lated
fo r co m p a riso n w ith seaw eed alg in ate a n d a c ety late d b ac teria l
a lg in a te . T he c o m p a riso n s w ere to d e te rm in e th e effects of
acety latio n o n the solution an d gelling p ro p e rtie s o f th e polym er.
P urified, ac ety late d b ac teria l alg inate was dissolved in d eio n ized
w ater a t a co n cen tratio n of 1 m g/m l. T hree volum es of this solution
w ere m ixed w ith o n e volum e of 1 N sodium h y d ro x id e solution.
A fter in c u b atio n for 20 m in u tes at ro o m te m p e ra tu re , w ith g entle
a g itatio n , o n e v o lum e o f 1 N h y d ro c h lo ric acid was a d d e d to
n e u tra liz e th e so lu tio n (fin al pH was a b o u t 7.0) a n d sto p th e
re a c tio n . T he d e a c e ty la te d b a c te ria l a lg in ate w as e x te n siv e ly
dialyzed against deionized w ater. The effectiveness o f th e process
w as d e te rm in e d fro m c o n c e n tra tio n s o f acety l g ro u p s in th e
preparation.
50
VII. Alginate Size an d Q uantity D eterm inations

M olecular w eig h ts w ere d e te rm in e d b y gel p e rm e a tio n
chrom atography (GPC) from alginate solutions [100 (ng/ml (w /v)] in
d eio n ize d w ater. A lginate sizes a n d p o ly d isp ersity in d ices w ere
d e te r m in e d b y m e a s u re m e n t o f m u ltia n g le lig h t scattering
in te n sitie s using a DAW N-Photometer (W yatt T echnology, S an ta
B arbara, CA). The DAWN GPC d e te c to r m easu res th e scatterin g
intensities of a sam ple at 15 different angles a n d tran sm its th e d a ta
to a c o m p u te r for digital co n v ersio n a n d su b se q u en t processing
u n d e r c o n tro l of th e ASTRA™ (o r ASTRA 202) so ftw are (W yatt
T echnology, S anta B arbara, CA). A cetone a n d cyclo h ex an e w ere
used for in stru m e n t calibration. C oncentrations w ere o b tain ed from
a W aters M odel 410 D ifferen tial R efractom eter (M illipore Corp.,
M ilford, MA). Alginate sizes w ere based on th e GPC calculation an d
th e following ex tern al stan d ard s: T10, T40, an d T500 (P harm acia
Co., Uppsala, Sweden) dextrans. Sample injection volum es w ere 100
jil a n d th e GPC colum n was an U ltrahydrogel Linear colum n (W aters,
M illipore Corp., Milford, MA). The ru n n in g buffer was 0.1 M NaNC>3 ,
a n d the te m p eratu re was 45 °G
VIII. Sugar Sensitivity to Acid

1. Thin Layer Chrom atography
S tandards of D-m annose an d L-gulose (Sigma Chemical Co., St.
Louis, MO) w ere p re p a re d a t a co n c en tratio n of 6 .6 6 m g /m l (w /v)
in deionized water. One m l of acid (1 N HC1 o r 1 N H 2 SO4 ) an d 1 ml
of sta n d a rd sugar solution w ere m ixed a n d th e solution h e a te d a t
100°C fo r 0.5, 1, 2, 3, a n d 4 h o u rs. Each so lu tio n was th e n
51
n eu tralized w ith 1 m l of 1 N NaOH giving a final sugar concentration

of 1.67 m g /m l (w/v) an d a final pH of betw een 6.0 an d 7.0.
T h in la y e r c h r o m a to g ra p h y w as p e r f o r m e d on th e
h y d ro ly s a te s on 0.25 m m p la te s c o a te d w ith silica gel 150A
(W hatm an Co., M aidstone, England), o r kieselgel 60 F254 (E. M erck
Co., D arm stadt, G erm any). The h y d ro ly ze d su g ar so lu tio n s w ere
spotted a t a co n cen tratio n of 50 fig su g ar/sp o t. T he ru n n in g solvent
was n -p ro p a n o l a n d w ate r in a ra tio of 85:15 (v /v ). T he p lates
w ere developed by spraying w ith 20% (v/v) H 2 SO4 in m eth an o l and
charrin g a t 100°C fo r 15-20 m inutes.
2. Ion C hrom atography

S tandards of D -m annose a n d L-gulose (Sigma Chem ical Co., St.
Louis, MO) w ere p re p a re d a t a c o n c en tratio n o f 1 m g /m l (w /v) in
deionized w ater. One m l of sugar solution was m ixed w ith 1 m l of
acid (1 N HC1 o r 1 N H 2 SO4 ). Each solution was h y d ro ly zed a t 100°C
fo r 0.5, 1, 2, 3, a n d 4 h o u rs. A fter h y d ro ly sis each so lu tio n was
n eu tralized w ith 1 m l of 1 N NaOH giving a final sugar concentration
of 0.33 m g /m l (w/v) an d a final pH betw een 6.0 a n d 7.0.
Q uantitative d eterm in atio n s of th e acid sen sitiv ities o f each
su g ar w ere m ad e w ith io n ch ro m ato g rap h y . Ion c h ro m a to g ra p h y
was p e rfo rm e d o n each h y d ro ly zed sta n d a rd , using a D ionex ion
chrom atography system (Dionex Corp., Sunnyvale, CA) fitted w ith a
C arbopac PA1 colum n (4 x 250 m m ). A 100 mM solution of NaOH
was used as e lu e n t a n d p u m p e d a t 0.5 m l/m in u te b y a g ra d ie n t
pum p. A 50 |il sam ple was in jected a n d th e signal was d etected by
a pulse am p ero m etric d etector. Integration was accom plished b y a
52
D ionex 4 4 0 0 in te g ra to r. T he rela tiv e p eak a re a s w ere u sed to

q u a n tita te th e p e r c e n t d e g ra d a tio n o f ea ch su g a r u n d e r the
experim ental conditions em ployed.
IX. Identification of th e Acid H ydrolysis Product of L-Gulose

1. Thin Layer C hrom atography
T hin lay er ch ro m ato g rap h y was used to h elp id en tify th e acid
hyd ro ly sis p ro d u c t of L-gulose. L-Gulose (Sigma Chemical Co., St.
Louis, MO) was acid h y d ro ly zed a n d se p a ra te d on a TLC p la te as
d escrib ed previously. At th e en d o f a ru n , th e TLC p late was air
d ried . T he silica gel in th e reg io n co rresponding to th e Rf v alue of
th e sp o t of in te re s t w as sc ra p e d fro m th e p la te a n d e lu te d in
deionized w ater for 10 m inutes. The solution was m icrocentrifuged
in a Sorval M icrospin 24S m icrocentrifuge (Du Pont Co., W ilmington,
DE) for th re e m inutes to p ellet the silica gel. The su p e rn a ta n t was
freeze d rie d in a Flexi-Dry freeze d ry ap p aratu s (FTS Systems, Stone
Ridge, NY).
T he fre eze d r ie d sam p le w as re s u s p e n d e d in 0.2 m l of
deionized w ater an d again sp o tted on a 0.25 m m TLC p la te co ated
w ith kieselg el 60 F254 (E. M erck Co., D arm stad t, G erm any) as
described previously. The sam ple was ru n in n -p ro p an o l a n d w ater
in a ra tio o f 85:15 (v /v ) w ith sam p les o f 1,6 a n h y d ro (3-D-
m a n n o p y ra n o s e , a n d 1,6 a n h y d ro -p -D -g lu c o p y ra n o se (Sigma
Chem ical Co., St. Louis, MO) a n d th e m ig ratio n of th e unknow n
com pared to th e know n stand ard s.
53
2. Stability of 1,6 A nhydro p-L-Gulopyranose

The stability of 1,6 an h y d ro |3-L-gulopyranose was m easu red
b y m o le cu lar m odeling o n SYBYL m o le cu lar m odeling softw are
v ersio n 6.2 (Tripos Assoc. Inc., St. Louis, MO). The m olecule was
d raw n using th e above softw are a n d th e n m inim ized to its low est
en erg y level at pH 7.0 w ith a dielectric co n stan t of 78.8 (equivalent
of w ater). Once the m olecule was a t its low est en erg y levels fo r the
above conditions, it was solvated in w ater b y co m p u ter a n d h ea ted
to 400°K for a total of 100,000 fem toseconds (1 0 "1 1 seconds). This
allow ed th e solvent to a tta in equilibrium . A d istan ce d e p e n d e n t
dielectric co n stan t was u sed to avoid th e con d itio n s of a vacuum .
Energy m easurem ents w ere m ade a t 250 fem tosecond intervals.
X. Alginate Reductions
A lginates w ere chem ically red u ced p rio r to acid hydrolysis of
th e polym ers. The m e th o d used fo r th e re d u c tio n o f th e u ro n ic
acids in alginates to th e co rresp o n d in g n e u tra l sugars was th a t of
T aylor e t al (140). An aqueous solution of alginate containing 100
m icroequivalents of carboxylic acid in 10 ml of deio n ized w ater was
ad ju sted to pH 4.75 w ith 0.1 M NaOH. One m illim ole of 1-eth y l-3 -
(3 -d im eth y lam in o p ro p y l)carb o d iim id e was a d d e d to th e alg in ate
solution to co n v ert the u ro n id es to esters. The pH of the reactio n
m ix tu re was m ain tain ed a t 4.75 b y titra tio n w ith 0.1 M HC1. The
reactio n was allow ed to continue u n til h y d ro g en ion u p tak e ceased
(45-60 m inutes). Then, 25 m l of a 3 M NaBH4 solution was ad d e d
dropw ise o ver a 1 h o u r p erio d to red u ce th e u ro n id es to th e m ore
read ily hydrolyzable n eu tra l polym ers. The pH was m a in ta in ed at
54
7.0 by titra tio n w ith 4 M HC1. 1-Propanol was a d d e d dropw ise, as

n ecessary to m inim ize foam ing. T he re a c tio n m ix tu re w as th e n
m ade slightly acidic to d estro y an y rem aining sodium b o ro h y d rid e,
a n d the solution was dialyzed exhaustively against deionized w ater.
Each re d u c e d alg in ate sam ple was th e n c o n c e n tra te d in a Buchi
M odel R110 ro ta ry ev ap o rato r (Buchi Lab., Flawil, Sw itzerland) an d
p recip itated b y ad d itio n of th ree volum es of iso p ro p an o l a n d dried
by w ashing in acetone.
XI. A lginate Com positions

Com position m easurem ents of th e alginates fro m M acrocystis
p yrifera (Sigma Chemical Co., St. Louis, MO), a n d P. syringae ATCC
19304 w ere m ad e b y ion ch ro m ato g rap h y of th e acid h ydrolyzed,
red u ced polym ers. Each alginate sam ple was p re p a re d in the sam e
m a n n e r as th e D -m annose, a n d L-gulose su g ar sta n d a rd s fo r ion
ch ro m ato g rap h y described previously. The re d u c e d sam ples w ere
m ixed a t a co n c en tratio n of 1 m g /m l a n d 1 m l o f so lu tio n m ixed
w ith 1 m l o f acid (1 N HC1 or 1 N H 2 9 0 4 ). T he sam ples w ere th e n
h ydroly zed a t 100°C for 0.5, 1, 2, 3, an d 4 hours. After hydrolysis,
50 [xl o f e a c h sa m p le w as in je c te d in to th e D io n ex io n
chro m ato g rap h y system described above. The resulting p eak areas
w ere th e n c o rre la te d a n d e x tra p o la te d b a c k to tim e z e ro to
d e te rm in e th e p erce n tag e of m an n o se a n d gulose p re se n t in th e
red u ced polym er. This com position was th e n directly co rrelated to
th e com position of b o th M acrocystis p yrifera an d P. syringae ATCC
19304 alginates.
55
XII. P roperties of Bacterial Alginates a n d Effects of A cetylation

1. Viscosity
The viscosities of M acrocystis pyrifera alginate a n d acetylated
a n d deacety lated P. syringae ATCC 19304 alg inate w ere m e asu red
as a fu n c tio n o f te m p e ra tu re , c o n c e n tra tio n , a n d d e g re e of
acety latio n . In ea ch case, v iscosities w ere d e te rm in e d b y th e
m e th o d o f A llison a n d M atthew s (1) using a sim p le U -sh ap ed
Ostwald capillary viscom eter designed for sm all volum es. The tim e
taken fo r a sam ple to fall a fixed distance u n d e r gravity (N), divided
b y th e tim e ta k e n b y w ate r to fall th a t sam e d istan ce (No) was
expressed as a m easure of com parative viscosity (Visc.COm = N/N 0).
The effect of te m p e ra tu re on alginate solution viscosity was
m e a su re d at alg in ate co n cen tratio n s o f 4 0 0 tig/m l (w /v) o v er a
te m p e ra tu re ra n g e o f 30°C to 85°C. T he effects of a lg in ate
co n c en tratio n o n viscosity was m easured a t 50°C a t co n cen tratio n s
ran g in g from 50 fxg/ml (w /v) to 1000 f.ig/ml (w /v). The effect of
a c e ty la tio n o n v isc o sity w as m e a s u re d a t 50°C a t a lg in a te
concentrations of 50 ng/m l (w /v), 100 ng/m l (w /v), a n d 200 ng/m l
(w /v) an d th e values averaged. D eacetylation of b acterial alginate
was as d escrib ed p rev io u sly w ith m in o r v ariatio n s. A 4 00 ng/m l
sam ple o f h ig h ly acety lated b acterial alginate was d ea ce ty lated to
v ario u s degrees b y varying the co n cen tratio n of NaOH u sed in the
re a c tio n a n d b y alterin g th e re a c tio n tim e. S odium h y d ro x id e
concentratio n s ran g ed fro m 0.25 M-1.0 M while reactio n tim es w ere
b etw een 5 m in u tes an d 20 m inutes. A cetylated seaw eed alg in ate
was o b ta in e d fro m Jin W. Lee (72) a n d p a rtia lly d e a c e ty la te d as
d escribed previously.
56
2. W ater Holding Capacity

W ater holding capacities of alginate gels w ere m e a su re d by
d eterm in in g th e am o u n t of w ater lost fro m th e gels u p o n drying.
A lginate gel b e a d s w ere m a d e fro m a 0.6% (w /v ) so lu tio n of
seaweed, acetylated o r d eacety lated bacterial alginate. Using a 5 ml
p ip e t tip, 4 m l of th e alginate solution was d rip p e d slowly in to 30
m l of CaCl2 , FeCl3 , o r PbCl2 solution. Each m etal solution was tested
at co n c en tratio n s of 0.05 M, 0.1 M, 0.25 M, o r 0.5 M. T he b ead s
w ere allow ed to form in each m etal solution fo r 15 m in u tes after
w h ic h th e y w ere w a sh e d th o ro u g h ly in d e io n iz e d w a te r (5
m in u tes). A fter a ir d ry in g fo r 5 m in u tes, 10 b e a d s fro m each
sam ple w ere p laced in to p re d rie d , ta re d alu m in u m w eigh dishes
a n d w eighed. The w eighing dishes w ere th e n p laced in a d ry in g
oven a t 100°C a n d th e sam ple d rie d u n til a c o n s ta n t w eight was
reac h ed . The dishes w ere th e n rew eighed a n d th e w ater hold in g
capacity of each b ead calculated as g w a te r/ g d ry alginate gel.
3. Surface Tension
The relativ e surface ten sio n s o f b ead s w ere m e asu red using
b e a d d ia m e te r, th e sm a lle r th e b e a d d ia m e te r th e h ig h e r th e
surface ten sio n o n th e b ead . Gel b ead s w ere m ad e as d escrib ed
p rev io u sly in 0.5 M CaCl2 . U pon fo rm atio n , 1 /3 of ea ch b ea d
sam ple was w ashed in d eionized w ater fo r 5 m in u tes a n d allow ed
to d ry fo r 5 m in u tes p rio r to d ia m e te r m e asu rem e n t w ith a dial
calip er (L. S tarre tte Co., Athol, MA). The second p o rtio n of each
sam ple was in cu b ated a t 4°C fo r 24 h o u rs in deionized w ater prior
to m easurem ent, an d th e final th ird of each sam ple was in c u b ate d
57
a t 4°C f o r 24 h o u rs in th e 0.5 M CaCl2 so lu tio n p r io r to

m easurem en t. W ater hold in g cap acity was d irec tly re la te d to the
relative surface tension o n each bead. A low er surface ten sio n on
th e b e a d re s u lte d in a la rg e r b e a d d ia m e te r a n d h ig h e r w ate r
holding capacity of the gel.
4. P recipitation b y Metal Ions

T he p re c ip ita tio n of seaw eed alg in ate a n d a c e ty la te d a n d
d e a c e ty la te d b a c te ria l a lg in a te b y m e ta l io n s was c o m p a re d .
P u rifie d a lg in a te s w ere d is s o lv e d in d e io n iz e d w a te r a t a
c o n c en tratio n o f 400 M.g/ml (w /v). M etal salts w ere d isso lv ed in
deionized w ater to p re p a re for the solutions w ith co n cen tratio n s of
0 to 25 o r 100 mM. T he m etal ions tested w ere: Csl+, Rbl+, Mg2+,
Ca^+, Sr2+, M n2+, Fe^+, Co2+, Cu2+, Zn2+, Pb2+, a n d U ^ . All m etal
salts w ere o b ta in ed from Sigma Chemical Co., St. Louis, MO, except
fo r u ra n y l a c e ta te (E astm an K odak Co., R o ch ester, NY). Four
volum es of seaw eed alginate solution o r acety lated o r deacetylated
b ac teria l alg in ate so lu tio n w ere m ixed w ith o n e v o lu m e of each
m etal solution, respectively. The m ixtures w ere in c u b a te d fo r 12
h o u rs a t ro o m te m p e ra tu re a n d ce n trifu g e d (17 ,0 0 0 x g fo r 30
m inutes) in a Sorval S upersp eed M odel RC-5B cen trifu g e (Du Pont
Co., W ilm ington, DE). The s u p e rn a ta n ts w ere se p a ra te d a n d th e
c o n c e n tra tio n o f a lg in a te re m a in in g in ea ch s u p e rn a ta n t w as
m e a su re d b y th e u ro n ic acid assay. T he am o u n ts o f alg in a te
se p a ra te d as a gel w ere calcu lated by d ifferen ce a n d those values
w ere u se d to d e te rm in e th e rela tiv e p re c ip ita tio n of th e alg in ate
solutions by th e m etal ions.
RESULTS
I. Production of Bacterial Alginate

1. Media Compositions an d Conditions
In b ro th cu ltu re, alg in ate p ro d u c tio n b y P. syringae ATCC
1 9 3 0 4 fo llo w ed th e g ro w th c u rv e o f th e o rg a n ism (Fig. 9).
M aximum cell m ass (d ry weight) a n d alginate yields w ere o b tain ed
48 h o u rs post inoculation. The type of carb o n source used affected
th e alginate yield of P. syringae ATCC 19304, as well as th e d egree
of acety latio n w ithin th e polym er (Table 5). P. syringae grew well
on glucose, sucrose, glycerol, a n d gluconic acid, b u t d id n o t grow on
fructose. Sucrose grown cells y ield ed an EPS, o nly 77% p e rc e n t of
w hich was uronic acid. Gluconic acid grown cells y ield ed th e m ost
alg in a te (ap p ro x im ately 2 0 0 i-ig/mg cell d r y w eig h t), w ith th e
h ig h est d eg ree of acetylation (approxim ately 100%). Gluconic acid
was th e ca rb o n source o f choice due to th e in creased alginate yield
of gluconic acid grown P. syringae.
P. syringae ATCC 19304 u tiliz e d am m o n ia as a n itro g e n
source, b u t was unable to utilize n itrate, n itrite, o r urea. T here was
a n in v e rse c o rre la tio n b etw een a lg in a te y ie ld a n d th e in itia l
c o n c e n tra tio n of am m o n ia in th e m ed ia. As in itia l am m o n iu m
c o n c e n tra tio n in c re a se d fro m 2 mM to 15 mM th e cell m ass
increased 2.4 fold, from 0.66 mg cell d ry w eig h t/m l to 1.58 mg cell
d ry w eight/m l. At th e sam e time, alginate yield d ecreased 2.4 fold,
fro m 1200 j.ig/ml to 500 ^g/m l (Fig. 10). This in d ic a te d th a t at
h ig h e r in itia l am m o n iu m c o n c e n tra tio n s th e c a rb o n n o rm a lly
d e s tin e d fo r alg in ate p ro d u c tio n was u sed b y th e cells fo r cell
58
59
2 1000
Cell mass (mg dry weight/ ml broth)
d
-800
o
*3
d
d
ad
tyig/ml broth)
-600
o
1 d
3
-400 d
d
tuo
d
-200
o
H
0
0 20 40 60 80 100
T im e (h r)
Figure 9. T he re la tio n sh ip betw een cell m ass (♦), a n d alg in ate

(u ro n ic acid) accu m u latio n (D) by P. syringae ATCC 19304 w ith
tim e in shake flask culture.
Table 5. Effects of C arbon Source on A lginate Yield from P. syringae ATCC 19304
Carbon Cell Yield Yield T otal EPS % A lginatee Yield A lginate % AcetylationS
Sourcea >b (m g /m l)c O ig/m l)d (ng/m g cell)f
Glucose 1.25 175 100 140 107 (±23)
Sucrose 1.23 211 77 132 4 (± 1)
Glycerol 0.96 134 100 140 84 (± 17)
Gluconic acid 1.04 213 100 205 97 (± 30)
a C ultured in DF b ro th su p p lem en ted w ith 2% carbon source, an d grow n for 48 hours a t 30°C
w ith shaking a t 180 rpm .
b Fructose was also te sted b u t th e re was no growth.
c Cell yield was m easu red as mg cell d ry w eig h t/m l b roth.
d Yield of total EPS was m e asu red as ng EPS/ml broth.
e The p erce n t of to tal EPS th a t was alginate.
f Yield of alginate was m easu red as [ig alg in ate/m g cell d ry weight.
8 The fimolar ratio (%) o f acetyl to uronic acid.
61
1400
3
©
* d
& 1200
©
-
S
a
s P
A
txo Is
na
'H - 1000
3 -
- 800 %
b©
a 00 a
rt5 "
w 0.8 -600
w -
3
rt ©
2 H
© 0.6 400
L> i 9 12 15
In itia l [A m m onium ] (mM)
Figure 10. The effect of the initial am m onium co n cen tratio n on cell
m ass (♦), a n d total alginate (uronic acid) accu m u latio n (□), by P.
syTingae ATCC 19304 at 48 hours.
62
division. As initial am m onium concentrations increased fro m 2 to 9

mM, th e specific yield of alginate d ecreased fro m 2100 fxg/mg cell
d ry w eig h t to 5 0 0 îg/mg cell d ry w eight. In itia l a m m o n iu m
concentrations above 9 mM resulted in a consistently low er specific
alginate yield (Fig. 11).
Initial pH of the DF b ro th an d te m p eratu re b o th affected th e
cell m ass an d alginate yield of P. syringae ATCC 19304. M axim um
grow th of P. syringae ATCC 19304 o ccu rred in DF b ro th w ith initial
pH 's betw een 6.4 a n d 7.2. A lginate yield was g reatest in DF b ro th
w ith initial pH 's b etw een 6.8 a n d 7.0. Above pH 7.0 th e alginate
y ield d eclin ed (Fig. 12). The o p tim u m te m p e ra tu re fo r alg in ate
p ro d u c tio n was 30°C. T h ere was little ch an g e in cell m ass w ith
te m p e ra tu re s b etw e en 25°C to 32°C. A lginate p ro d u c tio n was
extrem ely te m p e ra tu re d e p e n d e n t, w ith a sh a rp o p tim u m at 30°C
(Fig. 13).
2. Agar vs. Broth Culture

The ab ility of P. syringae ATCC 19304 to grow a n d p ro d u ce
a lg in a te w as c o m p a re d o n a g a r a n d in b r o th c u ltu re . Upon
successive tra n sfe rs in b ro th c u ltu re , alg in a te y ie ld d e c re a se d
dram atically. Initially, alginate yield reach ed a m axim um of 550 |ng
a lg in a te / mg cell d ry w eight a t 27 hours. A fter o n e tra n s fe r back
into b ro th culture, the y ield d ecreased to o nly 50 (.ig a lg in a te / mg
cell d ry weight. A fter a second tran sfer, th e re was less th a n 10 j.ig
alg in ate/ mg cell d ry weight. On ag ar m edia alginate p ro d u ctio n by
P. syringae ATCC 19304 was c o n siste n t u p o n tra n s fe r fro m one
63
3 00 0
Specific alginate accum ulation
tyig/mg cell dry w eigh t)
2000
1000
0 t '-----------1
----------- 1
------------'-----------1
------------'-----------r
2 5 y 12 15
In itia l [A m m onium ] (mM)
Figure 11. The effect o f th e in itial am m o n iu m co n cen tratio n on

specific yields of alginate (uronic acid) by P. syringae ATCC 19304
at 48 hours.
64
500
Cell mass (mg dry w eight/m l broth)
1.6 -
d
3
400 to
h
* 2
-3 0 0 « &
s a
.2
3>
0.8 -
CO
-200
3
0.6 o
H
0 .4 100
6.0 6.2 6 .4 6.6 6.8 7 .0
In itia l pH
Figure 12. The effect o f initial pH on cell m ass (♦), an d alginate

(uronic acid) accum ulation (0), by P. syringae ATCC 19304 a t 48
hours.
65
2 .7 5 500
fl 2 .5 0
©
P d
Xi 2 .2 5 ©
3
a 2.00
«i
qpag
s
•p
jd
&o 1.75
-4 0 0
as
d ©
© p
1 .5 0 -
s s
1 .25 -
p
t? 1.0 0 - d &o
&Q 300
a 0 .7 5 -
S 5'
rt
0)
CO 0 .5 0 - 5
©
a 0 .2 5 - H
© 0.00 —1" —r- —r~ —r~ 200
©
24 26 28 30 32 34
T em p. (°C)
Figure 13. The effect of te m p eratu re on cell m ass (♦), an d alginate

(uro n ic acid) accum ulation (0), by P. syringae ATCC 19304 a t 48
hours.
66
cultu re to an o th er. The organism averaged approxim ately 1870 jug

a lg in a te / mg cell d ry w eight over 5 successive transfers (Table 6).
Total a n d specific alginate yields w ere d eterm in ed in various
m edia. P. syringae was tested in b ro th an d on a n ag ar surface. DF
salts, n u trie n t m edia, p ep to n e m edia, an d b ee f ex tra ct m e d ia w ere
te ste d fo r th e ir ab ility to su p p o rt b ac teria l grow th an d p ro m o te
alginate p ro d u ctio n by P. syringae ATCC 19304 (Table 6). In b o th
b ro th a n d o n ag ar, n u tr ie n t m e d ia su p p o rte d th e h ig h e st to tal
alginate yield in a 48 h o u r perio d . The specific alg inate y ield was
1.5 to 2 fold g reater in b ee f extract m edia th a n in an y o f th e o th e r
m e d ia tested . In all cases, grow th o n ag a r m ed ia in creased the
specific yield of b acterial alginate by ab o u t 3 fold. In DF b ro th the
specific yield was a p p ro x im a tely 500 M-g/mg cell d ry w eight a n d
was c o n stan t fro m 24 h o u rs to 96 h o u rs. This in d ic ated th a t th e
to tal yield o f alginate in creased at a co n stan t ra te o ver this period.
On ag ar m ed ia specific alginate yields reach ed approxim ately 1850
ng/m g cell d ry w eight betw een 24 a n d 72 h o u rs a n d th e n beg an to
decrease after 72 hours.
F erric io n affected alg in ate p ro d u ctio n . By in creasin g the
initial concentrations of ferric ion fro m 0 mM to 1 mM, the specific
alginate yield b y P. syringae ATCC 19304 d ecreased by 87% (Fig.
14). This in d icated th a t iro n starvation m ay have a role as a trigger
for alginate production.
Table 6. A lginate Yield by P. syringae ATCC 19304 on D ifferent Media.
Yield in Liquid M edia Yield on Solid Media
M ediaa >b Specific Total Specific Total

(ng/m g cell)c (fig/m l)d (fig/mg cell)c (fig/cm 2 )e
DF Salts 550 480 1870 630
Beef Extract, 580 620 2030 980

Peptone
N utrient 590 640 2040 960
Peptone 680 540 2060 700
Beef Extract 1320 450 3000 550
a C ultures w ere grow n a t 30°C fo r 48 h o u rs. Liquid c u ltu re s w ere sh a k en a t 180 rp m . All
cultures w ere su p p lem en ted w ith 2% (w/v) gluconic acid.
t> Beef ex tract was u sed a t a 3 g/L co n cen tratio n an d Peptone was used a t a 5 g/L concentration.
Beef extract, Peptone, an d N u trien t m edia w ere from Difco Labs., D etroit, ML
c Specific p ro d u ctio n was m easu red as ng alg in ate/m g cell d ry weight.
d Total p ro d u ctio n in liquid cu ltu re was m easu red as fig alg in ate/m l broth.
e Total p ro d u ctio n on solid cu ltu re was m easured as fig alginate/cm 2 of ag ar surface.
68
2000
Specific alginate accum ulation
(pg/mg cell dry w eigh t)
1000 -
0.0 0.2 0 .4 0.G 0.8 1.0 1.2
In itia l [Fe+++] (mM)
Figure 14. Effect of in itial ferric ion (Fe^+) c o n c en tratio n o n the

specific yields of alginate (uronic acid) by P. syringae ATCC 19304
at 48 hours.
69
II. C haracterization of Bacterial Alginate

1. Recovery
A lginate p ro d u ced by P. syringae ATCC 19304 was recovered
fro m th e su rface o f DF ag a r plates su p p le m en ted w ith 2% (w /v)
gluconic acid. S everal d iffe re n t alcohols w ere te ste d fo r th e ir
ability to p recip itate bacterial alginate. Recoveries w ere com pared.
All of th e alcohols tested: m eth an o l, eth an o l, n -p ro p an o l, a n d iso
p ro p a n o l, p re c ip ita te d 100% of th e alg in ate a t a fin a l alco h o l
co n cen tratio n o f 50% (v /v ). In o rd e r to d e te rm in e th e effect o f
ac ety latio n on alcohol p recip itatio n , eq u al am o u n ts of ac ety late d
a n d d e a c e ty la te d a lg in a te w ere p r e c ip ita te d w ith v a r io u s
concentrations of iso-propanol. A cetylation d id n o t affect p ro d u c t
recovery.
2. M olecular W eight
T he w eig h t av erag e m o le cu lar w eig h t (Mw ) a n d n u m b e r
a v e ra g e m o le c u la r w e ig h t (Mn) w e re d e te r m in e d fo r b o th
acety lated a n d d eacety lated b acterial alginates b y gel p e rm e a tio n
c h ro m a to g ra p h y (GPC). T he Mw a n d th e Mn o f th e seaw eed
alginate w ere ap p ro x im ately 65% sm aller th a n th e n ativ e b acterial
alginate. D eacetylation w ith sodium h y d ro x id e d id n o t a lte r th e
m olecular w eights appreciably. Upon deacetylation, th e Mw of th e
bacterial alginate d ecreased by 6%, an d th e Mn d ec re ase d by 11%.
In e a c h case, th e p o ly d isp e rsity was ap p ro x im a tely 3.00. T his
in d ic a te d th a t th e re was a w ide ran g e of m o lecu lar sizes in each
alginate sam ple (Table 7).
70
Table 7. M olecular W eights of Alginates
A lginate sam ple Mn a (x 104 ) Mw b (x 104 ) Mw /M n c
M acrocystis 1.4 4.7 3.36

(± 1.5 x 103) (± 2.6 x 103)
A cetylated 4.3 12.7 2.95

P. syringae (± 8.2 x 103) (±7.2 x 103)
Deacetylated 3.8 11.9 3.13

P. syringae (± 7.7 x 103) (± 8.9 x 103 )
a N um ber average m olecular weight,

b W eight average m olecular weight.
c Polydispersity
71
3. Composition
T he d e te rm in a tio n of alg in a te co m p o sitio n re q u ire d th e
red u ctio n of the uronic acids to th e ir co rresponding n e u tra l sugars
p rio r to acid hydrolysis of th e polym er. This red u ctio n facilitated
the acid hydrolysis of th e glycosidic bonds by conversion of th e acid
resistan t glycosyluronic acid bonds to th e m ore acid labile glycosyl
bo nds. The acid sensitivities o f D -m annose a n d L-gulose (Sigma
C hem ical Co., St. Louis, MO) w ere s tu d ie d u sin g th in la y e r
chrom atography (TLC) an d ion chrom atography. TLC in d icated th at
D -m annose a n d L-gulose h av e m a rk e d d ifferen ces in th e ir acid
sensitivity. Acid h y d ro ly zed (HC1) D -m annose resu lted in only one
spot on TLC (Rf=.40). The in ten sity o f this spot rem a in ed co n stan t
b e tw e e n 0 a n d 4 h o u rs h y d ro ly sis tim e (Fig. 15). L-gulose
p ro d u ced a second spot (Rf=.56) afte r m ore th a n 1 h o u r hydrolysis.
T he in te n sity of this sp o t in creased w ith h y d ro ly sis tim e. At th e
sam e tim e, th e in te n sity o f th e gulose sp o t (Rf=.37) d e c re a se d
p ro p o rtio n a lly (Fig. 16). Both D -m annose a n d L-gulose re a c te d
sim ilarly in HC1 an d H 2 SO4 (Table 8 ).
The relative acid sensitivity of each sugar was m e asu red by
D ionex ion ch ro m ato g rap h y . D-M annose was acid stable. A fter 4
h o u rs o f HC1 o r H 2 SO4 h y d ro ly sis, 98% o f th e o rig in al su g ar was
recovered. L-gulose was relatively stable fo r 1 h o u r, afte r w hich it
began to rap id ly breakdow n. After 4 h o u rs hydrolysis, only 22% of
the original sugar was recovered (Fig. 17).
The acid breakdow n p ro d u c t of L-gulose was n o t definitively
identified. TLC of this com pound show ed a m igration (Rf=.58) equal
to 1,6 an h y d ro p-D -m annopyranose (Rf=.58), an d v ery close to 1,6
72
.4. .... *: ■ MW.",., ?A--' - 4 . ) > M»r-5)-St>i*>•
1 2 3 4 5 6 7
Figure 15. Thin layer chrom atograph of HC1 hydrolyzed D-m annose.
Lane 1= unhydrolyzed D -m annose in H20, Lane 2= u n h y d ro ly zed D-
m annose in HC1, Lanes 3-7= hydrolyzed D -m annose in HC1 fo r 0 .5 ,1 ,
2 ,3 , a n d 4 h o u rs respectively at 100°C
73
1 2 3 4 5 6 7
Figure 16. T hin lay er ch ro m ato g rap h of HC1 h y d ro ly ze d L-gulose.

Lane 1= u n h y d ro ly ze d L-gulose in H 2 O, Lane 2= u n h y d ro ly ze d L-
gulose in HC1, Lanes 3-7= hydrolyzed L-gulose in HC1 fo r 0.5, 1, 2, 3,
a n d 4 h o u rs respectively a t 100°G
74
Table 8 . Thin Layer C hrom atography (Rf values )a
Sample Spot A Spot B
D -m annose in HC1 .40 (± .03) N/AC
L-gulose in HC1 .37 (± .03) .56 (± .03)
D -m annose in EI2 SO4 .37 (± .04) N/A
L-gulose in H 2 SO4 .34 (± .03) .50 (± .04)
a Each Rf value was calculated by the distance of the sp o t from the

origin d ivided by the total distance traveled by the ru n n in g solvent,
b Each sam ple was h y d ro ly zed in an equal volum e of 1 N acid an d
hydroly zed for 4 h o u rs a t 100°G
c N/A= Not Applicable
75
120
100
H
W
kH 80
o
>
o
o
o
Pi GO
u
rt
oo
i/j 40
20
1 7 3 4
H y d ro ly sis T im e (h r)
Figure 17. The stability of m onom eric D -m annose a n d L-gulose in

HC1 an d H2904 u n d e r hydrolysis conditions at 100°C, as d eterm in ed
by ion chrom atography. L-gulose in HC1 (□), D -m annose in HC1 (♦),
L-gulose in H29D4 (■), D -m annose in H2 SO4 ( 0 ).
76
an h y d ro p-D-glucopyranose (Rf=.60, Fig. 18). This indicated th a t the

m olecu le m ay be a 1,6 a n h y d rid e . M olecular m o d elin g o f 1,6
an h y d ro p-L-gulopyranose (Fig. 19) show ed th a t this m olecule has a
low total energy (approxim ately -1400 kcal/m ol) an d th u s is stable
(Table 9). This len d ed em phasis to th e possibility of 1,6 an h y d rid e
form ation upon acid hydrolysis of L-gulose.
D estruction of th e re d u c e d sugars in th e alg in ates o n acid
hydrolysis p arallele d th e resu lts seen w ith th e D -m annose a n d L-
gulose m o n o m ers. A fter c o rre c tio n fo r gulose d e s tru c tio n by
extrapolation back to tim e 0, a com position of 60% m an n u ro n ic acid
a n d 40% guluronic acid was o b tain ed fo r M acrocystis alginate, an d
82% m an n u ro n ic acid a n d 18% guluronic acid fo r P. syringae ATCC
19304 a lg in ate (Fig. 20). This c o rre la te d well to th e re p o rte d
c o m p o sitio n fo r a lg in a te p ro d u c e d b y P. aeruginosa o f 80%
m a n n u ro n ic acid a n d 20% g u lu ro n ic acid, a n d fo r M acrocystis
alginate o f 60% m annuronic acid a n d 40% guluronic acid (150).
III. Functional Properties

1. Viscosity
The viscosity o f a solution d ep en d s o n th e m olecu lar w eight
a n d th e rig id ity of th e solute, as well as en v iro n m e n ta l factors,
especially tem p eratu re. The concentration, p ercen t acetylation, and
te m p e ra tu re all co n trib u ted to v ariatio n s in th e flow p ro p e rtie s of
alginates. At a n alginate co n cen tratio n of 0.1% (w /v) the viscosity
of acety lated b acterial alginate was 2.7 fold g reater th a n seaw eed
alginate. D eacetylated bacterial alginate was 2.0 fold m ore viscous
th a n seaw eed alginate. As alginate co n cen tratio n s in creased from
77
§ •
Figure 18. T h in la y e r c h ro m a to g ra p h o f th e acid h y d ro ly z e d

p ro d u c t o f L-gulose. Lane 1= u n h y d ro ly zed L-gulose in H 2 O, Lane
2= sta n d ard 1,6 an h y d ro p-D-m annopyranose, Lane 3= sta n d a rd 1,6
an h y d ro p-D-glucopyranose, Lane 4= acid h y d ro ly sis p ro d u c t o f L-
gulose.
Figure 19. C om puter d eriv ed im age of 1,6 an h y d ro p-L-gulopyranose. The im age is d epicted
cross stereo view.
T able 9. The Energetics a n d Stability of 1,6 A nhydro p-L-G ulopyranosea
Sam ple # Time Potential Kinetic Total T em perature

(Fem tosec)b Energyc Energy Energy (°K)
2 250 -2307.73 861.11 -1446.62 387.77
3 500 -2432.09 886.92 -1545.17 399.39
4 750 -2475.61 855.84 -1619.77 385.40
5 1000 -2512.66 878.87 -1633.79 395.77
7 1500 -2 5 4 2 .1 0 870.16 -1671.94 391.85
14 3250 -2554.33 865.87 -1688.46 389.92
31 7500 -2 512.04 820.92 -1691.11 369.67
33 8000 -2 5 6 9 .7 4 875.05 -1694.70 394.05
a V alues w ere o b ta in ed using SYBYL m o lecu lar m odeling softw are (Tripos Assoc. Inc., St. Louis,
MO).
b 1 fem tosecond equals 1 0 " 15 seconds.
c All en erg y values are m easu red in kcal/m ol.
80
50
40 -
■O
Im
o
► 30 -
o
o
4>
P4
to 20 -
©
53
O
M 10 -
I
4
H y d ro ly sis T im e (h r)
Figure 20. Gulose reco v ered , ex p ressed as a p e rc e n t of th e to tal

su g a r p r e s e n t in th e HC1 h y d ro ly z e d re d u c e d alg in ates, fro m
M acrocystis p yrifera (□), a n d P. syringae ATCC 19304 (♦), as
d eterm in ed by ion chrom atography.
81
0 jig/m l to 1000 (.ig/ml (w /v), th e com parative viscosity (N/No) of

each so lu tio n in c re ase d n o n lin early . In each case, th is in crease
exhibited non-new tonian flow dynam ics (Fig. 21). Seaw eed alginate
dev iated least fro m n ew to n ian flow. A cetylated b ac teria l alg inate
dev iated m ost. The d ifferen ce in th e viscosities o f acety lated a n d
d ea ce ty lated alginates in d ic a te d th a t acety latio n lin e arly affected
th e flow d y n am ics o f a lg in a te so lu tio n s. An a v e ra g e o f the
c o m p a r a tiv e v is c o s ity o f seaw eed a lg in a te m easu red at
c o n c e n tr a tio n s of 50 , 100, a n d 200 f-ig/ml (w /v ) in c re a s e d
approxim ately 8% p er a 50% increase in acetylation. An average of
th e co m p arativ e viscosity of b a c te ria l alg in ate m e a su re d a t th e
sam e c o n c e n tra tio n s , in c re a s e d 16% p e r a 50% in c re a s e in
a c e ty la tio n (Fig. 22). T his d ifferen ce was p ro b a b ly d u e to th e
in c re a s e d a v e ra g e m o le c u la r w eig h t o f th e b a c te ria l alg in ate
polym er, a n d its m ore ex ten d ed stru ctu re due to high am ounts of D-
m annuronic acid.
T em p eratu re also affected th e viscosity of alg in ate solutions.
Each a lg in a te so lu tio n r e s p o n d e d sim ila rly to th e affects o f
te m p e r a tu r e . T he v isc o sity o f e a c h a lg in a te so lu tio n a t a
c o n c e n tra tio n o f 4 00 f.ig/ml (w /v ) w as c o n s ta n t fro m 30°C to
ap p ro x im a tely 52°C. A t 52°C th e v isco sities o f e a c h a lg in a te
so lu tio n d e c re a se d d ra m a tic a lly . B etw een 52°C a n d 85°C th e
viscosities of th e seaw eed alginate sam ple d ecreased 3.4%, o n the
averag e, p e r °C. T he viscosities of th e a c e ty la te d b a c te ria l a n d
deacety lated bacterial sam ples d ecreased 9.2% p e r °C, a n d 6.5% p er
°C resp e ctiv e ly . At 85°C th e c o m p a ra tiv e v isco sities o f ea ch
alginate solution w ere approxim ately equal (Fig. 23).
82
10
(N/No)
8
Comparative Viscosity
0
0 200 400 600 800 1000 1200
A lg in a te C o n c e n tra tio n (îg/m l)
Figure 21. C om parative viscosity, (N/N0), o f M acrocystis alg inate

(□), a c e ty la te d P. syrin g a e a lg in a te (I), a n d d e a c e ty la te d P.
syringae alginate (♦), as a function of alginate concentration, (w/v).
83
2.0
(N/No)
1.6 -
1.4-
0 50 100 150
A c e ty la tio n (%)
Figure 22. The effects of acetylation on the com p arativ e viscosity,

(N/Nq), of Macrocystis alginate (D), an d P. syringae alginate (♦).
84
6
(N/No)
1
30 32 38 42 44 50 52 58 04 08 74 78 80 84
T e m p e ra tu re (°C)
Figure 23. The effects of tem p eratu re on the com parative viscosity,
(N/No), of Macrocystis alg in ate (0), acety lated P. syringae alginate
(I), a n d d eacety lated P. syringae alginate (♦).
85
2. Physical Effects
The affects of acetylation on gelation, w ater holding capacity,
surface tension, an d gel porosity w ere d eterm ined. Calcium induced
gelation was altered b y b o th acetylation, an d calcium concentration.
T he gels p r o d u c e d fro m a c e ty la te d b a c te ria l a lg in a te h e ld
approx im ately 100 g w a te r/g d ry alg in ate gel c o m p a re d to th e
deacety lated b acterial alg inate gel w hich h eld ap p roxim ately 3 2 g
w a te r/ g d ry alginate gel (Table 10). As th e co n cen tratio n of CaCl2
increased from 0.05 M to 0.50 M, the w ater holding capacity of each
Ca-alginate gel d ecreased linearly. The w ater holding capacities of
bacterial alginate gels m ad e w ith 0.50 M CaCl2 w ere approxim ately
54% th a t of gels m ad e w ith 0.05 M CaCl2. In co n trast, Ca-alginate
gels m ad e w ith seaw eed alginate show ed a 41% d ecrease in w ater
holding capacity over th e same calcium ion increase.
T he w ater holding capacities of b o th Fe-alginate (ferric), a n d
P b -a lg in ate gels w ere d e te r m in e d fo r seaw eed a lg in a te a n d
acety lated a n d d eacety lated b acterial alginates. The resu lts w ere
com pared to those o b tain ed from Ca-alginate gels. In each case, Fe-
alg in ate gels h e ld less w ater th a n th e ir C a-alginate counterparts,
b u t m ore th a n Pb-alginate gels. The w ater holding capacity of Fe-
a lg in ate (0.05 M FeCl3 ) gels m a d e w ith seaw eed a lg in a te w as
approxim ately 82% th a t of C a-alginate gels. P b-alginate (0.05 M
PbCl2) gels h eld w ater a t 67% th a t of Ca-alginate gels (Fig. 24). As
th e c o n c e n tra tio n of m etal (Fe^+ o r Pb2+) in c re ase d , th e w ate r
holding capacity of the resulting gels decreased. By increasing the
m etal con cen tratio n fro m 0.05 M to 0.50 M in increm ents of 0.05 M,
th e w ater h o ld in g cap acity in Fe-alginate gels d e c re a se d b y a n
86
Table 10. Effects of Calcium C oncentration o n the W ater Holding

Capacitiesa of Alginate Gels.
Calcium Concentration^
A lginate sam ple 0.05 M 0.10 M 0.25 M 0.50 M
M acrocystis 65 47 36 27
( ±3) (±4) ( ±4) ( ±3)
A cetylated 100 85 69 56
P. syringae (±6) ( ±5) (±5) ( ±5)
D eacetylated 32 26 24 17
P. syringae (±3) (± 2) (±2) (± 1)
a W ater holding capacities are calculated as g w ate r/g d ry alginate

gel.
b Calcium was d eriv ed from CaCl2 (Sigma Chem ical Co., St. Louis,
MO).
87
90
of Calcium)
80 -
70 -
{%
Water Holding
6 0 ■■
50 -
40
.0 5 M .1 M .25 M .5 M
M etal c o n c e n tr a tio n
F igure 24. P e rc e n t d iffe re n c e in w a te r h o ld in g c a p a c ity of

Macrocystis alginate gels m ade with ferric iron (D), a n d lead (♦), as
com pared to calcium alginate gels.
88
average of 2.2% p e r 0.05 M increase, a n d in Pb-alginate gels by an

average of 3.7% p e r 0.05 M increase. The w ater holding capacities
of Fe-alginate gels a n d Pb-alginate gels m ad e w ith acety lated a n d
d ea c e ty la te d b acterial alg in ate w ere sim ilar to th o se o f seaw eed
alginate. The w ater holding capacity of Fe-alginate (0.05 M FeCl3)
gels m a d e w ith a c e ty la te d a n d d e a c e ty la te d b a c te ria l alg in a te
averaged approxim ately 79% th a t of the corresponding bacterial Ca-
alg in ate gels. The w ater hold in g cap acity of Pb-alginate (0.05 M
PbCl2) gels m ade u n d er the same conditions averaged only 46% th a t
of th e corresponding b acterial Ca-alginate gels (Fig. 25, 26). As in
se aw ee d a lg in a te gels, th e w a te r h o ld in g c a p a c ity fo r both
acetylated an d deacetylated bacterial alginate gels d ecreased as the
m etal co n cen tratio n increased. The w ater holding capacities o f Fe-
alg in a te gels m ad e w ith a c e ty la te d a n d d e a c e ty la te d b a c te ria l
alginate d ecreased by a n average of 2.0% p e r 0.05 M in crease in
iro n co ncentration. The w ater holding capacity of Pb-alginate gels
d e c re a se d b y an av erag e o f 3.0% p e r 0.05 M in c re a se in le a d
concentration (Fig. 25, 26).
The b ea d size is a fu n ctio n of th e surface ten sio n of th e gel
th a t m akes up the bead, th e sm aller the b ead th e h ig h er th e surface
ten sio n . A cetylated b a c teria l alg in ate form s gel b e a d s in 0.5 M
CaCl2 th a t averaged 4.04 m illim eters in d iam eter (Table 11). These
b ead s a re 37% la rg e r th a n eq u iv alen t b ead s m ad e w ith seaw eed
a lg in a te , a n d 45% la rg e r th a n b e a d s m a d e w ith d e a c e ty la te d
bacterial alginate.
Incubation of the gel beads in deionized w ater o r 0.5 M CaCl2
a t 4°C fo r 24 h o u rs a lte re d th e size o f th e gel b ead s. Beads
89
90
(% of C alcium )
80 -
70 -
60 -
Water Holding
50 -
40 -
30
.05 M 1 M .25 M .5 M

acetylated P. syringae alginate gels m ade w ith ferric iro n (D), an d
lead (♦), as com pared to calcium alginate gels.
90
80
of Calcium)
70 -
60 -
{%
Water Holding
50 -
40 -
30
.05 M ] M .2 5 M .5 M

d eacety lated P. syringae alg in ate gels m ad e w ith fe rric iro n (□),
an d lead (♦), as com pared to calcium alginate gels.
91
Table 11. Effects of A cetylation on Bead Surface T ensiona
Bead Diam eters (mm.)
Time in W aterb T im einC aC l?c
A lginate sam ple 5 m inutes 24 h ours 24 h o u rs
M acrocystis 2.95 2.69 2.64

(± 0.05) (± 0.04) (± 0.06)
A cetylated 4.04 2.82 2.26

P. syringae (± 0.06) (± 0.04) (± 0.05)
Deacetylated 2.79 1.96 1.68

P. syringae (± 0.05) (± 0.04) (±0.05)
a T he rela tiv e surface te n sio n was m e asu red by b e a d d ia m e te r

(mm.), th e sm aller the b ea d th e h ig h er the surface ten sio n on the
bead.
b W ater values w ere m e a su re d afte r 15 m in u tes in 0.5 M CaCl2
a n d in cu b atio n in w ater at 4°C fo r the relevent times.
c CaCl2 values w ere m e a su re d after in cu b atio n in 0.5 M CaCl2 at
4°C for 24 hours.
92
in c u b a te d in d eio n ize d w a te r m a d e fro m a c e ty la te d b a c te ria l

alginate h a d d iam eters averaging 2.82 m illim eters, a d ecrease of
42% from th e initial size. These b eads w ere still 5% la rg e r th a n th e
seaw eed alg inate gel b ead s a n d 44% la rg e r th a n th e d ea c e ty la te d
bacterial alginate gel beads. This in d icated th a t the relativ e surface
ten sio n o f acety lated gels was less th a n d eacety lated alg in ate gels.
E xtended in cu b atio n (24 hours) of Ca-alginate gel b e a d s in 0.5 M
CaCl2 p ro d u c e d d iffe re n t results. The Ca-alginate gel b ead s m ade
fro m seaw eed alginate w ere th e larg est w ith an av erag e d ia m e te r
o f 2 .64 m illim e ters. T h ese b e a d s w ere 17% la rg e r th a n th e
acetylated alginate b eads a n d 57% larg er th a n th e b ead s m ad e from
d e a c e ty la te d b a c te ria l alg in ate. T his in d ic a te d th a t acetylation
d e c re a se d th e surface te n sio n o n th e C a-alginate gel b ea d s, a n d
ex ten d ed exposure to calcium d ecreased th e size of th e b ead s a n d
increased surface tension on th e beads.
T he re la tiv e p o ro sity of th e C a-alg in ate a n d F e-alg in ate
(ferric) gel beads w ere d eterm in ed from th e ra te of w ater loss w ith
tim e (Fig. 27). A large absolute slope in d icated a m o re ra p id w ater
loss resulting from larg er relative p o re sizes. Ca-alginate gels, m ade
fro m acety lated b ac teria l alginate, show ed th e m o st ra p id loss of
w ate r (ab so lu te slope= 6.8 x 1 0 '2 ). The ra te o f w ater loss in th e
gels m ad e from d eacety lated b acterial alginate (absolute slope= 5.2
x 10" 2) was 24% slower th a n th e acety lated b acterial p o ly m er a n d
13% faster th a n seaw eed alginate (absolute slope= 4.6 x 10"2). The
ra te s o f w ate r loss of Fe-alginate gels w ere also d e te rm in e d a n d
co m p ared as a p ercen tag e of C a-alginate gels. Both th e gels m ade
fro m seaw eed a lg in ate a n d d e a c e ty la te d b a c te ria l a lg in ate lo st
93
■N
fed Q
a •©
is
O
as 4
« ,£
rt
£ M
Im
&
to
3
0 10 20 30
T im e (m in.) a t 50 C
Figure 27. Rate of w ater loss of calcium alg in ate gels m ad e from
M acrocystis alginate, I slope l= 4.6 x 10"2 (□), ac ety late d P. syringae
alg in ate, I slope 1= 6.8 x 10"~ (♦), an d d e a c e ty la te d P. syringae
alginate, I slope 1= 5.2 x 10"2 (I), as a function of in cu b atio n tim e at
50°C.
94
w a te r a p p ro x im a te ly 2 fo ld f a s te r th a n th e ir C a -a lg in a te
co u n terp arts, indicating a m ore open structure. The gels p ro d u ced
by th e a c ety late d b a c te ria l alg in ate show ed a ra te of w ate r loss
a p p ro x im a tely eq u al to th a t of C a-alginate gel b ead s in d icatin g
com parable p ore sizes betw een these gels (Fig. 28).
3. Cation Precipitation by Alginates

P re c ip ita tio n o f a c e ty la te d a n d d e a c e ty la te d b a c te ria l
alginates, m easured as gelation, by cations was co m p ared to th a t of
seaw eed alg in ate. Tw elve m e tal ions w ere sc re e n e d a n d th e n
classified in to 3 groups, d ep en d in g o n th e ir ab ility to p re c ip ita te
acetylated a n d /o r d eacetylated bacterial alginate. The groups were:
1) those cations having th e ability to p recip itate h a lf th e available
bacterial alginate at a con cen tratio n of less th a n 20 mM m etal, (U6+,
Cu2+, Pb2+,Ca2+, Sr^+, a n d Fe3+), 2) those cations having the ability
to p recip itate h alf the available bacterial alginate at a co n cen tratio n
of g reater th a n 20 mM m etal, (Zn2+, Co^+, a n d Mn^+), a n d 3) those
cations un ab le to p recip itate h alf th e available b acterial alginate at
a concen tratio n u p to 100 mM m etal, (Mg2+, Csl+, an d R bl+).
Of all the cations tested, uranium , copper, lead an d ferric ions
p recip itated b o th acety lated an d d eacety lated b acterial alg inate a t
low concentrations. T hey were able to p recipitate m ore th a n 90% of
th e alginate a t m etal co n cen tratio n s less th a n 5 mM. A cetylation
d id n o t significantly affect th e ability of th ese ions to p re c ip ita te
b a c te ria l alg in ate. U ra n iu m p re c ip ita te d g re a te r th a n 90% of
d eacety lated b acterial alginate a t a m etal co n c e n tra tio n of 1 mM
a n d g re a te r th a n 90% o f th e a c e ty la te d b a c te ria l a lg in ate a t a
95
110
Ui
B
100 -
art d§
Q °
S3 90 -
tt o
rs W°i
WH
S3 4> 80 -
12 bo
2o rtd
28 70 -
JS S
fld
Et
GO
0 10 20 30
T im e (m in .)
Figure 28. Com parison of the w ater loss of ferric iro n alginate gels
of M acrocystis alg inate (D), acety lated P. syringae alginate ( ♦ h a n d
deacetylated P. syringae alginate (I), co m p ared to calcium alginate
gels as a function of incubation tim e at 50°G
96
concentration of 5 mM (Fig. 29). Cupric ion reac ted m uch the sam e
way, precipitating 98% of th e d eacetylated bacterial alginate a t 2.5
mM copper, an d g reater th a n 90% of acetylated b acterial alginate a t
a m etal co n cen tratio n of 5 mM (Fig. 30). Lead in d u c ed gelation of
b acterial alginates was least affected by acetylation. G reater th a n
90% o f b o th a c e ty la te d a n d d e a c e ty la te d b a c te ria l alginates
p recip itated in the presence of 1 mM lead chloride (Fig. 31). Ferric
io n s p re c ip ita te d b o th a c e ty la te d a n d d e a c e ty la te d b a c te ria l
alginate. A cetylation in creased th e p recip itab ility of th e bacterial
p o ly m e r b y fe rric ion. A p p ro x im ately 90% o f th e a c e ty la te d
b a c te ria l p o ly m e r p re c ip ita te d w ith 1 mM fe rric ch lo rid e. In
c o n tra st, 90% of th e d ea c e ty la te d b ac teria l alg in ate p re c ip ita te d
w ith 2 mM ferric chloride (Fig. 32).
A h ig h affinity of calcium ions for p o ly g u lu ro n ate resid u es is
th e basis fo r th e c u rre n t gelling th e o ry (the "egg box m odel") fo r
seaw eed alg in ates. As ex p ected , alm o st 100% of th e seaw eed
a lg in a te was p re c ip ita te d by 5 mM calcium c h lo rid e (Fig. 33).
C alcium p re c ip ita tio n w as less efficien t fo r b a c te ria l alg in ates,
p ro b a b ly d u e to th e ab sen ce of extensive po ly g u lu ro n ate blocks.
T he a m o u n t of b a c te ria l alg in ate p re c ip ita te d b y calciu m io n s
in c re ase d as th e calcium co n c en tratio n increased . A pproxim ately
95% of the deacetylated bacterial alginate an d 65% of th e acety lated
bacterial alginate precip itated with 60 mM calcium chloride.
As m ight be expected from its chem ical sim ilarity to calcium,
stro n tiu m was also an effective p re c ip ita n t of b o th acety lated an d
deacetylated bacterial alginate. As w ith calcium , stro n tiu m show ed
a g r e a te r a b ility to p re c ip ita te d e a c e ty la te d th a n a c e ty la te d
97
100
■0 80 -
$
B
a
60 -
o
k0H
&
3 40 -
rt
a
bjo
< 20 -
0 1 2 3 4 (> 7 8 9 10
U ra n iu m c h lo rid e (mM)
Figure 29. P recipitation of M acrocysns alg in ate (D), a c e ty la te d P.

syringae alginate (♦), a n d deacetylated P. syringae a lg in a te (I) by
u ran iu m ions. Relative precip itatio n is expressed as the % alginate
p recip itated by U&+- ions.
98
120
100 -
3
B 80 -
a
o
60 -
£
$
rt
a 40 -
ao
<
20 -
0+
0 2 4 6 8 10
C u p ric c h lo rid e (mM)
Figure 30. P recipitation of M acrocysns alg in ate (D), a c e ty la te d P.

syringae alginate (♦), a n d deacety lated P. syringae a lg in a te (I) by
co p p er ions. Relative p recip itatio n is expressed as th e % alg inate
precipitated by Cu2+ ions.
99
120
100 -
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$
B 80 -
‘8.
o
o
Wn GO -
&
B
«
a 40 -
GJO
<
20 -
0 ■*
0 1 2 4
L ead c h lo rid e (mM)
Figure 31. P recipitation of M acrocystis alg in ate (D), a c e ty la te d P.

syringae alginate (♦), an d deacetylated P. syringae a lg in a te (I) by
lead ions. Relative p re c ip ita tio n is ex p ressed as th e % alg in ate
precip itated by Pb^+ ions.
100
100
■o 80 -
B
$
Gk- 60 -
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<b
u
O h
O
4-> 40 -
(0
a
&0
VN
<
20 -
7 4 6 8 10
F e rric c h lo rid e (mM)
Figure 32. P recipitation of M acrocystis alg in ate (□), ac ety late d P.

syringae alginate (♦), a n d deacetylated P. syringae alg in a te (I) by
ferric ions. Relative p re c ip ita tio n is expressed as th e % alginate
p recip itated by F e ^ ions.
101
100
*0 80 -
B
&
Q. 60 -
o
O
b*
a,
Bca 40 -
d
bo
< 20 -
0 •#
0 20 40 60 80
C alciu m c h lo rid e (mM)
Figure 33. P recipitation of M acrocystis alg in ate (D), ac ety late d P.

syringae alginate (♦), a n d d eacetylated P. syringae alg in a te (I) by
calcium ions. Relative p recip itatio n is expressed as th e % alginate
p recip itated by Ca^+ ions.
102
bacterial alginate. S trontium p recip itated 100% of th e deacety lated

bacterial alginate a t a final concentration of 20 mM. A pproxim ately
78% of th e acety lated b acterial alginate was p re c ip ita te d w ith 60
mM strontium chloride (Fig. 34).
The second set of cations w ere those th a t p recip itated h alf the
available b acterial alg in ate a t a c o n c en tratio n o f g re a te r th a n 20
mM m e tah . Zinc io n s fell in to th is category. Zinc p re c ip ita te d
d e a c e ty la te d b a c te r ia l a lg in a te b e t te r th a n its a c e ty la te d
co u n terp art. The p recip itatio n of b o th acetylated a n d deacetylated
bacterial alginate d id n o t begin until zinc concentrations w ere above
10 mM. T he c o n c e n tra tio n o f p re c ip ita te d b a c te ria l alg in a te
in c re ase d as th e co n c en tratio n of zinc in creased . A pproxim ately
55% of th e acety lated polym er p recip itated in 60 mM zinc chloride,
a n d 95% of th e d e a c e ty la te d p o ly m er p re c ip ita te d a t th e sam e
m etal con cen tratio n (Fig. 35). It ap p ears th a t acetylation d ecreased
the precipitability of alginate by zinc ions.
M anganese a n d c o b a lt ions show ed a lim ite d a ffin ity for
d e a c e ty la te d b ac teria l alg inate a n d no affinity fo r th e acetylated
p o ly m e r. M an g an ese p re c ip ita te d 100% of th e d e a c e ty la te d
b acterial p o ly m er a t a co n c en tratio n of 75 mM (Fig. 36). C obalt
p re c ip ita te d a p p ro x im a te ly 87% o f th e d e a c e ty la te d b a c te ria l
p o ly m er a t th a t co n c e n tra tio n (Fig. 37). A cetylation com pletely
in h ib ite d th e a b ility o f m a n g a n e se a n d c o b a lt to p recipitate
b acterial alginate. N eith er acety lated n o r d ea ce ty lated b a c teria l
alg in ate p re c ip ita te d w ith cesium , ru b id iu m , o r m ag n esiu m ions
(Fig. 3 8 ,3 9 ).
103
120
100
T>
$
S 80
a,
o
<L>
3
rt
a 40
&o
<
M 20
0
0 20 40 GO 80
S tro n tiu m c h lo rid e (mM)
Figure 34. P recipitation of M acrocystis alg in ate (D), a c e ty la te d P.

syTingae alginate (♦), a n d d eacetylated P. syringae a lg in a te (I) by
stro n tiu m ions. Relative p recipitation is expressed as th e % alginate
p recip itated by Sr^+ ions.
104
120
100 -
•o
$
JS
•N
p 80 -
&
•H
o
O
lM 60 -
&
co
o 40 -
&
pN
o
<
20 -
0 20 40 60 80
Zinc c h lo rid e (mM)
Figure 35. P recipitation of Macrocystis alg in ate (0), a c e ty la te d P.

syiinga e alginate (♦), a n d deacety lated P. syTingae a lg in a te (I) by
zinc ions. Relative p re c ip ita tio n is ex p ressed as th e % alginate
p recip itated by Zn^+ ions.
105
120
100 -
x?
B 80 -
B
o 60 -
O
u
&
Brt 40 -
a4
•^
ao 20 -
<
- 20
0 50 100 150 200 250
M a n g a n e se c h lo rid e (mM)
Figure 36. Precipitation of Macrocystis alg in ate (0), a c e ty la te d P.

syringae alginate (♦), a n d d eacetylated P. syringae a lg in a te (I) by
m a n g a n e se ions. Relative p re c ip ita tio n is e x p re sse d as th e %
alginate p recip itated by Mn^+ ions.
106
120
100 -
•0
$
80 -
B
ft
•vM
o GO -
4
kn
&
40 -
&
rt
a
euo 20 -
<
- 20
0 50 100 150 200
C o b alt c h lo rid e (mM)
Figure 37. Precipitation of Macrocystis alg in ate (□), a c e ty la te d P.

syringae alginate (♦), a n d deacetylated P. syringae a lg in a te (I) by
cobalt ions. Relative p recip itatio n is expressed as th e % alg in ate
precipitated by Co2+ ions.
107
■e
is
5
»aoH
3
kH
6
is
a
&0
fN
1
<
1
0 20 40 60 80
C esium c h lo rid e (mM)
Figure 38. Precipitation of M acrocystis alg in ate (0), ac ety late d P.

syiringae alginate (♦), a n d deacetylated P. syTingae a lg in a te (i) by
cesium ions. Relative p recip itatio n is ex p ressed as the % alginate
p recip itated by Csl+ ions.
108
100
90
80
■o
8 70
8
a 60
o
O 50
a
40
8rt
30
a
CUD 20
<
M
0 10 20 30 40 50 60 70
M a g n esiu m c h lo rid e (mM)
Figure 39. P recipitation of Macrocystis alg in ate (□), a c e ty la te d P.

syringae alginate (♦), an d deacetylated P. syringae a lg in a te (I) by
m a g n esiu m ions. R elative p re c ip ita tio n is e x p ressed as th e %
alginate p recip itated by Mg 2+ ions.
109
The relative ability of these m etal ions to p recip itate alginates

w ere c o m p a re d to th e P i / 2 v alu es fo r seaw eed a lg in a te a n d
re c o rd e d as th e fold difference from those values (Table 12). The
P l / 2 v alu e is d e fin e d as th e c o n c e n tra tio n of m e tal io n s (mM)
re q u ire d to p recip itate 50% of a p o ly m er fro m a 400 iig /m l (w /v)
so lu tio n . T he re la tiv e o rd e r of p re c ip ita tio n o f a c e ty la te d a n d
d e a c e ty la te d b a c te ria l alg in ate b y th ese ions, as d e te rm in e d b y
P l / 2 , w ere as follows:
A cetylated b acterial alginate: Pb2+=Fe3+=u6'1- > Cu2+ > Sr2+ > Ca2+ >
Zn^+ > Mg 2+=Co2+=Cs 1+=Mn 2+ =Rb 1+
D eacetylated bacterial alginate: Pb2+=Fe3+=U(5+ > Cu2+ > Sr2+ > Ca2+ >
Zn2+ > Mn2+ > Cq 2+ > Mg 2+ =Cs 1+=Rb 1+
110
Table 12. The Precipitation of M acrocystis Alginate a n d A cetylated

a n d D eacetylated P. syringae A lginate by M etal Ions
Fold Increase fro m P i / 2 a

o f M acrocystis A lginate^
Ions Periodic M acrocystis A cetylated D eacetylated

Group Alginate P. syringae P. syringae
( P l/2 ) c Alginate A lginate
Csl+ IA No affinity No affinity No affinity

Rbl+ No affinity No affinity No affinity
Mg 2+ II A No affinity No affinity No affinity

Ca2+ 3.1 3.4 1.7
Sr2+ 1.8 3.7 1.1
Mn2+ VII A 30.0 No affinity 0
pe 3f VIII A 1.8 -0.7 -0.6

Cb2+ 9.6 No affinity 3.7
Cu2+ IB 0.5 1.6 2.0
Zn2+ II B 7.0 4.3 2.3
Pb2+ IV B 0.5 0 0.2
U&f Actinide 0.9 -0.2 -0.4

m etal
a P l / 2 is th e c o n c e n tra tio n o f m e ta l io n s (mM) re q u ire d to

p re c ip ita te 50% (w /v) a lg in a te fro m 4 0 0 [.ig/ml (w /v) alg in ate
solutions.
b V alu es e x p re s se d a r e th e fo ld d iffe re n c e fro m P l/2 o f
M acrocystis alg in ate (positive v alu es = less ab ility to precipitate,
Negative values = g reater ability to p recip itate, zero values = equal
ability to precipitate).
c " No affinity" signifies th a t the ion d id n o t p recip itate 50% of the
alginate sam ple up to 100 mM ion concentration.
DISCUSSION
A lginate biosynthesis is a com m on characteristic of a m ajority

of p seu d o m o n ad s in rRNA-DNA hom ology group I (94). This group
in c lu d e s a ll th e f lu o r e s c e n t and a few n o n f lu o r e s c e n t
pseudom onads. Until recently, o nly certain strains of P. aeruginosa
isolated alm ost exclusively from cystic fibrosis p atien ts, an d strains
of A. vinlandii w ere know n to p ro d u ce O -acetylated alginate as EPS
(67). The fluorescent pseudom onads, P. flu o rescen s, P. m endocina,
a n d P. p u tid a , w ere su b seq u en tly fo u n d to p ro d u c e alginate, b u t
o n ly u n d e r c o n d itio n s o f stre ss (b a c te rio c in , b a c te rio p h a g e ,
a n tib io tic , 53, 58). W ithin th e p a s t sev en y ea rs, m a n y p la n t
p a th o g en ic flu o re sce n t p se u d o m o n ad s h av e b e e n reco g n ized as
alginate p ro d u cers u n d e r the ap p ro p riate conditions. This strongly
in d ic a te s th a t th e a b ility to sy n th e siz e a lg in a te m a y h a v e a n
im p o rta n t evolutionary role.
L ittle is k n o w n a b o u t th e sy n th e sis o f a lg in a te b y th e
p h y to p a th o g e n ic p se u d o m o n a d s . P se u d o m o n a s syringae pv
phaseolicola ATCC 19304 p ro d u ces h ig h am o u n ts of O -acetylated
alginate in vitro. The am o u n t o f alginate, as well as th e d eg ree of
acetylation of th e polym er, v aried w ith carb o n source. P. syringae
ATCC 19304 grown on sucrose p ro d u ced a m ixed p o p u latio n of EPS.
A lthough alginate was th e p re d o m in a n t EPS p ro d u ced , P. syringae
ATCC 19304 also p ro d u ced levan (C-2 -» C-6 fru c ta n backbone) on
sucrose (57). Levan p ro d u ctio n indicates th a t th e b ac teriu m also
h as the ability to synthesize levansucrase. Sucrose was n o t utilized
as th e carb o n source in this research since th e p resence of lev an in
111
112
th e exo p o ly m er ex tracts m ad e p u rificatio n o f th e alg in ate m ore

difficult.
Gluconic acid was chosen as th e ca rb o n source in this w ork
because cells grown on gluconic acid p ro d u ced approxim ately 21%
m o re alginate th a n cells grow n on glucose. It is well esta b lish e d
th a t th e p rim a ry ro u te o f glucose catabolism in p seu d o m o n ad s is
th ro u g h th e E ntner-D oudoroff pathw ay, w hich p ro d u ces p y ru v ate
a n d g ly cerald eh y d e 3-p h o sp h ate (52). Glucose co n v ersio n to 6-
p h o sp h o g lu c o n a te is re q u ire d p rio r to e n try in to th e E n tn er-
D oudoroff pathw ay. This conversion can be accom plished in one of
two ways. Glucose can be p h o sp h o ry lated to glucose 6 -p h o sp h ate
by a hexokinase a n d subsequently oxidized to 6-phosphogluconate
b y a glucose 6-p h o sp h ate deh y d ro g en ase (148), o r glucose can be
c o n v e rte d to gluconate a t th e surface of th e cell b y a n NAD(P)
d e p e n d e n t glucose d eh y d ro g en ase follow ed b y gluconolactonase.
G luconate is th e n o x idized to 2 -k eto g lu co n ate b y a m e m b ra n e
b o u n d gluconate dehydrogenase during tra n sp o rt of th e su g ar into
the cell. Once inside the cell the 2-ketogluconate is p h o sp h o ry lated
to 6-phosphogluconate (47). Most pseudom onads, i.e., P. aeruginosa,
P. fluorescens, a n d P. p u tid a oxidize glucose to gluconate using the
second m echanism p rio r to tra n sp o rt of th e sugar into the cell (52).
The in creased p ro d u ctio n of alginate by gluconate grown cells over
glucose grow n cells p ro b a b ly re su lts fro m a d e c re a se d en erg y
re q u ire m e n t fo r th e enzym atic oxidation of glucose to g luconate
p rio r to tra n sp o rt into the cell.
In P. aeruginosa, th e C-6 o f g lu c o n ate in c o rp o ra te s into
alginate (13). C arbon atom s 1, 2, an d 3 of gluconate are converted
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to p y ru v ate th ro u g h the 2-keto 3-deoxyphosphogluconate aldolase

reac tio n a n d are ev en tu ally lost as CO2 a n d acety l CoA. Carbon
a to m s 4, 5, and 6 a re c h a n n e le d in to a lg in a te th ro u g h
g ly cerald eh y d e 3 -p h o sp h ate. G ly cerald eh y d e 3 -p h o sp h a te can
co n d e n se w ith d ih y d ro x y aceto n e p h o sp h a te to p ro d u c e fru cto se
1,6-diphosphate a n d u ltim ately fructose 6 -p h o sp h ate, w hich is the
starting m aterial fo r alginate biosynthesis (7, 78). Besides being a
source of fructose 6-phosphate, gluconic acid can also be oxidized to
acetyl CoA (7). Acetyl CoA is re p o rte d ly th e source of th e O-acetyl
groups on th e m annosyl resid u es of x a n th a n gum (66), a n d is the
prob ab le source of acetyl in bacterial alginates (136).
P. syringae ATCC 19304 utilized am m onia as a n itro g en source
fo r grow th. It was u n a b le to use n itra te , n itrite , o r u rea. All
p seu d o m o n ad s p ro d u c e e n e rg y b y re sp ira tio n . In som e cases,
n itra te can be u sed as a n a lte rn a te e le c tro n a c c e p to r allow ing
grow th to o ccu r a n a ero b ica lly . T hose p se u d o m o n a d s th a t can
c o n d u c t n itra te re s p ira tio n can re d u c e n itra te b e y o n d th e toxic
n itrite stage to m olecular n itro g en by "denitrification." P. syringae
was u n a b le to utilize n itra te as a n itro g en source a n d th e re fo re
lacks the cellular m akeup to co n d u ct n itra te resp iratio n . This m ay
be d u e to th e absence o f cytochrom e C in th e electro n tra n sp o rt
ch a in of P. syrin g a e (9 4 ). In p la n ts, fre e am m o n iu m io n s are
p re s e n t a t v e ry low levels. A m m onium is toxic to th e p la n ts,
because it in h ib its th e p ro d u ctio n of ATP in the m ito ch o n d rial an d
ph o to sy n th etic electro n tra n sp o rt system s. Most n itro g en p rese n t
in plan ts is fo u n d associated w ith organic com pounds. In plants,
am m onium is converted in to organic com pounds p rim arily through
114
th re e d ifferen t reactions: 1) form ation of glutam ic acid b y reactio n

w ith a-k eto g lu tarate, 2) fo rm atio n o f g lu tam in e b y re a c tio n w ith
glutam ic acid, an d 3) form ation of carbam yl p h o sp h a te in arg in in e
b io sy n th e sis a n d p y rim id in e b io sy n th e sis. P. sy rin g a e is a
proteolytic bacterium (94). This b acteriu m p ro b ab ly p ro v id es itself
w ith am m o n ia th ro u g h d eam in atio n o f th e co m p o n en t am ino acids
allowing the survival of th e bacterium o n the h o st leaf surface.
Polysaccharide p ro d u ctio n by m icroorganism s is e n h a n ce d in
n itro g en lim ited, high ca rb o n co n ten t m ed ia (147). The changing
c a r b o n /n itr o g e n ra tio e x h ib ite d th e sam e e ffe c t o n a lg in a te
p ro d u c tio n in P. sy rin g a e th a t h a s b e e n r e p o r te d fo r o th e r
p se u d o m o n a d s. H ig h e r y ie ld s w e re o b ta in e d w ith h ig h
c a rb o n /n itro g e n ratio s (low n itro g e n co n ten t, h ig h g luconate). It
has b een pro p o sed th a t lim itation of essential n u trien ts, in this case
n itr o g e n , in h ib its g ro w th a n d d ir e c ts th e c o u rs e o f to ta l
p o ly sa cc h arid e b io sy n th esis fro m cell w all m a te ria l (LPS a n d
peptidoglycan) to extracellu lar polysaccharide synthesis (1 3 3 ). It
was fu rth e r p o stu lated th a t d u rin g active grow th th e sam e pool of
iso p ren o id lipid ca rrie rs involved in p o ly sacch arid e synthesis are
u tilized by cell w all a n d LPS p re c u rso rs (133). T hese co n d itio n s
d ire c t a lg in ate b io sy n th esis to w ard p ro d u c tio n as a se c o n d a ry
m e ta b o lite . In th e case o f P. syrin g a e ATCC 19304, a lg in ate
biosynthesis d id n o t fully fit this hypothesis. A lginate p ro d u c tio n
by P. syringae is necessary for successful colonization of p la n t host
tissue (113). A lthough alg in ate p ro d u c tio n d id in c re a se as th e
c a rb o n /n itro g e n ratio in creased , a lg in a te w as p ro d u c e d b y P.
syringae coincidental w ith cellular reproduction.
115
Bacteria secrete EPS for m an y d ifferen t reasons. Bacterial EPS

m ay aid colonization (16, 50), p rev en t desiccation, store energy, or
help co ncentrate a n d take u p ch arg ed m olecules, p articu larly m etal
ions (11, 17, 46). In cystic fibrosis p atien ts P. aeruginosa secretes
alg in ate to h e lp colonize th e lungs a n d to p ro te c t itself against
phagocytosis. P. syringae p ro d u ces alginate to aid in colonization
a n d parasitizatio n of leaf surfaces (30, 138).
T he a b ility o f P. syringae to p ro d u c e alg in a te d e c re a se d
r a p id ly u p o n su ccessiv e tra n s fe rs in liq u id m e d ia. A lg in ate
p ro d u c tio n re m a in e d stab le u p o n tra n sfe r o n solid m ed ia. The
pathogenicity of P. syringae is d ep e n d en t upon its ability to produce
alginate (113). Colonization of a leaf surface by P. syringae causes
h alo blig h t (leaf spots) o f th e leaf. A lginate m u st be p re s e n t to
p ro d u c e leaf spots (34). These leaf spots are p ro b a b ly d u e to an
in te rr u p tio n of th e p h o to sy n th e tic p a th w a y in th e le a f. All
p seudom o n ad s respire, a n d thus m u st have a source of ferric iro n
fo r synthesis of cytochrom es. On th e leaf surface, th e sources of
ferric iro n are lim ited to ferredoxin an d the cytochrom e b f complex.
T he b re a k d o w n o f fe rre d o x in o r th e c y to c h ro m e b f co m plex
in te rru p ts p h o to sy n th esis b y stopping electro n tra n sp o rt a n d aids
in le a f sp o ttin g . A cety lated P. syrin g a e a lg in a te s h a v e a h ig h
affinity for ferric iron. By increasing the ferric iro n co n cen tratio n in
the grow th m edia alginate p ro d u ctio n decreased. The p arasitization
o f P. syrin g a e ATCC 19304 m ay be d u e to th e a b ility o f th e
acety lated alg in ate to scavenge th e ferric iro n fro m ferred o x in o r
the cytochrom e b f complex. By scavenging the iron, photosynthesis
116
w o u ld be in te r r u p te d a n d le a f sp o ts w o u ld o c c u r d u e to th e
inability of th e p lan t to m ake energy for chlorophyll biosynthesis.
T h e n a tiv e a lg in a te s o f P. sy rin g a e ATCC 1 9 3 0 4 w ere
p o ly d isp erse. This in d ic a te d th a t th e re w as a h ig h d e g re e o f
variability in size of th e alginate p ro d u ced by th e biological system.
T he Mw fo r th e n ativ e b a c te ria l p o ly m er was 1.3 x 1 0 $. U pon
deacetylation, th e Mw d ro p p ed approxim ately 6 % to 1.2 x 105. This
d ro p was d u e to th e loss o f th e acetyl groups at C-2 a n d /o r C-3 of
th e m a n n u ro n ic acid resid u es w ithin th e polym er. By m onitoring
th e difference in the acid stability of D -m annose a n d L-gulose from
th e acid h y d ro ly ze d , re d u c e d alg in ates, m o re a c c u ra te a lg in ate
com positions w ere o b tain ed fo r th e bacterial polym er. The alginate
p ro d u ced b y P. syringae ATCC 19304 h ad a final com position of 82%
m an n u ro n ic acid an d 18% guluronic acid. This com position was in
co n tra st w ith th e p reviously re p o rte d com position o f g re a te r th a n
95% m a n n u ro n ic acid a n d less th a n 5% g u lu ro n ic acid w h ere th e
relative acid sensitivity of each sugar was n o t considered (38).
Both th e solution a n d gelling p ro p erties of b acterial alginate
w ere a lte re d b y th e d eg ree o f acety latio n o n th e p o ly m er. T he
v is c o s ity o f th e a c e ty la te d p o ly m e r w as h ig h e r th a n th e
d e a c e ty la te d b a c te r ia l o r se a w e e d a lg in a te s a t e q u iv a le n t
concentrations. The n ativ e b acterial alginates w ere acety lated w ith
a n a v e ra g e o f 1.2 ac ety l u n its p e r u ro n ic acid re sid u e . T his
p ro d u c e d a 6 p e rc e n t in c re a se in Mw a n d a 26% in c re a se in
viscosity a t a co n c e n tra tio n of 1 m g /m l. A cetylation re p o rte d ly
increases th e viscosity of alginate solutions (129). T hese in creases
m a y be d u e to th e in c re a se in to ta l m o le c u la r w eig h t o f th e
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p o ly m e r, o r a lte re d co n fo rm atio n s of th e p o ly m e r in aq u eo u s
solution. A lginates h igh in m an n u ro n ic acid ex h ib it a flat rib b o n
like con fo rm atio n . High am o u n ts of g u lu ro n a te p ro d u c e a m ore
p u ck e re d stru c tu re . It is possible th a t th e in tro d u c tio n of acetyl
gro u p s shifts th e co n fo rm atio n al en e rg y w ith o u t in creasin g th e
accessible surface of th e m olecules, eventually p roducing a flexible
p olym er w ith an in creased n u m b e r of conform ations. Because the
a c e ty la te d p o ly m e r show ed a 4 fo ld in c re a se in v isco sity o v er
m olecular w eight, it is p ro b ab le th a t b oth factors play a role.
A cetylation a lte re d th e affin ity o f th e p o ly m e r fo r m a n y
cations, in clu d in g calcium . Except fo r ferric a n d to som e e x te n t
co p p er ions, th e ability of m ultivalent cations, p articu larly calcium,
to in d u c e g elatio n of th e alg in ate was re d u c e d in th e ac ety late d
polym er. In p arallel, th e re was a m ark ed decrease in th e stren g th
of th e gels. The effects o f acety latio n o n the gelling p ro p e rtie s of
b ac teria l alg in ates w ere seen in th e w ater h o ld in g capacities, th e
surface tensions, a n d th e relative porosities of the Ca-alginate gels.
A cetylation p ro fo u n d ly affected the rigidity of the Ca-alginate
gels a n d th e ir ab ility to h o ld w ater. G enerally, th e v o lum e of an
ionic gel is d e p e n d e n t u p o n a p o sitiv e osm otic p re ssu re . The
osm otic p ressu re of th e gel is d u e m ainly to th e positive e n tro p y of
th e mixing of co u n terio n s w ith w ater, w hich is c o u n terb ala n ced at
e q u ilib riu m b y a n eg ativ e p re ss u re d u e to th e elasticity of th e
n etw o rk (139). For C a-alginate gels, w hich a re en th alp ic r a th e r
th a n e n tro p ic (3), th e elasticity d e p e n d s o n th e n u m b e r a n d
stren g th of th e crosslinks. Since th e in tro d u ctio n of acetyl groups
im p airs th e co o p erativ e b in d in g of calcium ions, th e n u m b e r of
118
dissociated counterions p e r polym er chain increases w ith increasing

degree o f acetylation. The high n u m b er of dissociated co u n terio n s
e n h a n c e s th e positive o sm otic p re ssu re . At th e sam e tim e, it
w eakens th e forces holding th e netw ork together. R eduction in the
cooperative binding of calcium ions red u ces b o th th e stre n g th an d
th e n u m b e r o f crosslinks in th e netw ork. Fewer crosslinks lessen
th e surface tension on th e gel a n d as a re su lt allows h ig h e r w ater
holding capacities w ithin th e gel netw ork. This was o b serv ed w ith
th e P. syringae alginate. A cetylated b acterial alg in ate gels h a d a
m u c h low er su rface te n sio n , a n d as a re s u lt a w a te r h o ld in g
c a p a c ity th a t w as a p p ro x im a te ly 6 8 p e rc e n t h ig h e r th a n its
d e a c e ty la te d c o u n te rp a rt. Fe-alginate gels a n d P b -alg in ate gels
show ed th e sam e general properties, how ever n e ith e r th e ferric n o r
lead alginate gels h eld as m uch w ater as th e Ca-alginate gels. These
differences are attrib u ta b le to the relative affinities o f each catio n
to th e alg in ate. Ferric a n d lead ions h a d a h ig h e r affin ity th a n
calcium ions for b o th acetylated an d deacetylated alginates.
Because of its h ig h e r viscosity a n d low er affin ity fo r m a n y
polyvalen t cations, acety lated b acterial alginate m ay be a possible
substitu te fo r seaw eed alginate in m any applications. M ore viscous
so lu tio n s ca n be m a d e w ith low er c o n c e n tra tio n s o f bacterial
a lg in a te th a n w ith seaw eed a lg in a te , re d u c in g th e p o ly m e r
re q u ire m e n ts in a given ap p licatio n . By v ary in g th e d e g re e of
acetylation, it should be possible to pro d u ce alginates w ith designer
properties.
As a polyelectrolyte, alginate has th e p o ten tial to concentrate
toxic, heav y a n d /o r v alu ab le m etals from th e e n v iro n m e n t. T he
119
re m o v a l o f m e tals b y m icro o rg an ism s re p o rte d ly d e p e n d s on

p h y sic o c h e m ic a l a d s o rp tio n to c e llu la r c o m p o n e n ts su c h as
polysaccharides a n d p ro te in s (84). Because o f th e h ig h n eg ativ e
c h a rg e on th e ce ll w alls o f b a c te r ia , fu n g i, a n d a lg a e ,
m icroorganism s have b een used to co ncentrate m etal ions including
u ran iu m , co p p er, m anganese, cadm ium , m o ly b d en u m , gold, and
m e rc u ry (20, 56, 65). T he p o ly an io n ic n a tu re o f alg in ates also
allows th e binding an d co ncentration of heavy m etals th ro u g h m etal
in d u ced gelation o f the polym ers. The form ation of alginate gels in
th e presen ce of p o ly v alen t cations is a lte re d b y acety latio n of the
alginate, as well as th e n ativ e co n fo rm atio n of th e p o ly m er. The
physical binding o f calcium ions to gulu ro n ate residues in seaw eed
alginate is d u e to charge charge in teractio n s betw een th e positive
charges from calcium a n d the negative charges fro m th e carboxyl
groups. The size of calcium ions is such th a t th ey fit in to th e space
fo rm ed b y the p o lyguluronate stretch es of seaw eed alginates (83).
Random ly organized b acterial alginate also gels in th e p resen ce of
m a n y p o ly v alen t cations. This casts som e d o u b t on th e p rin cip al
te n a n t of th e "egg-box" theory; th a t only polyguluronate residues in
a lg in a te p la y a key ro le in g elatio n . T he a b ility o f b a c te ria l
alginates, w ith o u t p o ly g u lu ro n ate blocks, to gel in th e p resen ce of
calcium indicates a reevaluation of this th eo ry is necessary.
M ultivalent cations in te ra c t d iffe re n tly w ith alg in ates (59,
60). Each c a tio n ex am in ed show ed v a ria tio n s in its a b ility to
p re c ip ita te seaw eed a n d b acterial alginate. W ith th e exception of
f e r r ic a n d u r a n iu m io n s, p o ly v a le n t c a tio n s m o re re a d ily
p re c ip ita te d seaw eed alg in ate o v er its b a c te ria l c o u n te rp a rt. In
120
m ost instances, deacetylation of th e bacterial polym er en h an ced its

c a tio n p re c ip ita b ility . This a b ility to a d ju s t th e affin itie s of
alginates fo r specific p o ly v alen t cations in creases th e feasibility of
using th is p o ly m er to rem ove toxic m etals, i.e., lead o r u ra n iu m
from drinking w ater.
A cid h y d ro ly s is is u s e d to d e p o ly m e riz e p o ly m e rs for
co m p o sitio n al analysis. Ideally, h y d ro ly sis goes to co m p letio n
w ith o u t loss o f the com ponents. Practically, how ever, som e sugar
loss occu rs a n d m u st b e a c c o u n te d fo r to accu rate ly d eterm in e
polysaccharide com positions. C om positional analysis of alginates
re q u ir e s th e re d u c tio n o f th e u ro n ic a c id re s id u e s to th e ir
c o rre sp o n d in g n e u tra l su g ars p rio r to acid h y d ro ly sis. T h is
red u ctio n facilitates th e acid hydrolysis of th e glycosidic b o n d s by
converting th e acid resistan t glycosyluronic acid b o n d s to the m ore
acid labile glycosyl bonds. R eduction of alginate polym ers liberates
D -m annose an d its C-5 ep im er L-gulose a fte r acid hydrolysis. Both
D -m annose a n d L-gulose react differently u n d e r acid conditions. D-
M annose is re la tiv e ly acid stab le. L-gulose is m a rk e d ly acid
s e n sitiv e . In g en e ra l, ald o se c o n ta in in g p o ly sa c c h a rid e s a re
com pletely h y d ro ly zed w ith m inim al sugar loss b y 1 M H 2 SD4 a t
100°C for 5 h ours (119).
At e q u ilib riu m D -m annose re sid u e s fa v o r th e c h a ir
c o n fo rm a tio n w hile th e L-gulose re sid u e s a d o p t th e IC 4 c h a ir
co n fo rm atio n b e c a u se th e b u lk y g ro u p a t C-5 o rie n ts to a n
e q u a to ria l p o sitio n . T h ese su g a rs a re n o t lo ck ed in to th e se
conform ations. In so lu tio n th ese m o n o m ers exist in a dyn am ic
equilibrium . This equilibrium allows altern atio n betw een b o th ^C \
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a n d IC 4 co n fo rm atio n s, as well as th e boat, h alf-ch air, a n d o p en

conform ations. The op en chain conform ation allows an om erization
aro u n d C -l producing b o th th e a a n d p anom er. This eq u ilib riu m
allow s n u m e ro u s co n fo rm atio n al possibilities fo r each m o n o m er
includin g th e possible fo rm atio n of 1,6 an h y d ro h ex o p y ran o ses in
acid solutions.
Sugars o f th e gulo., ido., a n d altro . co n fig u ratio n s undergo
sp o n tan eo u s conversion to 1,6 an h y d rid e s in acids. Sugars of the
gluco., m anno., a n d galacto. configurations p ro d u ce v ery little 1,6
an h y d rid e a t eq u ilib riu m a n d are alm ost com pletely h y d ro ly zed to
th e free aldoses u n d e r acid conditions (4, 81). This is because of the
o rie n ta tio n of th e h y d ro x y l groups a t C-2, C-3, a n d C-4. Axially
o rien te d hydroxyl groups, p articu larly a t C-3, destabilize a n h y d rid e
fo rm a tio n . E q u a to ria lly o rie n te d h y d ro x y l g ro u p s fa v o r th e
fo rm atio n of 1,6 an h y d rid es. The equilibrium is d e p e n d e n t on th e
steric a rra n g e m e n t of h y d ro x y l groups w hich d o n o t tak e p a r t in
th e re a c tio n . O nce a 1,6 a n h y d r id e b o n d is fo rm e d , th e
co nform atio n al eq u ilibrium is shifted so th e sugar n o lo n g er exists
in a d y n am ic state. T he fo rm atio n o f a seco n d rin g w ith in th e
m olecule locks it into th a t p articu lar conform ation.
D -M annose, w hose fre e e n e rg y fa v o rs th e 4 q l c h a ir
conform ation, does n o t read ily form the 1,6 an h y d rid e b o n d w hen it
is in th e I-C4 ch air conform ation a n d th e hydroxyl group atta c h e d to
th e a n o m e ric c a rb o n is in th e p p o s itio n . A lth o u g h th is
conform ation brings C-6 a n d th e anom eric hydroxyl group in to axial
positions aro u n d th e ring an d into proxim ity, this conform ation also
brings th e hydroxyl groups a t C-2 an d C-3 into axial positions w hich
122
destabilize a n d in h ib it a n h y d rid e form ation. The axial h y d ro x y l

g roup a t C-3 in teracts w ith th e p o ten tial a n h y d rid e b o n d blocking
its form atio n . In th e p conform ation D-m annose, D-glucose, an d D-
galactose all have axial hydroxyl groups a t C-3 a n d th erefo re do n o t
readily fo rm 1,6 anhydrides.
T h e f re e e n e rg y o f L -g u lo se fa v o rs th e 1Gj. c h a ir
conform ation. At dynam ic equilibrium it can also exist in th e 4 c i
co n fo rm atio n , w hich p laces th e b u lk y g ro u p a t C-5 in a n axial
o rien tatio n . If the hydroxyl group a t the anom eric ca rb o n is in the
p position th e C-6 a n d th e anom eric hydroxyl group com e in to close
eno u g h proxim ity to allow 1,6 an h y d rid e form ation. An exception
is th a t th e a n h y d rid e b o n d form s below th e p lan e of th e rin g w ith
L-gulose ra th e r th a n above it. This o rien tatio n places th e hydroxyl
groups a t C-3, an d C-4 in eq u ato rial positions a ro u n d th e ring an d
th e h y d ro x y l g roup a t C-2 axial. In this p o sitio n th e free en erg y
favors 1,6 an h y d rid e form ation due to the absence of a destabilizing
axial hydroxyl group a t C-3.
U pon a c id h y d r o ly s is , L -gulose lo c k e d in to a new
co n fo rm a tio n . This was seen b y th e in c re ase in in te n s ity o f a
second spot on TLC over tim e. A lthough th e acid hydrolysis product
o f L-gulose w as n o t d e fin itiv e ly id e n tifie d , th e m o lecu le was
te n tativ ely id e n tified as a 1,6 a n h y d rid e because it h a d th e sam e
m igration as 1,6 an h y d ro p-D -m annopyranose, a n d 1,6 a n h y d ro p-
D -glucopyranose on TLC. C om puterized m olecular m odeling of 1,6
a n h y d ro p-L-gulopyranose using SYBYL softw are in d ic ated th a t the
m olecule h as a low total energy, fav o rab le fo r its p ro d u c tio n an d
stability.
123
Existing m e th o d s fo r d e te rm in a tio n of th e co m p o sitio n of

alginates reco m m en d hydrolysis in 1 M H2 SO4 fo r 90 m in u tes at
100°C (38). Since L-gulose is m ore susceptible to co n fo rm atio n al

m od ificatio n u n d e r these con d itio n s th a n is D -m annose, lack of
accounting for gulose d estru ctio n has resu lted in in accu rate rep o rts
of com position.
Analysis of acid hydrolyzed red u ced alginates p aralleled those
o b ta in e d w ith th e D -m annose a n d L-gulose m o n o m ers. W h en
correctin g fo r gulose d estru ctio n , by ex trap o latio n back to tim e 0 ,
th e results o f th e seaw eed alginate analysis co rrelated exactly w ith
th e p u b lish ed resu lts of 60% m an n u ro n ic acid a n d 40% guluronic
acid as d eterm in ed b y th e reductive cleavage m eth o d of Zeller and
G ray (1 5 0 ). T he a lg in a te s p ro d u c e d b y th e p h y to p a th o g e n ic
p se u d o m o n a d s a p p a re n tly h a v e a h ig h e r c o n c e n tra tio n o f L-
guluronic acid th a n h a d b ee n previously re p o rte d . T he re p o rte d
com position of th e P. syringae ATCC 19304 alginate is g reater th an
95% D -m annuronic acid a n d less th a n 5% L -guluronic acid (38).
A fte r c o rre c tin g fo r th e re la tiv e acid s e n s itiv itie s o f each
corresponding n eu tral sugar, the actual com position o f the bacterial
alg in ate was 82% D -m annuronic acid an d 18% L-guluronic acid.
T his ra tio c o rre la te s w ell to th e re p o r te d c o m p o sitio n o f P.
aeruginosa alginate of 80% D -m annuronic acid a n d 20% L-guluronic
acid (150).
P. syringae ATCC 19304 prod u ced large am ounts of acetylated
alg in ate w hen grow n on gluconic acid. W hile p ro d u c tio n of this
p o ly m e r d e c re a se d d ram a tic a lly o n su b cu ltu re in liq u id m edia,
p ro d u c tio n was stable on solid m edia. M axim um p ro d u c tio n was
124
d e p e n d e n t o n th e pH, te m p e ra tu re , a n d a h ig h carb o n /n itro g en

ra tio w ithin th e grow th m edia. The Mw of th e b a c teria l polym er
was approxim ately 1.7 tim es larg er th a n th e seaw eed c o u n te rp a rt
w ith a com position of 82% m annuronic acid a n d 18% guluronic acid,
a n d an average of 1.2 acetyl groups p e r m onom er. These physical
characteristics allowed for m uch h ig h er solution viscosities a t equal
c o n c en tratio n s, a n d c a tio n in d u c e d gels w ith in c re a s e d w a te r
holding capacity. By co ntrolling th e d eg ree o f ac ety latio n it was
p o ssib le to c o n tro l th e so lu tio n a n d gelling p ro p e rtie s of the
p o ly m er to som e ex ten t. The poly an io n ic n a tu re o f th e p olym er
allows fo r th e binding of m any toxic a n d h eav y m etals. Ultim ately,
it should be possible to create inexpensive d esig n er alginates whose
p ro p e rtie s a re c o n tro lle d b y th e d e g re e o f a c e ty la tio n o n th e
polym er.
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V IT A
Richard David A shby was b o m in Kansas City, Kansas o n May

6, 1962. A fter g raduating from Frem ont High School in Sunnyvale,
California in 1980, he a tten d e d Brigham Young U niversity w here he
o b tained his Bachelor of Science degree in M icrobiology in 1987. In
A ugust 1988, h e b eg an his g ra d u a te tra in in g a t L ouisiana State
U niversity in Baton Rouge, Louisiana.
141
DOCTORAL EXAMINATION AND DISSERTATION REPORT
candidate: R i ch ard David Ashby
Major Field: M i c r o b i o l o g y
Title of Dissertation: j h e P r o d u c t i o n and C h a r a c t e r i z a t i o n o f A l g i n a t e

P ro du ced by Pseudomonas s y r i n q a e
Appro?
f % K._
Major Professor and Chaxrman
Dean of the Gracîate School
EXAMINING COMMITTEE:
y g.-y
r, . ■v/M
^.u f H 'L Jk l
j2 /^ P u ,
Date of Examination:
June 2 1 , 1994

PHD Study With Same Method As B-1

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The Production and Characterization of Alginate Produced by

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T he p rod u ction and characterization o f algin ate produced by

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Subm itted to the G raduate Faculty of the

The D epartm ent of Microbiology

I w o u ld like to ex p ress m y d e e p e st a p p re c ia tio n to m y

II. C haracterization of Bacterial A lginate.............................. 69

Table 2. Some food applications of alginates................................ 16

Table 5. Effects of carb o n source o n alginate yield from

Table 11. Effects of acetylation o n b ead surface ten sio n 91

Figure 2. The block structures of alginate..................................... 11

Figure 10. The effect of the initial am m onium co ncentration

Figure 11. The effect of the initial am m onium co ncentration

Figure 12. The effect of initial pH on cell mass, an d

Figure 14. The effect of initial ferric ion (Fe^+) concentration

Figure 20. Gulose recovered, expressed as a p ercen t of

Figure 21. Com parative viscosity, (N/No), of M acrocystis

Figure 22. The effects of acetylation on th e com parative

Figure 27. Rate of w ater loss of calcium alginate gels m ade

Figure 29. Precipitation of M acrocystis alginate, acetylated

Figure 30. Precipitation of M acrocystis alginate, acetylated

Figure 31. Precipitation of M acrocystis alginate, acetylated

Figure 32. Precipitation of M acrocystis alginate, acetylated

Figure 34. Precipitation of M acrocystis alginate, acetylated

Figure 35. Precipitation o f M acrocystis alginate, acetylated

Figure 36. Precipitation o f M acrocystis alginate, acetylated

Figure 37. Precipitation of M acrocystis alginate, acetylated

Figure 38. Precipitation of M acrocystis alginate, acetylated

Figure 39. Precipitation of M acrocystis alginate, acetylated

A lginate h a s m a n y in d u s tria l uses b ecau se o f its u n iq u e

A lginate is a w ater-soluble gum used in the food an d chem ical

p ro d u ces alg inate only d u rin g en cy stm en t a n d does n o t p ro d u ce

I. Bacterial Exopolysaccharides: General Characteristics.

com m on. The p resen ce of n eg ativ e ch arg es is a fe a tu re o f some

II. Alginate: Overview

Species Polysaccharide S tructure

Leuconostoc m esenteroides D extran ( lit 6 glucose)n

W ithin the last 30 years, alginate was fo u n d to be p ro d u c e d

Figure 1 . Structures of the uronic acids of alginates.

m a rk e d ch an g e in th e th re e d im en sio n al co n fo rm atio n of th ese

Figure 2. T he block stru c tu re s of alg in ate. A. T he rib b o n -lik e

p roductio n by P. aeruginosa was related to resistance to antibiotics,

Figure 3. The "egg box" m odel for C a ^ induced gelation of poly «- L-

effects w ere n o t seen w ith p o ly m an n u ro n ate blocks o r altern atin g

alg in ate is b a se d are: 1 ) th e fo rm a tio n of viscous so lu tio n s a t

Table 2. Some Food Applications of A lginatesa

P roperty Product Perform ance

W ater holding Frozen foods M aintains texture d uring

Gelling Puddings Firms body an d texture

Emulsifying Salad dressings Emulsifies an d stabilization

Stabilizing Beer M aintains b ee r foam

a Table was ad ap ted from Sandford an d Baird (117).

Table 3. Some Industrial Applications of A lginatea

P roperty Product Perform ance

W ater holding Paper coating Controls rheology of coatings