Agric. Biol Chem.

, 50 (9), 2301 -2307, 1986 2301

Screening for and Characteristics of Microbial Flocculants*

Ryuichiro Kurane, Kiyoshi Takeda and TomooSuzuki
Fermentation Research Institute, Agency of Industrial Science & Technology,
Yatabe, Tsukuba NewScience City, Ibaraki 305, Japan
Received March 12, 1986

Fourteen strains of the genera Rhodococcus, Nocardia, and Corynebacterium were found to
produce substances which flocculate kaolin clay.
Rhodococcus erythropolis produces a kind of the microbial flocculant. It had very interesting
and unique flocculating characteristics. It could efficiently flocculate all suspended solids in aque-
ous solution tested. Amongthose effectively tested were microorganisms such as E. coli and
alcohol yeast, activated sludge, Microcystis aeruginosa (AOKO), kaolin clay, muddy water, river
dredging muddywater, river bottom sediment (HEDORO),ash from a steam-power station,
and chacoal. These results indicated that the flocculant from R. erythropolis had a wide floc-
culating activity against both organic and inorganic materials. Cationic ions such as Ca2+
also significantly increased the flocculating activity.

Organic polymers have been used for many Corynebacterium sp.,2) Aspergillus sojae,3)
years to flocculate colloids. Vegetative agents Dematium sp.,4) and Paecilomyces sp.5)
were first used to clarify water.1} Nowadays, In our previous study of microbial degra-
manykinds of flocculating agents are used in a dation ofphthalate esters,6) the prevention and
wide range of industrial fields such as waste recovery of deflocked sludge was effectively
water treatment, dredging, fermentation proc- achieved by inoculation of Rhodococcus eryth-
esses, and improvement of industrial proc- ropolis (formerly Nocardia erythropolis) S-l
esses. For example, inorganic materials such into activated sludge, in addition to the com-
as polyaluminium chloride (PAC) are used for plete removal of phthalate esters from waste
waste water treatment, and organic synthetic water. This microorganism was isolated and
high polymers such as polyacrylamide de- characterized as a phthalate ester rapidly de-
rivatives are frequently used as economical grading microorganism.7) This microorganism
and powerful flocculating agents for dredging seems to produce someagents which flocculate
fields, plant waste water treatment, and im- bacteria or prevent deflocculation of sludge. It
provement of industrial systems. However, it has also been reported that Rhodococcuseryth-
has been also reported that the monomerof ropolis {Nocardia erythropolis) was isolated as
acrylamide is a strong carcinogen. Therefore, a petroleum-assimilating bacterium from oil-
biodegradable, safe flocculants, which cause fields and soils8'9) and that this bacterium was
no problems in environmental pollution, are widely distributed in soils in Japan as a major
attracting wide interest and are urgently called gram-positive bacterium.10) Moreover, we
for. Especially, there is a potential need for have tested the pathogenicity of isolate S-l of
biodegradable, safe flocculants in food and R. erythropolis for animals such as mouseand
fermentation fields. Recently, it was reported guinea pig by injection of the cells and culture
that some microorganisms2~5) produced floc- broth.7) This isolate S-l showed no patho-
culating substances. These organisms were genicity. R. erythropolis is also classified as

Microbial Flocculants. Part I.

2302
R. Kurane, K. Takeda and T. Suzuki

a non-pathogenic bacterium in Bergey's cultivation at 30°C, and whole crude flocculant was in-
Manual. oculated into 100ml of the bulking-sludge. The sludge
This paper deals with the screening of volume (SV30) was measured in the sludge mixed liquor
flocculant-producing microorganisms using after 30min of standing in a 100-ml cylinder. The mixed
liquor suspended solid (MLSS) withdrawn from the mixed
bacteria, which we kept as phthalate ester liquor was measured by the dry sludge weight (ppm) at
105°C overnight after centrifugation at 3,000rpm for
assimilating bacteria, because they are more 5min and washing. The sludge volume index (SVI) was
easily handled in cultivation than fungi, and
also deals with someproperties of a flocculat- calculated by the following formula.
ing agent produced by Rhodococcus eryth- SVI = [SV30 (ml)/MLSS (ppm)] x 10,000
ropolis S-l.
2) Effects of cation on flocculation. Ninety-three milli-
liters of kaolin solution (5,000 ppm) in distilled water was
mixed with 2ml of the culture fluid of R. erythropolis S-1
MATERIALS AND METHODS and 5ml of various cation solutions (1%). Then the pH
each mixture was adjusted to pH 7.0, and it was left to
Screening for flocculant producing microorganisms. flocculate. After that, flocculating activity was measured.
Thirty strains, which were previously isolated and charac- Flocculating activity was indicated as the precipitation
terized as phthalate ester assimilating bacteria,7'11* were volume(%) of kaolin clay and the clarification of the
screened for flocculant production. For the screening upper phase (supernatant), measuring the optical density
medium, a complete denned medium containing phthalate at 660nm.
esters as a sole carbon source12) and nutrient broth were
used. The above microorganisms were grown in 20ml of
the culture medium in 100-ml flasks using a rotary shaker Table I. Microorganisms Producing Substances
at 30°C for 4~7 days. Flocculant activity for all 30 that Flocculate Kaolin Clay
cultures was estimated against kaolin clay. That is, floc- Flocculating activity
culation was measured by adding 0.5 g of kaolin clay and
1.0ml or 2.0ml of cultures (cells and culture broth) to M icroorganism P htha late
distilled water in a 100-ml graduated cylinder (final total ester. cultureculture
volume; 100ml). After mixing, this was left to stand and
whether flocculation occurred or not was observed after 5
minutes. Rhodococcus erythropolis
S- l , 26 0 - 2 ( 2 st r a i ns ) + + +
R hodococcus ery th rop olis
Cultivation of Rhodococcus erythropolis. According to 23-1, 23-3, 48-1, 62-1, 71-1,
the "Approved list of bacterial names" presented by the 242-1, 2 54-1, 256-2 (8 strains)
Ad Hoc Committee of the Judical Commission of the
International Committee on Systematic Bacteriology No c ar d ia re s tr ic t a F ER M -P 39 1 9 + +
(ICBS) in 1980, Nocardia erythropolis in the 8th edition of Nocardia calcarea FERM-P39 16
Bergey's Manual was classified as Rhodococcus erythro- Nocardia rhodnii DF-3
polis. Corynebacterium sp. FERM-P3527
Corynebacterium sp.
The medium was l.Og of glucose (or fructose), 0.5g of 62-3, 240-4, 242-2
(NH4)2SO4, 0.5g ofK2HPO4, 0.2g ofKH2PO4, 0.02g of
Mycobacterium sp. 6F-3
MgSO4-7H2O, and 0.01g of NaCl, in 1 liter of distilled Bacillus brevis DF-F
water, with the pH adjusted to pH 7.5. After preculti-
Pseudomonas putida (Biotype A)
vation on slant agar, five loops of cells were inoculated D F -2
into 500-ml flasks containing 100 ml of the above medium, Pseudomonas putida (Biotype B)
and cultivated with a rotary shaker at 30°C for 4~7 days. D F -1
Pseudomonas acidovorans 256- 1
Flocculation tests. P seudom onas cep acia 254-4
1) Sedimentation of bulking-sludge. Bulking-sludge P seudom onas sp . D E H P -F
from a kanzo waste water treatment system, which used Unidentified gram positive
the lagoon method with sludge in a private company in bacteri um DEH P-4, DE HP-C3,
Chiba Prefecture and caused bulking due to high BOD, DBP-C3, DBP-C4, 1-3, C-3
was used. Kanzo, which is a kind of grass, has long been
used as a Chinese medicine. Crude flocculant was obtained -, no flocculation observed with 2ml of cultures; +,
by adding cold ethanol to a final concentration of60% to 1 flocculation observed with 2ml of cultures; + +, floc-
liter of culture broth of R. erythropolis S-l after 10 days culation observed with 1 ml of cultures.
 2303
Screening for and Characteristics of Microbial Flocculants
3) Flocculation of various suspended solids in aqueous water (pH 9.5) was adjusted to pH 7.0 with HC1. Eighty
solution. Ninety-three milliliters of various suspended so- milliliters of the adjusted waste water was mixed with
lids in aqueous solution was mixed with 2ml of cultures 10ml ofCaCl2 solution and l ~ 10ml of culture broth of
(cells and culture broth) of R. erythropolis S-l and 5 ml of R. erythropolis S-l in a 100-ml graduated cylinder.
CaCl2 solution (1%) in a 100-ml graduated cylinder, and Flocculating activity after lOmin was estimated by
adjusted to pH 7.0. Controls using the medium without measuring the precipitated volume (%) and clarification of
cultures or without CaCl2 solution were examinedsimul- the supernatant with the optical density at 660nm. Total
taneously. Flocculating activity after 1 min and 3 min was organic carbon (TOC) of the supernatant was estimated
measured by the method mentioned above. with a Beckman TOC analyzer (model 915-B). Total
For the very viscous concentrated sludge, that activity nitrogen (TN) was measured by the Kjeldahal method.
after 20min and 60min was indicated as the clear super-
natant volume (%) and clarification of supernatant RESULTS
(OD.660). The concentrated sludge (8 w/v %) from the
waste water treatment plant was used. Screening for flocculant producing micro-
4) Application to the waste water from livestock in-
dustry. Urine and excrement waste water from pigs after organ isms
solid-liquid separation was used. This stockbreeding waste Cultures of 30 strains, which were isolated
as phthalate ester assimilating bacteria, were
Table II. Sedimentation of Bulking-sludge with tested for the ability to flocculate kaolin clay.
Flocculant (NOC-1) Produced The results are summarized in Table I.
by R. erythropolis Flocculation was found in Rhodococcus eryth-
ropolis (Nocardia erythropolis), Nocardia re-
SVI
stricta, Nocardia calcarea, Nocardia rhodnii,
Bulking-sludge* 290 and Corynebacterium sp. The microorganism
Bulking-sludge + NOC- 1 ** 50 which had the strongest flocculating activity
Bulking-sludge from kanzo wastewater treatment
amongthose tested was Rhodococcus eryth-
was used.
ropolis S-l and 260-2.
Flocculant (NOC-1) was obtained by adding 1 liter
of culture broth after 10 days of cultivation at 30°C Characteristics of theflocculant ofRhodococcus
with 60% cold EtOH, and whole crude flocculant ery th ropo lis
was inoculated into 100 ml of bulking-sludge. 1) Sedimentation of the hulking-sludge.

Table III. Effect of Cations on Flocculation of Kaolin Clay with Culture Broth (NOC-1)
OF R. erythropolis

Flocculating activity

Precipitate volume Clarification of supernatant

(% ) (O D 660)
1 min 3 min 1 min 3 min

K aolin 1.90 1.90

Kaolin + Culture medium 1.90 1.90
Kaolin + Culture medium + cation 1.90 1.90
Kaolin+ Culture broth (NOC-1) (+ ) 1.90 1.35
Kaolin + Mg2 1.90 1.90
Kaolin+ Mg2+ +NOC-1 10 1.80 1.20
Kaolin+ Fe2 + 1.90 1.90
Kaolin+Fe2+ +NOC-l 1.85 1.40
Kaolin + Ca2 1.90 1.90
Kaolin+Ca2+ +NOC-l 27 20 0.30 0.15
K aolin+ A 13 1.90 1.90
Kaolin+Al3+ +NOC-l 25 18 0 .0 5 0. 0 2

Reaction mixture: (100 ml graduated cylinder). Kaolin (5,000 ppm), 93 ml; Culture broth (NOC-1), 2 ml; Cation (1 °/c
solution), 5ml.
2304
R. Kurane, K. Takeda and T. Suzuki

Table IV. Flocculation of Various Suspended Solids in Aqueous Solution

with Culture of R. erythropolis S-l
For the detailed reaction condition, see Materials and Methods.
F loccu latin g activity

Su spended solids Pre cipitate volume Clarification of supernatant

(% ) (O D 660)
1 mi n 3 mi n 1 m in 3 min

M icroorganisms
E. coli
E. coli 1.00 1.00
E. coli+Ca2* 1.00 1.00
E. coli+Ca2+ +cultures 16 0. 4 5 0 .1 8
A lcoho l yeast (S accharom y ces sp .)
Y east 1.00. 1.00
Yeast + Ca2 1.00 1.00
Yeast + Ca2 + +cultures 14 0 .15 0. 05
Microcystis aeruginosa (AOKOV
A OKO 3.8 3.8
A O K O + C a24 3.8 3.8
AOKO +Ca2 + + cultures 75 38 0.42 0.17
Soil solid liquid (Muddy water) b
Muddy w ater >2.00 >2.00
Muddy water + Ca2 >2.00 >2.00
Muddy water + Ca2 + +cultures 15 20 0.85 0.10
River dredging muddy water (RDMW)C
RD M W 1.80 1.80
R D M W + C a2 1.80 1.80
RDMW + Ca2 + 4-cultures 20 18 0.10 0.06
River.botto m sediment (H EDORO)d
H ED O R O > 2 .0 0 >2.00
HEDORO +Ca2 >2.00 >2.00
HEDORO + Ca2 + +cultures 55 19 0.35 0.25
Coal ash from a steam-power station6
C o alash 1. 15 1. 15
C oal ash + C a2 1. 15 1. 15
Coal ash+Ca2+ +cultures 16 12 0.01 0.01
Activated charcoal powder'
C harcoal 7.4 7.4
Charcoal + Ca2 7.4 7.4
Charcoal+ Ca2 + +cultures 0.82 0.35

Microcystis aeruginosa from Kasumigaura Lake during summertime was used.

Soil solid liquid was prepared as followings; 500g of soil was added and stirred in 1 liter of distilled water, and
then upper phase after standing for 3min was used as soil solid liquid.
River dredging muddy water was estimated to contain 1.4w/v% of mud.
River bottom sediment "HEDORO" from the Yodo river was used.
Concentration of coal ash in distilled water was 1,000ppm.
Activated charcoal powder, which was finely ground in a mortar with a pestle, was suspended in distilled water
andused.
 2305
Screening for and Characteristics of Microbial Flocculants

Flocculation and sedimentation of bulking-
sludge by the crude flocculant from R. eryth-
ropolis S-l was examined. As shown in Table
II, the value of sludge volume index (SVI) was
rapidly decreased by the addition of the floe-
A B C flocculant from R. erythropolis had a wide
Fig. 1. Photograph after 3min of Standing of Kaolin
Clay Flocculated with R. erythropolis Flocculant.
A, with R. erythropolis S-l flocculant; B, with R. eryth-
ropolis 260-2 flocculant; C, control.
Concentration of kaolin clay was 5,000 ppm.
culant, while that of the uninoculated control
bulking-sludge was very high. That is to say,
the bulking of sludge was improved and the
sedimentability of the sludge was restored.
2) Effects of cationic ions on flocculation.
Table III shows the effects of the addition of
cations into the reaction mixture on the floc-
culation of kaolin clay with culture broth from
R. erythropolis S-l. The flocculation rate of
kaolin clay was significantly increased by the
addition ofCa2+ and Al3+. On the other hand,
the activity was not affected by Mg2+, and it
was slightly stimulated by Fe2+.
3) Flocculation of various suspended solids.
The rates of flocculation of various suspended
solids in aqueous solution with the cultures of
R. erythropolis S-l are summarized in Table
IV. The flocculant from R. erythropolis S-l
flocculated all materials tested, including
microorganisms such as E. coli and alcohol
yeast (Saccharomyces sp.), activated sludge,
Microcystis aeruginosa, a main constituent of
"AOKO" and causes serious problems es-
pecially in lakes and marshes during the sum-
mer, kaolin clay, muddywater, river dredging
muddy water, river bottom sediment called
"HEDORO"that causes serious environmen-
tal problems, ash from a steam-power station,
and charcoal. This results indicated that the
flocculating activity against both organic and
inorganic materials.
Cultures of R. erythropolis S-l were used on
highly viscous concentrated sludge (8 w/v%)
from the waste water treatment plant. The

Table V. Dehydration of Concentrated Sludge from Waste Water Treatment Plant

with Cultures (Cells and Broth) of R. erythropolis
Dehydrating activity

Clear supernatant volu me Clarification of supernatant

(% ) (O D 660)
20 m i n 60 m i n 20 min 60 min

C o ne, sludge 0.25

C o ne, slu d ge + C a2 0.30
Cone, sludge +Ca2+ +cultures 13 0.0 5 0.05

Reaction mixture: (100ml graduated cylinder). Concentrated sludge,* 95 ml; Ca solution (1%), 5 ml; cultures, 2ml.
* Concentration rate of sludge was 8w/v%.
2306
R. Kurane, K. Takeda and T. Suzuki

Table VI. Flocculation of Pig Urine and Excrement Waste Water with Culture Broth
of R. erythropolis S-l
Supernatant after lO min
Precipitate volum e
(% ) TOC TN C larif
ication
(ppm ) (m g/liter) (O D 660)

Waste water 0 1420 420 8.60

蝣
0 1420 420 8.60
Waste water + Ca2
Waste water+Ca2+ +culture broth*
1.Om l 3 1250 310 6.90
5.Om l ll 425 2 15 0 .02> **
10.Oml 13 504 213 0 .02> **

1.0ml of this culture broth contained about 0.1 mg crude flocculant.

This supernatant was colorless and clear.

viscous concentrated sludge was not sediment- The microorganisms that produced flocculant
ed by the cultures of R. erythropolis S-l in a were coryneform bacteria, genera of
few minutes, but it was sedimented after 20 Rhodococcus, Nocardia, and Corynebacterium,
and 60min. As shown in Table V, this shows amongthem tested. In our previous paper13)
that the cultures can aggregate highly viscous which dealt with the use of fluorescent anti-
solutions. body tracers, strain 260-2 of R. erythropolis
4) Application to the waste water from live- was only stained with anti-7?. erythropolis S-l
stock industry. The flocculant produced by R. fluorescent antibody aside from the ho-
erythropolis S-l was used for the primary mologus strain, and none of the other strains
treatment of the pig urine and excrement waste of R. erythropolis were stained. Therefore, it is
water. As shown in Table VI, pig urine and interesting that strains with stronger activity
excrements did sediment effectively with the than the other strains amongR. erythropolis
culture broth of R. erythropolis. This results tested were strain S-l and 260-2. Work on the
show the feasibility of the use of the flocculant components of the flocculant has not been
of R. erythropolis to waste water treatment for finished, and so the relationship between the
domestic stockbreeding. componentsof cell surface structure as immu-
nogens and those of the flocculant is un-
DISCUSSION certain.
In the screening, nutrient mediumappeared
Some studies 2 ~5) on flocculating substances more favorable than the complete defined me-
from microorganisms have been reported from dia such as phthalate medium. In the next
different viewpoints, such as coagulation of paper, we will report on the culture conditions
kaolin clay and removal of microorganisms in for producing the flocculant of R. erythropolis.
the fermentation industry. Among these re- The deflocculation of activated sludge was
ports, Nakamuraet al.3) reported serial studies efficiently prevented and restored by the ad-
on an Aspergillus sojae flocculant. Takagi et dition of R. erythropolis into the activated
al.5) reported that Paecilomyces flocculant had sludge, as reported in our previous paper.6)
a unique ability to flocculate almost all sus- In the microbial population of activated
pended solids in aqueous solution. The above sludge,11} the ratio of R. erythropolis cells to
mentioned microorganisms that produce the total cells in the corn steep liquor-fed acti-
strong flocculant seem all to be fungi. In this vated sludge was normally about 2 or 3%.
paper, we screened for bacteria producing Greater numbers of R. erythropolis cells were
flocculants, because of their easier cultivation. counted in the activated sludge, which had
 2307
Screening for and Characteristics of Microbial Flocculants
high sedimentability, than those in sludge waste water of domestic stockbreeding is said
in bad conditions such as deflocked sludge. to be one of the most difficult waste waters for
The widespread distribution of R. erythro- treatment, because of its high BODand deep
polis in soils as a major gram positive bacte- coloration. Manyprocesses and systems were
rium10* and activated sludge11} means that used on the waste water from the livestock
humans have been in contract with this micro- industry. Some synthetic high polymers have
organism for a long period in the open en- been used as flocculants to treat that waste
vironment. Although its safety is not certified water. However, synthetic flocculants are, gen-
at the same level as Bacillus natto, it might erally speaking, unfavorable for recycling to
be this flocculant from R. erythropolis has an farm-land. Therefore, livestock waste water
advantage in its application to open systems precipitated with the flocculant of R. eryth-
such as waste water treatment. The wide- ropolis may be used as an organic natural
spread distribution in natural circum- manure on farm-land.
stances10'1^ and non-pathogenicity7) of R. The supernatant after treatment with the
erythropolis mayalso suggest that the floccu- flocculant of R. erythropolis became colorless
lant from R. erythropolis is very useful in and clear, as shown in Table VI. This suggests
downstreamprocessing such as a separation that the flocculant of R. erythropolis is also
of cells in the fermentation industry. able to remove coloring material from an
The situation of floc-formation by the floc- aqueous solution in addition to the floccu-
culant of R. erythropolis depended on the lation of suspended solids.
suspended solids tested. For example, kaolin REFERENCES
clay was flocculated immediately after the
addition of the flocculant, and sedimented in 1) J. M. Cohen,G. A. Rourkeand R. L. Woodward, /.
an aggregate. On the other hand, the floe ofE. Am. Water Works Assoc, 50, 463 (1958).
2) J. E. Zajic and E. Knettig, "Developments in
coli by the flocculant was gradually formed Industrial Microbiology," American Institute of
and sedimented in drops. Biological Science, Washington D. C, 1971, pp.
This Rhodococcus flocculant had interest- 87-98.

ing and unusual aspects. That is to say, the 3) J. Nakamura, S. Miyashiro and Y. Hirose, Agric.
Biol. Chem., 40, 377 (1976).
results of the experiments described here dem- 4) S. Shinohara, Japanese Patent (Kokai Koho) S54-
onstrate that the flocculant produced by R.
55798 (1979) (in Japanese).
erythropolis could efficiently flocculate all sus- 5) H. Takagi and K. Kadowaki, Agric. Biol. Chem., 49,
pended solids in aqueous solution tested and 3151 (1985).

had a wide flocculating activity against both 6) R. Kurane, T. Suzuki and Y. Takahara, Agric. Biol.
organic and inorganic materials. Takagi et 7) Chem.,R. Kurane, 43, 421 (1979).
T. Suzuki, Y. Takahara and K.
al.5M) reported that the Paecilomyces floc- Komagata, Agric. Biol. Chem., 41, 1031 (1977).
culant had also the ability to flocculate all 8) I. Komura, K. Komagata and K. Mitsugi, /. Gen.
suspended solids from organic materials such Appl. Microbiol., 19, 161 (1973).
as microorganisms to inorganic materials such 9)Microbiol.,
H. Iizuka and K. Komagata, /. Geri. Appl.
10, 207 (1964).
as aluminium oxide and that the Paecilomyces 10) K. Katoh and T. Suzuki, Nogihen-Hokohu (B)
flocculant was a polysaccharide composed of (Bulletin National Inst. Agric. Science, (B)), 30,
galactosamine. Therefore, the unusual charac- 73 (1979). (in Japanese)
teristics of the Rhodococcus flocculant arouse ll) R. Kurane, T. Suzuki and Y. Takahara, Agric. Biol.
our interests in its chemical structure. Chem., 43, 907 (1979).
Artificial fertilizer has contributed to make 12) R. Kurane, T. Suzuki and Y. Takahara, Agric. Biol.
Chem., 41, 2119 (1977).
abundant harvests in agricultural products in 13) R. Kurane, T. Suzuki, Y. Takahara, N. Kurita and
recent years. On the other hand, the farm-land M. Miyaji, Agric. Biol. Chem., 43, 2093 (1979).
became sterile and lost softness by its excess 14) H. Takagi and K. Kadowaki, Agric. Biol. Chem., 49,
3159 (1985).
use. Organic manure is now reconsidered. The