You are on page 1of 9

African Journal of Microbiology Research Vol. 5(9), pp.

1037-1045, 4 May, 2011


Available online http://www.academicjournals.org/ajmr
DOI: 10.5897/AJMR11.008
ISSN 1996-0808 2011 Academic Journals




Full Length Research Paper

Evaluation of different pH and temperatures for
bacterial cellulose production in HS (Hestrin-Scharmm)
medium and beet molasses medium

Esin Poyrazolu oban* and Halil Biyik

Biology Department, Faculty of Science and Letters, Adnan Menderes University, 09010, Aydin, Turkey.

Accepted 7 April, 2011

Acetobacter pasteurianus HBB6 and Acetobacter lovaniensis HBB5 strains used in this study was
isolated from vinegar in Turkey. Bacterial cellulose was produced from HS (Hestrin-Scharmm) medium
and beet molasses using A. pasteurianus HBB6 and A. lovaniensis HBB5. In this study, the effects of
different pH (2.5, 3.5, 4.5, 6.5, 7.5 and 8.5) and temperatures (4, 22, 30 and 37C) were examined. The
highest yield was obtained at pH 6.5 (0.040/0.035 g/L in HS medium, 0.029/0.021 g/L in beet molasses)
and the lowest yield was obtained at pH 3.5 (0.007/0.006 g/L) and 4.5 (0.017/0.013 g/L) in HS medium or
beet molasses, respectively. While the optimum temperature was obtained at 30C, the lowest yield
was obtained at 22C in HS medium and beet molasses medium. Besides, cell growth and bacterial
cellulose yield were not observed in pH 2.5 or temperature of 4C in HS medium and beet molasses.

Key words: Bacterial cellulose, pH, temperature, (Hestrin-Scharmm) medium, beet molasses


INTRODUCTION

Bacterial cellulose is an exopolysaccharide produced by
various species of bacteria, such as those of the genera
Gluconacetobacter (formerly Acetobacter),
Agrobacterium, Aerobacter, Achromobacter, Azotobacter,
Rhizobium, Sarcina, and Salmonella (Shoda and
Sugano, 2005). Production of cellulose from Acetobacter
xylinum was first reported in 1886 by Brown (Brown,
1886).
Medium costs limit commercial use of bacterial
cellulose, so low-cost substrates are being used, such as
molasses from the sugar cane process with a content of
55 to 60% sucrose, or corn step liquor (Yoshinaga et al.,
1997; Raspor and Goranovic, 2008). These are
preferable to glucose because it is converted to gluconic
acid with a consequent rapid fall in pH (Sutherland,
1996). Food process effluents such as potato effluents,
cheese whey permeate and sugar beet raffinate have
also been studied as substrates for bacterial cellulose
production (Thompson and Hamilton, 2001).



*Corresponding author. E-mail: epoyrazoglu@adu.edu.tr. Tel:
+90 256 2182000. Fax: +90 256 2135379.
Bacterial (BC) cellulose is relatively expensive to
produce, and as such is unlikely to replace traditional
sources of cellulose. One way to reduce the cost of
producing bacterial cellulose is to use cheap and readily
available substrates. Molasses, as runoff syrup, is a by-
product of the final crystallization stage in the process of
sugar production. Because molasses is one of the most
economical carbon sources, it is widely used as a
substrate in microbial fermentation, e.g., in production of
lactic acid (Kotzamanidis et al., 2002),
polyhydroxybutyrate (Beaulieu et al., 1995), ethanol
(Sheoran et al., 1998). Besides, BC production using
molasses as a carbon source has been reported (Keshk
and Sameshima, 2006; Bae and Shoda, 2005).
The main responsible culture conditions for the
production of BC are cultivation method, carbon source,
nitrogen source, Ph and temperature (Jung et al., 2003;
Chawla et al., 2009). The objective of this work was to
produce low cost environmentally friendly cellulose
utilizing beet molasses as a sole cheap carbon source in
Hestrin-Scharmm (HS) medium. Therefore, we tested
different pH and temperatures for bacterial cellulose
production using Acetobacter pasteurianus HBB6 and
Acetobacter lovaniensis HBB5 in beet molasses or HS
1038 Afr. J. Microbiol. Res.



medium.


MATERIALS AND METHODS

Microorganisms

A. pasteurianus HBB6 and A. lovaniensis HBB5 strains used in this
study was isolated from vinegar in Turkey.


Media and culture conditions

The standard medium used in this study comprised the following:
2.0% (w}v) glucose, 0.5% (w}v) yeast extract, 0.5% (w}v)
polypeptone, 0.675% (w}v) Na2HPO4.12H2O and 0.115% (w}v) citric
acid monohydrate in distilled water (pH 6.0). For the preculture,
stock culture was inoculated into 50 ml of the basal medium in a
250 ml Erlenmeyer flask and culture was allowed to proceed at
30C for 48 h under static conditions. A static culture for cellulose
production was carried out using a 250 ml conical flask. The culture
was started by inoculating 5% of the culture supernant of a
preculture. For static-flask fermentation, cultivations were
performed at 30C for 7 days under static conditions (Son et al.,
2001).


Cellulose yield

After cultivation, the culture medium was separated into the
supernatant and the cellulose floccules by centrifugation at 3000 x
g (Heraeus Sepatech Labofuge 200, Germany) for 15 min at room
temperature. The cellulose floccules were washed with distilled
water to remove medium components and treated with 4% (w/v)
sodium hydroxide solution at 80C for 1 h to eliminate bacterial
cells. The bacterial cellulose was rinsed extensively with 6% acetic
acid and then distilled water until the pH of water became neutral
(Ishihara et al., 2002). The purified bacterial cellulose was dried to
constant weight at 105C and then weighed (Son et al., 2001).


Analytical methods

In this study, the bacterial cellulose concentration in the culture
broth was calculated from the dry weight of purified cellulose. This
was determined by the phenol/sulfuric acid method for total sugar
using glucose as standard and absorption measurements at 470
nm (Dubois et al., 1956; Kim et al., 1994). Output coefficient of
specific product is calculated by the formula, according to Gerhardt
and Drew (1994).


RESULTS

A. pasteurianus HBB6 and A. lovaniensis HBB5 cells
were incubated in 250 ml conical flasks with broth
volumes of 100 ml, and the production of cellulose at
different pH and temperatures was investigated. The cells
grown in 75 ml of medium produced the highest level of
cellulose as measured at 7 days. Therefore 75 ml of
medium was used in the following studies. The effects of
various pH (2.5, 3.5, 4.5, 6.5, 7.5 and 8.5) and various
temperatures (4, 22, 30 and 37C) were examined using
HS medium and beet molasses.
At different pH, roducellulose pcing output of




A. pasteurianus HBB6 and A. lovaniensis HBB5 strains in
HS medium and beet molasses medium are shown in
Tables 1 and 2.
The bacterial cellulose yields in HS broth for A.
pasteurianus HBB6 was in the range 0.007 to 0.040 g/L
and for A. lovaniensis HBB5 was 0.006 to 0.035 g/L,
respectively. The bacterial cellulose yields in beet
molasses for A. pasteurianus HBB6 was in the range of
0.006 to 0.029 g/L and for A. lovaniensis HBB5 0.008 to
0.021 g/L, respectively. Cell growth and bacterial
cellulose yield only were not observed in pH 2.5 in HS
broth (Figures 1a and b). However, cell growth and
bacterial cellulose yield were not observed in pH 2.5 and
3.5 in beet molasses (Figures 2a and b). According to the
results of this assay, the best pH range was 6.5.
At different temperatures, cellulose producing output of
A. pasteurianus HBB6 and A. lovaniensis HBB5 strains in
HS medium and beet molasses medium are shown in
Tables 3 and 4.
The bacterial cellulose yields in HS broth for A.
pasteurianus HBB6 was in the range 0.016 to 0.043 g/L
and for A. lovaniensis HBB5 was 0.011 to 0.038 g/L,
respectively. The bacterial cellulose yields in beet
molasses for A. pasteurianus HBB6 was in the range of
0.015 to 0.030 and 0.009 to 0.023 g/L for A. lovaniensis
HBB5, respectively. Cell growth and bacterial cellulose
yield were not observed at 4C in HS broth (Figures 3a
and b) and beet molasses (Figures 4a and b). According
to the results of this assay, the best temperature range is
30C.


DISCUSSION

Bacterial cellulose is synthesized in the form of film on
the surface of the HS medium solution by Acetobacter sp.
The yield of the biosynthesis process depends on many
factors, that is, pH, temperature, time, and the relation of
the surface area to the volume of substrate (Concalves
and aszkiewicz, 1999). The comparison between the
results of different cultural conditions used by different
researchers is shown in Table 5. We examined effect of
these factors for bacterial cellulose produced of A.
pasteurianus HBB6 and A. lovaniensis HBB5.
During the cultivation, the appearance of the two acids,
gluconic acid and 5-keto-gluconic acid are responsible for
the decrease of the pH value of the culture medium
during the first cultivation days. Monosaccharides are
also converted by membrane-bound Acetobacter
dehydrogenase into (keto) gluconic acids. The
conversion of glucose to (keto) gluconic acids is not
beneficial for overall cellulose productivity. The sharp
decrease in the medium-pH (final pH value of 3.5)
probably not only cellulose formation, but also lowers the
medium pH to suboptimal levels for cell viability and
cellulose synthesis (Klemm et al., 2001). Acetobacter
xylinum had been shown to be distinctly acid tolerable
capable of growing at pH as low as 3.5 (Lapuz et al.,
Coban and Biyik 1039



Table 1. Effect of different pH on dry weight of cells or cellulose production by A. pasteurianus HBB6 and A. lovaniensis HBB5 in
HS medium.

pH
Dry weight of cell
(g/L)
Polysaccharide quantify
according to standard curve
at 470 nm (g /L)
Dry weight of
raw cellulose
(g/L)
Y(p/x)
% Cellulose
producing
output
A. pasteurianus HBB6
2.5 - - - - -
3.5 0.22 6650 0.007 0.032 3.2
4.5 0.25 1251 0.025 0.10 10.0
6.5 0.26 1985 0.040 0.154 15.4
7.5 0.18 7130 0.011 0.061 6.1
8.5 0.18 6870 0.009 0.05 5.0

A. lovaniensis HBB5
2.5 - - - - -
3.5 0.15 6430 0.006 0.04 4.0
4.5 0.28 1248 0.023 0.082 8.2
6.5 0.31 1622 0.035 0.113 11.3
7.5 0.20 9230 0.014 0.07 7.0
8.5 0.17 6650 0.007 0.041 4.1

Cells were cultivated in a flask-static at 30C for 7 days. Y(p/x): Output coefficient of specific product. p: Dry weight of bacterial cellulose
(g/L,) x: Dry weight of cell (g/L).



Table 2. Effect of different pH on dry weight of cells or cellulose production by A. pasteurianus HBB6 and A. lovaniensis HBB5 in
beet molasses.

pH
Dry weight of
cell (g/L)
Polysaccharide quantify
according to standard curve
at 470 nm (g/L)
Dry weight of raw
cellulose (g/L)
Y(p/x)
% Cellulose
producing
output
A. pasteurianus HBB6
2.5 - - - - -
3.5 - - - - -
4.5 0.28 1180 0.017 0.061 6.1
6.5 0.36 1264 0.029 0.081 8.1
7.5 0.25 7050 0.010 0.040 40
8.5 0.20 5960 0.006 0.030 3.0

A. lovaniensis HBB5
2.5 - - - - -
3.5 - - - - -
4.5 0.32 9110 0.013 0.041 4.1
6.5 0.33 1208 0.021 0.064 6.4
7.5 0.30 7060 0.010 0.033 3.3
8.5 0.26 6510 0.008 0.031 3.1

Cells were cultivated in a flask-static at 30C for 7 days. Y(p/x): Output coefficient of specific product; p: Dry weight of bacterial
cellulose (g/L); x: Dry weight of cell (g/L).



1967) and Verschuren et al. (2000) reported pH 4.0 and
5.0 to be ideal for the development of cellulose.
Therefore, it is important to control the pH within the
optimal range.
Studies indicate that the optimal pH range for cellulose
production by A. xylinum is 4 to 6 (Fontana et al., 1990)
while Galas et al. (1999), demonstrated pH 4 to 7 as
optimum. Most researchers used pH 5 (Ishikawa et al.,
1995; Fiedler et al., 1989; Joris et al., 1990) or 6 (Hestrin
and Schramm, 1954; Masaoka et al., 1993). Jonas and
1040 Afr. J. Microbiol. Res.





Figure 1a. Effect of different pH on dry weight of cells or cellulose production by A.
pasteurianus HBB6 in HS medium. Cells were cultivated in a flask-static at 30C for 7 days.





Figure 1b. Effect of different pH on dry weight of cells or cellulose production by A.
lovaniensis HBB5 in HS medium. Cells were cultivated in a flask-static at 30C for 7 days.





Figure 2a. Effect of different pH on dry weight of cells or cellulose production by A. pasteurianus
HBB6 in beet molasses. Cells were cultivated in a flask-static at 30C for 7 days.
Coban and Biyik 1041





Figure 2b. Effect of different pH on dry weight of cells or cellulose production by A.
lovaniensis HBB5 in beet molasses. Cells were cultivated in a flask-static at 30C for 7 days.



Table 3. Effect of different temperature on dry weight of cells or cellulose production by A. pasteurianus HBB6 and A.
lovaniensis HBB5 in HS medium.

Temperature
(C)
Dry weight
of cell (g/L)
Polysaccharide quantify
according to standard
curve at 470 nm (g/L)
Dry weight of raw
cellulose
(g/L)
Y(p/x)
% Cellulose
producing
output
A. pasteurianus HBB6
4 - - - -
22 0.22 9850 0.016 0.073 7.3
30 0.26 2169 0.043 0.165 16.5
37 0.23 1258 0.028 0.122 12.2

A. lovaniensis HBB5
4 - - - -
22 0.19 7130 0.011 0.058 5.8
30 0.31 1642 0.038 0.123 12.3
37 0.26 1226 0.021 0.081 8.1

Cells were cultivated in a flask-static at 30C for 7 days. Y(p/x): Output coefficient of specific product; p: Dry weight of bacterial
cellulose (g/L); x: Dry weight of cell (g/L).



Table 4. Effect of different temperature on dry weight of cells or cellulose production by A. pasteurianus HBB6 and A.
lovaniensis HBB5 in beet molasses.

Temperature
(C)
Dry weight
of cell (g/L)
Polysaccharide quantify
according to standard
curve at 470 nm (g/L)
Dry weight of raw
cellulose (g/L)
Y(p/x)
% Cellulose
producing output
A. pasteurianus HBB6
4 - - - -
22 0.28 9540 0.015 0.053 5.3
30 0.33 1265 0.030 0.091 9.1
37 0.31 1234 0.022 0.071 7.1

A. lovaniensis HBB5
4 - - - -
22 0.25 6850 0.009 0.036 3.6
30 0.32 1247 0.023 0.072 7.2
37 0.28 9230 0.014 0.050 5.0

Cells were cultivated in a flask-static at 30C for 7 days. Y(p/x): Output coefficient of specific product; p: Dry weight of bacterial
cellulose (g/L); x: Dry weight of cell (g/L).
1042 Afr. J. Microbiol. Res.





Figure 3a. Effect of different temperature on dry weight of cells or cellulose
production by A. pasteurianus HBB6 in HS medium. Cells were cultivated in a flask-
static at 30C for 7 days.





Figure 3b. Effect of different temperature on dry weight of cells or cellulose production by A.
lovaniensis HBB5 in HS medium. Cells were cultivated in a flask-static at 30C for 7 days.





Figure 4a. Effect of different temperature on dry weight of cells or cellulose production by A.
pasteurianus HBB6 in beet molasses. Cells were cultivated in a flask-static at 30C for 7 days.
Coban and Biyik 1043





Figure 4b. Effect of different temperature on dry weight of cells or cellulose
production by A. lovaniensis HBB5 in beet molasses. Cells were cultivated in a flask-
static at 30C for 7 days.



Table 5. The comparison between the results of different cultural conditions used by different researchers (Pourramezan et al.,
2009).

Volume (L) Yield (g/L) System Temp. (C) pH Time (h) References
- 9.7 Shaking culture - - 7 Tsuchida and Yoshinaga (1997)
30 20 Shaking culture 30 5 42 Kouda et al. (1998)
2 15 Shaking culture 30 5.5 50 Hwang et al. (1999)
Tubes 3 Static culture 30 5.6-7.5 4 (weeks) Ishihara et al. (2002)
0.075 16.4 Shaking culture 30 5.6 192 Son et al. (2003)
0.61 21 Shaking culture 30 5 50 Naritomi et al. (2002)
0.1 12.8 Shaking culture 30 5 72 Bae et al. (2004)
0.03 - Static culture 28 6 168 Keshk and Sameshima (2005)
0.03 11.98 Static culture 30 7 192 Pourramezan et al. (2009)



Farah (1998) suggested that, for the industrial production
of Biofill and Gengiflex, pH 4 and 4.5 gave better results,
especially to avoid contaminations.
According to Wlochowicz (2001), the highest water
binding capacity of cellulose is determined in Hestrin-
Schramm (S-H) medium at pH 4.8 to 6.0
According to Son et al. (2001), a high level of cellulose
production was observed over a broad pH range of
between 4.5 and 7.5, and was maximum at pH 6.5. It is
generally accepted that the optimal pH range for cellulose
production by A. xylinum is 4.0 to 7.0 (Delmer and Amor,
1995). Pourramezan et al. (2009) studied the effects of
various pH (4 to 8) were examined using HS medium.
The highest yield was obtained at pH 7 and the lowest
yield was obtained at pH 4.
In addition to the pH of the nutrient broth, the yield of
bacterial cellulose is also temperature dependent.
Studies revealed that the optimal growth temperature for
cellulose production is 25 to 30C (Cannon and
Anderson, 1991). Most authors used the temperature
range from 28 to 30C (Romano et al., 1989; Geyer et al.,
1994). The variation in temperature caused changes of
cellulose degree of polymerisation and water-binding
capacity, whereas, the bacterial cellulose synthesized at
30C had a lower DP (~10,000) and a higher water-
binding capacity (164%) in comparison to that produced
at 25 and 35C (Schmauder et al., 1992). Jonas and
Farah (1998) stated that the optimal growth temperature
for cellulose production is 25 to 30C, although most
researchers used 28 to 30C. With 24C, Jonas and
Farah observed 50% better cellulose yields during 72 h
cultivation; but for industrial purpose, it did not produce
the desired results (Jonas and Farah, 1998). Son et al.
(2001) examined the effects of various temperatures (20
to 40C) using HS medium. The optimum temperature for
cellulose production was observed at 30C. There was no
significant difference in cellulose production at 25C.
However, cellulose production decreased above 35C
(Son et al., 2001). According to Pourramezan et al.
(2009), the optimum temperature was obtained at 30C
1044 Afr. J. Microbiol. Res.



and the lowest yield was obtained at 45C.
Ishihara et al. (2002) found that 3 g/L bacterial cellulose
was produced at 30C and pH 5.6 to 7.5 during 4 weeks
in static culture, while in this research, 0.26 to 0.31 g/L
bacterial cellulose was produced at 30C and pH 6 during
1 week in static culture. Keshk and Sameshima (2005)
showed that 0.03 g/L bacterial cellulose was produced at
28C and pH 6 during 168 h in static culture, while in this
research, 0.18 to 0.20 g/L bacterial cellulose was
produced at 30C and pH 7.5 during 1 week in static
culture. Another research found that 11.98 g/L bacterial
cellulose was produced at 30C and pH 7 during 192 h in
static culture, while in this research, 0.17 to 0.37 g/L
bacterial cellulose was produced at 37C and pH 8.5
during 1 week in static culture (Pourramezan, 2009).
From Tables 1 to 3 and Figures 1 to 4, it is shown that
bacterial cellulose produced was affected by different pH,
temperature, carbon source and incubation time.
According to these factors, we observed better cellulose
yield with pH 6.5 at 30C in HS medium and beet
molasses. The results obtained from this report should
help design a better strategy for the production of
bacterial cellulose.


ACKNOWLEDGMENTS

This work was supported by the Adnan Menderes
University, Research Foundation (ADU- FEF 04002 and
TUBITAK 106T448). We are grateful to Dr. Kubilay Metin
for helping us on analytical methods.


REFERENCES

Bae S, Shoda M (2005). Statistical optimization of culture conditions for
bacterial cellulose production using box-behnken design. Biotechnol.
Bioeng., 90: 20-28.
Bae S, Sugano Y, Shoda M (2004). Improvement of bacterial cellulose
production by addition of agar in a jar fermentor. J. Biosci. Bioeng.,
97: 33-38.
Beaulieu M, Beaulieu Y, Mlinard J, Pandian S, Goulet J (1995).
Influence of ammonium salts and cane molasses on growth of
Alcaligenes eutrophus and production of polyhydroxybutyrate. Appl.
Env. Microbiol., 61: 165-169.
Brown AJ (1886). On an acetic ferment which forms cellulose. J. Chem.
Society. Faraday. Trans., 49: 432-439.
Cannon RE, Anderson SM (1991). Biogenesis of bacterial cellulose.
Crit. Rev. Microbiol., 17: 435-447.
Chawla PR, Ishwar BB, Survase SA, Singhal RS (2009). Fermentative
production of microbial cellulose. Food Technol. Biotechnol., 47: 107-
124.
Concalves M, aszkiewicz B (1999). Celu1loza bakteryjna biosynteza,
waciwoci i zastosowanie. Przegl Papiern 55: 657-661, in polish.
Delmer DP, Amor Y (1995). Cellulose biosynthesis. Plant Cell, 7: 987-
1000.
Dubois M, Gilles AK, Hamilton JK, Rebers PA, Smith F (1956).
Colorimetric method for determination of sugars and related
substances. Anal. Chem., 28: 350-356.
Fiedler S, Fssel M, Sattler K (1989). Production and application of
bacterial cellulose: I. A survey on state of research and investigations
concerning fermentation kinetics. Zentralblatt. Fur. Mikrobiol., 144:
473-484.




Fontana JD, de Sousa AM, Fontana CK, Torriani IL, Moreschi JC,
Gallotti BJ, de Sousa SJ, Narcisco GP, Bichara JA, Farah LF (1990).
Acetobacter cellulose pellicle as a temporary skin substitute. Appl.
Biochem. Biotechnol., 25: 253-264.
Galas E, Krystynowicz A, Tarabasz-Szymanska L, Pankiewictz R,
Zyska M (1999). Optimization of the production of bacterial cellulose
using multivariable linear regression analysis. Acta Biotechnol., 19:
251-260.
Gerhardt P, Drew SW (1994). Growth yield calculations, pp. 244-246.
Methods for general and molecular bacteriology, Gerhardt P., et al.
(eds)., American Society for Microbiol., Washington, D.C., p. 584.
Geyer U, Heinze T, Stein A, Klemm D, Marsch S, Schumann D,
Schmauder HP (1994). Formation, derivatization, and applications of
bacterial cellulose. Int. J. Biol. Macromol., 16: 343-347.
Hestrin S, Schramm M (1954). Synthesis of cellulose by Acetobacter
xylinum: II. Preparation of freeze-dried cells capable of polymerizing
glucose to cellulose. Biochem. J., 58: 345-352.
Hwang JW, Yang YK, Hwang JK, Pyun YR, Kim YS (1999). Effects of
pH and dissolved oxygen on cellulose production by Acetobacter
xylinum BRC5 in agitated culture. J. Biosci. Bioeng., 88: 183-188.
Ishihara M, Matsunaga M, Hayashi N, Tisler V (2002). Utilization of D-
xylose as carbon source for production of bacterial cellulose. Enzyme
Microb. Technol., 31: 986-991.
Ishikawa A, Matsuoka M, Tsuchida T, Yoshinaga F (1995). Increasing
of bacterial cellulose production by sulfoguanidine-resistant mutants
derived from Acetobacter xylinum subsp. sucrofermentans BPR2001.
Biosci. Biotechnol. Biochem., 59: 2259-2263.
Jonas R, Farah LH (1998). Production and application of microbial
cellulose. Poly. Deg. Stab., 59:101-106.
Joris K, Billiet F, Drieghe S, Brachx D, Vandamme E (1990). Microbial
production of -1,4 glucan. Meded. Fac. Landbouwwet Rijksuniv
Gent., 55: 1563-1566.
Jung JY, Park YH, Park JK (2003). Effect of medium composition on the
bacterial cellulose production by Gluconacetobacter hansenii PJK.
Korean J. Biotechnol. Bioeng., 18: 94-99.
Keshk MASS, Sameshima K (2005). Evaluation of different carbon
sources for bacterial cellulose production. Afr. J. Biotechnol., 4: 478-
482.
Keshk S, Sameshima K (2006). Influence of lignosulfonate on crystal
structure and productivity of bacterial cellulose in a static culture.
Enzyme Microb. Technol., 40: 48.
Kim SK, Kong IS, Kwon KJ, Rha C, Sinskey AJ, Kong JY (1994).
Exopolysaccharides produced by Z.ramigera mutants and analysis of
structural change by solutions properties. Biotechnol. Lett., 16: 789-
794.
Klemm D, Schumann U, Udhardt U, Marsch S (2001). Bacterial
synthesized cellulose - artificial blood vessels for microsurgery. Prog.
Polym. Sci., 26: 1561-1599.
Kotzamanidis CH, Roukas T, Skaracis G (2002). Optimization of lactic
acid production from beet molasses by Lactobacillus delbrueckii
NCIMB 8130. World J. Microbiol. Biotechnol., 18: 441-448.
Kouda T, Naritomi T, Yano H, Yoshinaga F (1998). Inhibitory effect of
carbon dioxide on bacterial cellulose production by Acetobacter in
agitated culture. J. Ferment. Bioeng., 85: 318-321.
Lapuz MM, Gallardo EG, Palo MA (1967). The nata organism cultural
requirements, characteristics and identity. Philippine J. Sci., 96: 91-
108.
Masaoka S, Ohe T, Sakota N (1993). Production of cellulose from
glucose by Acetobacter xylinum. J. Ferment. Bioeng., 75: 18-22.
Naritomi T, Kouda T, Yano H, Yoshinaga F (2002). Influence of broth
exchange ratio on bacterial cellulose production by repeated-batch
culture. Process Biochem., 38: 41-47.
Pourramezan GZ, Roayaei AM, Qezelbash QR (2009). Optimization of
culture conditions for bacterial cellulose production by Acetobacter
sp. 4B-2. Biotechnol., 8: 150-154.
Raspor P, Goranovic D (2008). Biotechnological applications of acetic
acid bacteria. Critic. Rev. Biotechnol., 28: 101-124.
Romano M, Franzosi G, Seves A, Sora S (1989). Study of the
production of cellulose gel and cellulose by Acetobacter xylinum.
Cellulose Chem. Technol., 23: 217-223.
Schmauder HP, Einfeldt L, Geyer U, Klemm D (1992). Biosynthesis of
cellulose by Acetobacter xylinum using glucose and modified carbon-




sources. Med. Fac. Landbouww Univ. Gent., 57/4a: 1797-1799.
Sheoran A, Yadav BS, Nigam P, Singh D (1998). Continuous ethanol
production from sugarcane molasses using a column reactor of
immobilized Saccharomyces cerevisiae HAU-1. J. Basic Microbiol.,
38: 123-128.
Shoda M, Sugano Y (2005). Recent advances in bacterial cellulose
production. Biotechnol. Bioprocess. Eng., 10: 1-8.
Son HJ, Heo MS, Kim YG, Lee SJ (2001). Optimization of fermentation
conditions for the production production of bacterial cellulose by a
newly isolated Acetobacter sp. A9 in shaking cultures. Biotechnol.
Appl. Biochem., 33: 1-5.
Son HJ, Kim HG, Kim KK, Kim HS, Kim YG (2003). Increased
production of bacterial cellulose by Acetobacter sp. V6 in synthetic
media under shaking culture conditions. Bioresour. Technol., 86: 215-
219.
Sutherland IW (1996). Extracellular polysaccharides. In: Biotechnol: A
multi-volume comprehensive treatise, 2nd ed., Rehm HJ, Reed G
eds. VCH, Weinheim, Germany, pp. 613-657.






















































Coban and Biyik 1045



Thompson DN, Hamilton MA (2001). Production of bacterial cellulose
from alternate feedstocks. Appl. Biochem. Biotechnol., 91: 503-513.
Tsuchida T, Yoshinaga F (1997). Production of bacterial cellulose by
agitation culture systems. Pure Appl. Chem., 69: 2453-2458.
Verschuren PG, Carodona TD, Nout MJR, de Gooijer KD, van den
Heuvel JC (2000). Location and limitation of cellulose production by
Acetobacter xylinum established from oxygen profiles. J. Biosci.
Bioeng., 89: 414-419.
Wlochowicz A (2001). Personal communication, pp. ????.
Yoshinaga F, Tonouchi N, Watanabe K (1997). Research progress in
production of bacterial cellulose by aeration and agitation culture and
its application as a new industrial material. Biosci. Biotechnol.
Biochem., 61: 219-224.