Food Hydrocolloids Vol.8 no.

5 ppA07-4l8, 1994

Bacterial cellulose. I. Factors affecting the production of cellulose

by Acetobacter xylinum

Milda E.Embuscado, Jay S.Marks and James N.BeMiller

Department of Food Science, Purdue University, West Lafayette, IN 47907-1160,
USA

Abstract. Cellulose production by a strain of Acetobacter xylinum (the nata organism) was affected
by the type and concentration of sugar, nitrogen source and pH. Fructose, fructose + sucrose,
fructose + lactose and sucrose gave the highest yields (5.25-7.38 gil). Glucose gave low yields when
used alone or in combination with other sugars. The pH of all glucose-containing media decreased
from pH 5.0 to ~3.0. When fructose alone or in combination with sucrose was used at 1,5 or 15%
concentrations,S and 15% total sugar concentrations gave the highest, and similar yields. Organic
sources of nitrogen were the most favorable for cellulose biosynthesis. Among the organic sources
tested, yeast extract in combination with peptone gave the best product yield. Higher yields of
cellulose were obtained at pH 4.5 than at 5.5 and its production was completely inhibited at pH 3.5.

Introduction
Cellulose and cellulose derivatives are versatile multi-functional food
ingredients. Wood pulp and cotton linters are the raw materials for the
production of microcrystalline cellulose and cellulose derivatives. The cellulose
produced by Acetobacter xylinum is an alternative source. Unlike the cellulose
from wood pulp, bacterial cellulose is devoid of other contaminating poly-
saccharides (1), and its isolation and purification are relatively simple, not
requiring energy- or chemical-intensive processes. Further, environmental
problems due to by-products of wood pulping given an added impetus to study
unexplored sources of cellulose.
Acetobacter xylinum has been known to produce pure cellulose for more than
100 years. Most investigations with this microorganism have dealt primarily with
the elucidation of cellulose biosynthesis, but most of the strains studied so far
produce only a skin or pellicle under non-agitated conditions. An excellent
review on the biogenesis of bacterial cellulose is that by Cannon and Anderson
(2).
One strain of A.xylinum is known to produce cellulose 'gels' 5-7 em thick or
nata (3,4), but it has never been used for bacterial cellulose production. It could
appropriately be classed as a cellulose overproducer (2).
A.xylinum has been isolated from rotting sugary fruits, vegetables and
fermenting coconut water (5). Thirty-three nata-forming microorganisms have
been found. They produce nata with different textures, ranging from soft to
tough when cooked in syrup (5).
Recent bacterial cellulose studies have focused on the optimization of the
cellulose 'gel' or nata production (6), the processing of nata for food use (7), and
its applications (7-9). These researchers used A.aceti while the present study
employed A.xylinum, the nata organism, which was isolated directly from a nata
fermenting medium.
407
M.E.Embuscado, J.S.Marks and J.N.BeMilIer

Nata is a favorite dessert delicacy native to the Philippines and produced

mainly in the coconut regions of Laguna and Quezon provinces. The cellulose
'gel' or nata is synthesized by a strain of A xylinum grown in coconut water or
fruit juices (pineapple, tomato, etc.) supplemented with sucrose. The 'gel' is cut
into one-inch cubes, boiled in water several times to remove the acidic flavor and
odor, then cooked in sugar solution to obtain the desirable chewy texture. The
final product is a thick, translucent, white or cream-colored gelatinous substance
that is often eaten with ice cream or mixed with fruits and cream.
The objective of the present work was to investigate the potential of a strain of
A.xylinum, the nata organism, for cellulose production by the batch method
under non-agitated conditions. In particular, the important factors affecting
bacterial cellulose production were examined and identified.

Materials and methods

Organism
The microorganism used (a strain of A xylinum ) was isolated from actively
fermenting broth obtained from a nata producer in Laguna, Philippines.
Isolation and purification of the culture was accomplished through pour plate,
serial dilution and repeated streaking technique (10,11). The isolated organism
was identified by the method of Carr and Passmore (12). A sample of the isolate
was also sent to the American Type Culture Collection (ATCC, Rockville, MD)
for purity assessment and identification. The isolate was positively identified as a
pure cellulose-producing strain of A xylinum by ATCC (Project Report SC
2430, June 14, 1990).
The microorganism was maintained on tomato serum agar slants (11) and was
transferred monthly. The inoculum was grown in 1 I of tomato serum broth (70 g
sucrose,S g peptone, 3 g yeast extract, 1 g K zHP0 4 , 0.2 g MgS0 47H zO, 0.1 g
NaCI and 100 ml tomato serum in 1 I of deionized water, pH 5.0). After 72 h of
static growth, the medium was blended for 3 min using a Stomacher blender,
then aseptically filtered through a fine-glass filtering crucible to remove most of
the cellulose fibers or 'gel'. The filtrate was centrifuged; the pellet was washed
three times and suspended in sterile phosphate buffer (pH 5.0) in a flask
containing glass beads. The cell suspension was refrigerated and held at 5C.
This culture was generally used within a week, but the cells remain viable for
three weeks.
The rate of cellulose production in tomato serum broth was checked regularly
to determine that mutation of the microorganism had not taken place. The
culture was also plated regularly to detect the presence of cellulose-less mutants
or other contaminants.

Optimum carbon source for cellulose production

A basal medium as described above, but without sucrose, was used and
supplemented with four individual sugars (fructose, glucose, sucrose, lactose)
and six combinations (1:1 wt/wt). Sugar solutions were sterilized separately and
408
 Bacterial cellulose. I

mixed with the basal medium (7% final concentration) prior to inoculation.
Portions (160 ml) of the medium were distributed in 250 ml Erlenmeyer flasks.
Four flasks were used for each carbon source.

Nitrogen source essential for cellulose production

Seven nitrogen sources and their 16 combinations were compared for their effect
on cellulose production in the same basal medium supplemented with 7%
sucrose but without yeast extract and peptone. The nitrogen sources tested were
(NH4hS04, NH 4H2P0 4, KN0 3 . urea, peptone, tryptone and yeast extract.

Effect of pH on cellulose production

The effect of three pH levels (3.5, 4.5 and 5.5) on cellulose production was also
examined using the same basal medium with 7% sucrose.

Assay
Weight of crude cellulose. The cellulose 'gel' was blended in a stainless steel
Waring blender for 3 min. The solids were strained through a sieve (40 mesh)
and washed repeatedly with distilled water until the filtrate became clear to
remove bacterial cells, residual sugars, salts and other metabolites. The solids
were transferred to a preweighed aluminum dish and frozen in a contact freezer.
The samples were then freeze-dried (48 h or until completely dry) in a Virtis 50-
SRC freeze dryer set at 60 millitorr and -59C. The samples were further dried
to constant weights, and the dry weight of the samples was taken as the crude
cellulose yield (gil of the medium).

pH. The pH of the medium before and after cellulose production was
determined using an Orion Research 601A Digital Ionalyzer.

Total viable count. The number of viable organisms in the inoculum and at the
end of fermentation was determined by the pour plate technique with 0.1 %
peptone (pH 5.0) as the diluent and tomato serum agar as the medium. Colonies
were counted after 4 days of incubation at 30C using a Bactronic Colony
Counter (Model no. 110, New Brunswick Scientific).

Sugars. The broth was analyzed for sugar content using a Waters High
Performance Liquid Chromatograph equipped with a differential refractometer
(Model R401) and a Shimadzu C-R3A Chromatopac integrator. The column
used was a Waters Dextro-Pak Cartridge (10 micron) in a RCM-lOO Radial
Compression Module. The mobile phase was distilled water purified using a
Milli-RlQ Water Purifier to at least 2 megaohm resistivity. The flow rate was set
at 1 ml/min (~1000 p.s.i.).
Before injection into the HPLC port, the sample was first filtered through a
coarse-glass filtering crucible. Samples containing> 10% sugar were diluted to
5% before filtration, passed through a Sep-Pak C I 8 cartridge (Waters), and
409
M.E.Embuscado, J.S.Marks and J.N.BeMiIler

filtered again using a 0.20 urn membrane filter to remove any remaining
particles.
The injection volume used was 20 j.11. The identity of the sugars and their
concentrations were determined through comparison with the retention times
and peak areas of standard sugar solutions.

Results and dicussion

Influence of type and concentration of sugars on cellulose production
Preliminary experiments revealed that synthesis of cellulose is affected by the
kind of sugar present in the medium (Table I). Fructose gave the best yield,
followed by a combination of fructose + lactose. Sucrose and a combination of
fructose + lactose also gave good yields. These four carbohydrate sources gave
significantly better yields than any of the other sources. Fructose has been
reported to be the most favorable substrate for cellulose synthesis by organisms
of the Acetobacter genus (13,14). The presence of glucose in the medium alone
or in combination with other sugars produced lower amounts of cellulose. In
addition, the final pH of all fermentation media containing glucose was 1-2 pH
units lower than any of the others, even when it was used in combination with a
'high-cellulose yielding' sugar (e.g. fructose). A.xylinum is known to convert
glucose to gluconic acid (15). Presumably, gluconic acid production was the
primary biochemical reaction in the glucose-containing media.
The cellulose yields from sucrose, although lower than those from fructose,
are contrary to those reported by Tarr and Hibbert (14), who observed much
lower yields for all disaccharides (sucrose, lactose, maltose). Similarly, Dudman
(16) found that A.acetigenum could not fully utilize sucrose for cellulose
synthesis. The strain of A.xylinum used in this study is unique because it has
always been maintained in sucrose-containing medium and nata has always been
produced using sucrose as the carbon source. Several have reported the presence

Table I. Effect of sugars on the yield of cellulose by Acetobacter xylinum"

Sugar" Cellulose" Final pHd

(gil)

Fructose 7.38 0.38 4.40

Glucose 1.00 0.05 3.01
Sucrose 5.25 0.28 4.77
Lactose 1.62 0.D7 5.00
Fructose + glucose 1.00 0.D3 3.09
Fructose + sucrose 6.38 0.32 4.42
Fructose + lactose 5.44 0.32 4.50
Glucose + sucrose 1.50 0.04 3.04
Glucose + lactose 0.75 0.03 3.08
Sucrose + lactose 2.50 0.06 4.88

a Incubated for 16 days at 30C.

bTotal sugar concentration was 7.0%. For a mixture of two sugars, the concentration of each was
3.5%.
"Homogenized. washed and freeze-dried.
dInitial pH 5.0

410
 Bacterial cellulose. I

of invertase in some strains of A.aceti and A.xylinum (15). The presence of such
enzyme would seem to be required for metabolic utilization of sucrose. Perhaps
some of the glucose formed by hydrolysis of sucrose is used for gluconic acid
production, decreasing the efficiency of carbohydrate to cellulose conversion.
The effect of the two sugars which gave the highest yields of cellulose were
then examined in more detail. Fructose at 1, 5 and 15% concentrations and
fructose + sucrose (1:1) at 1, 5 and 15% concentrations were used (Figure 1).
The rate of cellulose production decreased after 8 days of incubation, except
when 1% fructose or 1% fructose + sucrose was used (Figure 1). About 70% of
the cellulose was produced in the first 8 days in all media except those containing
only 1% total carbohydrate. Tarr and Hibbert (14) found that, for the strain they
studied, low amounts of cellulose were produced after the tenth day of
incubation. In the studies conducted by Dudman (16) on A.acetigenum in
diluted molasses, the rate of cellulose synthesis was very rapid during the first 9
days of incubation, the amount of cellulose produced in this time accounting for
>75% of the total final cellulose production. In that case, the first phase was fast
and was followed by a much slower rate of production, although a small increase
in total yield was observed up to the 46th day of incubation.

8.-------------------,

61-

.lfi- -*
I I

5 10 15 20
Incubation period (day)
Fig. 1. Cellulose production during the course of fermentation using different concentrations of
sugars: (D) 1% fructose, (6) 5% fructose, (0) 15% fructose, (*) 1% fructose + sucrose, (.) 5%
fructose + sucrose, and (&) 15% fructose + sucrose.

411
M.E.Embuscado, J.S.Marks and J.N.BeMiller

Statistical analysis of the data using SAS (17) indicated that sugars and their
concentrations were significant factors in cellulose production (P < 0.01). Using
the Student-Newman-Keuls (SNK) multiple range test to rank the sugars and
their concentrations in terms of their yields, 5% fructose, 5% fructose + sucrose
and 15% fructose + sucrose belong to the first group (highest yields), followed
by 15% sucrose. Lowest yields were given by 1% fructose and 1% fructose +
sucrose. These findings are consistent with the results obtained by Tan and
Hibbert (14) and by Dudman (16). It appears that an appropriate level of sugar
is necessary for optimum cellulose production. Sugar utilization decreased as

Table II. Sugar utilization by A.xy/inurn during tbe course of cellulose production

Sugar(s) in the medium Sugar utilized" Sugar converted to cellulose"

(%) (%)
(IS - FS)/IS x IOO Y/(IS - FS) x 100

I% fructose 37.38 18.25

5% fructose 13.36 68.82
15% fructose 5.57 69.30
1% fructose + sucrose (I: I) 29.15 12.95
5% fructose + sucrose (1:1) 21.45 41.29
15% fructose + sucrose (1:1) 22.90 38.24

"IS = initial sugar concentration (gil).

"y = crude cellulose yield (gil).
FS = final sugar concentration (gil).

Table III. Changes in the sugar concentration, total viable count (TVC)" and pH during the course of
fermentation at different sugar concentrations

Sugar Parameter Incubation (days)

0 4 8 14 16

1% Fructose Fructose (gil) 10.70 10.20 8.20 8.10 6.70

TVC (cfu/ml) 7.50 29.5 12.2 9.30 4.62
pH 4.88 4.77 5.01 5.15 5.19
5% Fructose Fructose (gil) 50.90 48.60 47.10 45.85 44.10
TVC (cfu/ml) 7.50 35.20 40.20 9.00 6.62
pH 4.88 4.81 4.68 4.53 4.54
15% Fructose Fructose (gil) 139.28 138.88 137.08 135.78 133.71
TVC (cfu/ml) 7.50 1.00 2.34 23.60 13.70
pH 4.88 4.74 4.65 4.42 4.40
1% Fructose + sucrose Fructose (gil) 6.09 6.50 5.31 5.06 3.47
Sucrose (gil) 5.30 5.20 5.00 5.00 4.60
TVC (cfu/ml) 7.50 40.70 4.25 2.15 3.00
pH 4.88 4.74 4.97 5.22 5.21
5% Fructose + sucrose Fructose (gil) 27.35 24.12 22.78 23.95 23.55
Sucrose (gil) 25.93 25.70 24.17 19.88 18.30
TVC (cfu/ml) 7.50 36.50 39.10 56.00 7.88
pH 4.88 4.83 4.79 4.59 4.64
15% Fructose + sucrose Fructose (gil) 74.87 72.38 78.47 83.39 80.29
Sucrose (gil) 75.86 75.65 67.20 62.07 58.49
TVC (cfu/ml) 7.50 24.90 71.00 13.50 11.70
pH 4.88 4.78 4.68 4.06 4.06

"TVC: x 105 .

412
 Bacterial cellulose. I

sugar concentration increased, but percent sugar converted to cellulose

appeared to level off at or before 5% concentration (Table II), as there was no
difference in percent sugar converted to cellulose when the concentration was
increased from 5 to 15%. From Table II, it is apparent that sugar levels >5%
would not be an advantage because they do not increase the efficiency of
cellulose production.
The changes in the sugar concentration, pH and total viable count (TVC)
during the course of fermentations are given in Table III. Bacterial growth was
delayed when the initial fructose concentration was 15%. There was a slight
decrease in the pH of the media as the fermentation proceeded, except for those
containing 1% sugar, which registered a slight increase in pH after an initial drop
during the first 4 days.

Influence of nitrogen source on cellulose production

In agreement with the results of Tan and Hibbert (14), a preliminary search for

Table IV. Effect of nitrogen source on cellulose yield

Nitrogen source Cellulose yield

(gil)

A. Inorganic/
(NH 4)S04 [1] 2.38 0.10
(NH 4H z)P0 4 [2] 2.44 0.11
KN0 3 [3] 0.38 0.03
B. Inorganic + inorganic"
[I] + [2] 2.38 0.20
[1] + [3] 1.12 0.18
[2] + [3] 1.75 0.08
c. Organic"
Peptone 5.12 0.10
Tryptone 3.88 0.18
Yeast extract 3.88 0.20
Urea 1.25 0.05
D. Organic + organic"
Peptone + yeast extract 5.25 0.30
Peptone + tryptone 4.75 0.32
Urea + tryptone 2.25 0.15
Urea + peptone 2.12 0.20
E. Inorganic + organic"
[I] + triptone 2.62 0.22
[1] + peptone 2.94 0.20
[1] + urea 2.00 0.20
[2] + tryptone 3.00 0.15
[2] + peptone 3.25 0.18
[2] + urea 2.62 0.10
[3] + tryptone 2.88 0.12
[3] + peptone 3.500.14
[3] + urea 1.56 0.06

a Nitrogen concentration was 0.08%.

bTwo substrates were used to give a total of 0.08% nitrogen with each source contributing -0.04%
nitrogen.

413
M.E .Embuscado , J .S.Marks and J .N.BeMiller

the proper nitrogen source for cellulose production revealed that organic
nitrogen sources, with the exce ption of urea , gave higher yields th an inorganic
so urces (Table IV). Amon g the organic sources, peptone gave the best yield,
followed by tr yptone and yeas t extract. Tarr and H ibbert ( 14) found th at
pepton e was not a desirabl e source bec ause of the for ma tio n of zoo gleal
substa nce instead of a distinct cellulose membrane , which co uld be due to th e
stra in of Acetobacter and carboh ydr at e sour ce(s) used in their study .
Inor gan ic compounds gave relati vely low to mod er at e yields of cellulose , with
ammo nium ph osph ate and ammo nium sulfate giving the highest yields and
pot assium nitr ate the lowest yield.
Co mbina tion of an orga nic and an ino rga nic nitrogen source sho wed a mark ed
improve me nt in yield compa red with an ino rgan ic source alone . A combination
of two inorganic compounds result ed in little or no increase in th e amount of
cellulose produced .
Based on the results of the pr eliminary experiment, yeast extract alone and
yeast extract supplemented with two levels of peptone wer e chosen for more
det ailed examination (Fig . 2) .
In decreasing order, sta tistical analysis by the SNK multi ple range test gave
th e following ranking of th e nit ro gen sources: (a) yeast extract supplemented

8 r-------------------,

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QI

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, .i
2 '." .I

'-: ...i
;:.; .~

0 ....---'----'---....1...----'-----'-'
o 4 8 12 16
Incubation period (day)
Fig. 2. Ce llulose produ ction during the course of fermen tation at differ ent co ncentrations of nitrogen
so urces: (* ) no nitrogen added , (e) yeast extrac t, (.) yeast extract + 0.08% nitrogen from peptone ,
and ( .to) yeast ex tract + 0.16% pept one .

414
 Bacterial cellulose. I

with 0.08% nitrogen from peptone, (b) yeast extract + 0.16% nitrogen from
peptone and yeast extract alone, (c) no nitrogen source. Neither excess nor
limited nitrogen increased cellulose yield. Dudman (18) likewise found that
cellulose formation by A.acetigenum was not enhanced by a limiting amount of
nitrogen in the presence of an excess of the carbon source. The production of
polysaccharides by other bacteria such as Aerobacter aerogenes (19), Pseudo-
monas (20), Pullularia pullulans (21) and Xanthomonas campestris (22) were
enhanced by limited nitrogen.
The changes in percent nitrogen, pH, total viable count and the initial and
final sugar concentrations are shown in Table V. The growth of A.xylinum was
not affected by different levels of the nitrogen sources. Percent nitrogen and pH
of the medium decreased slightly on incubation.

Influence of pH on cellulose synthesis

pH is an important factor in microbial growth and polysaccharide synthesis.
Although a microorganism can regulate its internal hydrogen ion concentration
quite efficiently, maintenance energy for pH control is necessary when the
microorganism is exposed to pH levels beyond its optimum range (23). In
addition, the pH of the environment has an important effect on the structure and
permeability of the cell membrane (23), which could greatly influence the
biochemical activities of the microorganism. The production of polysaccharides
such as xanthan by Xanthomonas campestris (24-26) and the exopolysaccharides
of Pseudomonas (20) are all affected by pH.

Table V. Changes in total viable count (TVC)", % nitrogen, pH and sugar concentration during the
course of fermenation at different initial nitrogen levels

Nitrogen Parameter Incubation (day)

0 4 8 12 16

None Sucrose (gIl) 64.35 NO NO NO 62.05

TVC (cfu/ml) 3.08 25.10 17.60 13.60 45.00
pH 4.97 4.71 4.86 4.96 4.77
Nitrogen (%) 0 0 0 0 0
YE h Sucrose (gIl) 69.12 NO NO NO 64.63
TVC (cfu/ml) 3.08 40.80 46.00 43.50 74.10
pH 4.97 4.73 4.82 4.84 4.82
Nitrogen (%) 0.021 0.019 0.016 0.010 0.010
YE + Sucrose (gil) 72.20 NO NO NO 58.98
0.08% TVC (cfu/ml) 3.08 42.10 40.70 60.20 45.10
PEc pH 4.97 4.96 5.02 4.96 4.70
Nitrogen (%) 0.111 0.104 0.102 0.096 0.086
YE + Sucrose (gIl) 71.46 NO NO NO 61.73
0.16% TVC (cfu/ml) 3.08 37.50 45.60 87.00 58.88
PE pH 4.97 5.12 5.13 4.99 4.78
Nitrogen (%) 0.155 0.148 0.136 0.133 0.130

"TVC: X 105.
hYE = yeast extract.
cpE = peptone (0.08 or 0.16% nitrogen from peptone).
NO = not determined.

415
M.E.Embuscado, J.S.Marks and J.N.BeMiller

12

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10

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e:, -:, 1:.:.:::::::...'

0
0 4 8 12 16
Incubation period (day)

Fig. 3. Cellulose production during the course of fermentation at different initial pH values: (e) pH
3.5. (.) pH 4.5. and (.) pH 5.5.

Table VI. Changes in total viable count (TV C)" . sugar concentration and pH during the course of
fermenation at different initial pH levels

Initial pH Parameter Incubation (days)

of medium 0 4 8 12 16

3.5 Sucrose (gil) 67.12 NO NO NO 61.09

TVC (cfu/ml) 3.84 0.34 4.53 2.27 3.74
pH 3.47 3.46 3.41 3.41 3.38
4.5 Sucrose (gil) 71.71 NO NO NO 63.02
TVC (cfu/ml) 3.84 57.40 64.90 34.90 9.75
pH 4.52 4.35 4.35 4.35 4.30
5.5 Sucrose (gil) 66.28 NO NO NO 51.81
TVC (cfu/ml) 3.84 4.25 1.54 20.4 29.9
pH 5.42 5.28 5.22 5.0 4.84

"TVC: X 105 .
NO = not determined.

416
 Bacterial cellulose. I

The optimum pH for growth of Acetobacter species is between 5.4 and 6.3
(27). This may not necessarily be the same optimum pH range for cellulose
production. Although various studies have been conducted on cellulose
synthesis by A.xylinum (14,28-31), the pH for its optimum biosynthesis has not
been heretofore investigated fully.
pH 4.5 gave the highest yield followed by pH 5.5 (Figure 3). Cellulose was not
produced at pH 3.5. Growth of the microorganism was also adversely affected
by the low pH (Table VI). However, bacterial growth recovered on the 8th day
of incubation and remained constant until the end of the incubation period. The
pH of the medium changed slightly during incubation. A.xylinum can survive at
low pH values.
Among the three initial pH values studied, pH 4.5 was best for bacterial
growth and cellulose production (Table IV). There was only a slight reduction in
the pH of the medium at the end of fermentation. The final pH in the carbon
source experiment (Table I) for the sugars that were favorable for the
production of cellulose was reduced to -4.5.

Acknowledgement
Dr R.L.Merson, Chairman of the Department of Food Science and Technology,
University of California, Davis and his staff are thanked for assistance and for
providing the equipment and facilities for this research project.

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417
M.E.Embuscado, J.S.Marks and J.N.BeMiller

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Received on November 26, 1993; accepted on March 14, 1994

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