Anaerobic Production of Extracellular Polysaccharide by Butyrivibrio fibrisolvens nyx
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 385-391 Vol. 58, No. 1
0099-2240/92/010385-07$02.00/0
Copyright C) 1992, American Society for Microbiology
Anaerobic Production of Extracellular Polysaccharide by
Butyrivibrio fibrisolvens nyx
DANIEL E. WACHENHEIMt AND JOHN A. PATTERSON*
Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907
Received 15 August 1991/Accepted 21 October 1991
Anaerobic production of extracellular polysaccharide (EP) was examined, using a previously uncharacter-
ized, obligately anaerobic rumen isolate, Butyrivibrio fibrisolvens nyx, which produced an EP that was
rheologically similar to xanthan gum. The main objectives were to determine the nutritional requirements and
conditions which promoted EP production by strain nyx. Strain nyx was grown anaerobically in defined and
semidefined media. In addition to carbohydrate and nitrogen sources, strain nyx required acetic acid, folic
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acid, biotin, and pyridoxamine. Strain nyx produced similar amounts of EP at 35 to 40°C. Conditions that
improved growth usually improved EP production. Of the carbohydrates tested, glucose supported the fastest
growth and most EP production, followed by sucrose, xylose, and lactose. Strain nyx utilized ammonium
sulfate, urea, or vitamin-free casein hydrolysate as nitrogen sources for growth and EP production. At 2 and
20 g/liter, respectively, ammonium sulfate and vitamin-free casein hydrolysate supported about the same rates
of growth and EP production. EP was not produced in the lag or stationary phases, and EP production was
exponential during exponential cell growth. Based on the results of this work, anaerobic EP production with
B. fibrisolvens nyx could reduce energy costs for industrial EP production compared with the cost of aerated
systems. Finally, this work demonstrated that, under appropriate growth conditions, a gastrointestinal tract
(ruminal) microorganism produced high levels of EP.
Just as microbial growth changes the pH, redox state, optimal nutrient concentrations) on EP production must be
chemical constitution, and turbidity of the microbial ecosys- determined individually for the species in question.
tem, the associated microbial production of extracellular Butyrivibrio fibrisolvens nyx, a previously uncharacter-
polysaccharides (EPs) increases the viscosity and surface ized rumen isolate, was selected for this investigation on the
tension of aqueous systems (4, 14, 25). These properties basis of its rheological properties. The overall objective was
allow microbially produced polysaccharides to be used in to characterize the nutritional (sources of nitrogen, carbo-
many applications that involve modifying the flow properties hydrates, and acetate) conditions and temperatures that
of foods, pharmaceuticals, and other industrial products (19, supported accumulation of EP by the obligately anaerobic
31, 34). However, the high viscosity resulting from polysac- bacterium B. fibrisolvens nyx.
charide production in aerobic industrial systems hinders heat (This article is from the thesis submitted by D. E. Wachen-
transfer and mass transfer of oxygen and results in high heim in partial fulfillment of requirements for the Ph.D.,
energy requirements to agitate the broth (4, 25, 30, 33). This Department of Animal Sciences, Purdue University.)
work addresses a biological alternative (use of an anaerobic
microorganism) to a typical biochemical engineering chal-
lenge (greater agitation and higher oxygen pressures) (30, MATERIALS AND METHODS
33). In addition to its potential for commercial applications, Culture maintenance and growth media. B. fibrisolvens nyx
this investigation is also one of the first to thoroughly was selected from the author's (J.A.P.) culture collection.
examine conditions which support extracellular polysaccha- Strain nyx was originally isolated from the rumen of a steer
ride production by an obligately anaerobic, nonpathogenic fed an alfalfa-cottonseed hull diet. The isolation medium
gastrointestinal tract (ruminal) bacterium. contained xylan as the sole carbohydrate source. For long-
Carbohydrate and nitrogen nutrition, cofactors, tempera- term storage, strain nyx was grown overnight in a modified
ture, and aeration (for aerobes) influence microbial EP medium 98-5 of Bryant and Robinson (8), which contained
production. However, the effects of these conditions are not volatile fatty acids and hemin as described by Leedle and
generally predictive for unrelated species. For example, Hespell (24); glucose, maltose, cellobiose, and trypticase at
nitrogen limitation increases EP production by Xanthomo- 5 g/liter each; and 1.0 g of yeast extract per liter. The
nas campestris B1459 (39) and Zoogloea ramigera (28) and cultures were stored at -18°C with added glycerol as de-
decreases EP production by Porphyridium sp. strain scribed by Teather (41). For routine storage, strain nyx was
UTEX637 (2). Similarly, higher-than-optimal growth tem- grown overnight in medium A (Table 1) and stored at 2°C for
perature increases EP production by X. campestris (37) but up to 3 weeks. Inocula were prepared by growing strain nyx
lower-than-optimal growth temperature increases EP pro- overnight in medium A.
duction by Klebsiella aerogenes (16). Thus, the effects of Strict attention was given to anaerobic technique for all
these conditions (and other growth conditions, such as culture manipulations (5, 12). Oxygen was removed by
boiling media under a stream of oxygen-free CO2 (5). Media
were then sealed, autoclaved (120°C, 18 lb/in , 15 min),
*
Corresponding author. cooled, and transferred into an anaerobic glovebox (Coy
t Present address: College of Veterinary Medicine, Oregon State Laboratories, Ann Arbor, Mich.) (12) containing oxygen-
University, Corvallis, OR 97331. free CO2 with approximately 5% H2. Sterile reducing agents
385386 WACHENHEIM AND PATTERSON APPL. ENVIRON. MICROBIOL.
TABLE 1. Media for maintenance of B. fibrisolvens nyxa added to the supernatant. After chilling overnight at 2°C, the
resultant floc was removed and dialyzed (molecular weight
Compound or Amt/liter cut-off, 6,000 to 8,000) against six changes of deionized,
solution Medium A Medium B distilled water. The dialysate was freeze-dried and stored in
Glucose 15.0 g 40.0 g a dessicator. The resultant EP cake was tested for purity by
Sodium acetate 6.0 g 2.0 g analysis for protein, free glucose, turbidity, and total carbo-
K2HPO4 0.72 g 0.0 g hydrates (20, 38). For analysis of EP in growth studies,
KH2PO4 0.72 g 8.2 g samples were removed from serum vials and weighed in
(NH4)2SO4 0.72 g 1.9 g tared 15-ml centrifuge tubes. An equal amount of deionized,
NaCl 0.72 g 0.48 g distilled water was added, and the samples were centrifuged
MgSO4. 7H20 0.09 g 0.10 g as described above. The supernatants were then frozen.
CaCl2. 1H20 0.06 g 0.06 g Thawed supernatants were dialyzed against three changes of
Resazurin 0.001 g 0.001 g deionized, distilled water. The glucose oxidase test was used
Trace metalsb 10.0 ml 10.0 ml to ensure the effectiveness of dialysis. The dialysates were
HEPESC 0.012 g 0.012 g
Biotin 0.00025 g 0.00025 g diluted and analyzed for polysaccharide by the phenol-
Folic acid 0.00025 g 0.00025 g sulfuric acid procedure (20). Measurement of EP by phenol-
Pyridoxamine 0.002 g 0.002 g sulfuric acid was compared with measurement by dry
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Na2CO3d 4.0 g 4.0g weight, and there was no difference between the two meth-
Na2S 9H2Od 0.25 g 0.25 g ods (P > 0.6). Phenol-sulfuric acid was used because this
" Media were prepared anaerobically, as described in the text. The pH was method was more rapid and required less sample than
adjusted to 7.0 by using 2 M KOH before the media were boiled under a measurement by dry weight.
stream of anaerobic CO2. The final pH of the media was 7.0, with CO2 or Rheology. The EP was dissolved in saline (1 g of NaCl per
C02-H2 (95:5) gas phases, after addition of Na2CO3. Medium A and Medium liter) at concentrations from 0.1 to 10.0 g/liter. EP solutions
B were modified from the media described in references 8, 13, and 39. were stored overnight at 2°C and warmed to 25°C prior to
b The trace metals solution was modified from references 24 and 36 and
contained (in grams per liter): disodium EDTA, 0.43; FeSO4 7H20; 0.20; rheological testing. Bubbles were removed by subjecting
MnSO4. 1H20, 0.17; ZnSO4 .7H20, 0.01; H3BO3, 0.03; CoCI2 6H,O, samples to a vacuum. A Brookfield RVT viscometer,
0.012; CuCI2 2H20, 0.001; NiCI2 6H20, 0.002; and NaMoO4. 2H20, 0.003. equipped with a UL adaptor (Brookfield Co., Stoughton,
' HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) was used Mass.), was used at 25°C to measure viscosity as centipoise
to buffer the vitamin solution. The vitamin solution was filter sterilized and
stored anaerobically until used. Vitamins were added aseptically to cooled, (cP), using 16 ml of EP solution. All measurements were
sterilized media. performed twice. Xanthan gum (Sigma Chemical Co., St.
d Added to cooled, sterile media as a separately prepared, autoclaved, Louis, Mo.) was used as a reference without additional
anaerobic solution. purification because of the negligible effect of additional
purification of xanthan shown by others (43).
Assays. Glucose was determined with glucose oxidase
and carbonate buffer were added to cooled, autoclaved (Sigma), using a glucose standard curve (38). Carbohydrates
medium as separately prepared, anaerobic solutions. Vita- were determined by the phenol-sulfuric acid method (20).
mins were added as a filter-sterilized anaerobic solution. Purified strain nyx EP was used for the standard curve
Media were dispensed into sterile serum tubes or serum because different carbohydrate types resulted in different
vials, which were then stoppered with butyl rubber serum slopes. Protein was determined by the Coomassie brilliant
stoppers and aluminum crimp closures. blue dye-binding reaction, with bovine serum albumin (Sig-
Experimental treatments. Medium B (Table 1) was modi- ma) used for the standard curve (20).
fied to test experimental treatments (types and amounts of Fermentation acids. Samples were centrifuged as de-
carbohydrates and nitrogen sources; amount of acetate). scribed above, and 0.2 ml of 25% (wt/vol) H3P03 was added
Sterile, anaerobic treatment solutions were dispensed into to 1.0 ml of supernatant. A glass rod was used to remove
sterile 50-mi serum vials, to which 25 ml of the basal solution gelled polysaccharide. From the remaining liquid, 0.5 Jl was
was added. The basal solution was inoculated with 1% analyzed by gas-liquid chromatography (GLC). A 6-ft (ca.
(vol/vol) of an overnight culture of strain nyx prior to being 2-m)-long column, packed with SP-1200 (Supelco, Belle-
dispensed into the treatment vials. All dispensing was per- fonte, Pa.), was used in a Varian 3700 GLC with a flame
formed in the anaerobic glovebox. The vials were stoppered ionization detector. The temperature of the oven was 130°C
with sterile, black rubber serum stoppers (Bellco Inc., (isothermal), that of the injector was 170°C, and that of the
Vineland, N.J.), removed from the anaerobic glovebox, and detector was 180°C, with carrier gas (N2) flowing at a rate of
incubated in a waterbath at the appropriate temperature 30 ml/min. Lactate was initially determined colorimetrically
(37°C, unless otherwise stated). (3), but in subsequent analyses, lactate was determined by
Initial characterization of B. fibrisolvens nyx. Strain nyx GLC concomitantly with acetate and butyrate.
was characterized by morphology (Gram stain, phase con- Cell density. Culture samples were weighed into tared test
trast microscopy), fermentation acids, and substrate utiliza- tubes and diluted (final OD,VOL. 58, 1992 ANAEROBIC POLYSACCHARIDE BY BUTYRIVIBRIO FIBRISOLVENS 387
qualitative variables (treatment types) and quantitative var-
iables (treatment amounts), as well as interaction effects (27,
29).
RESULTS
Identification of strain nyx. Strain nyx was identified as B. a. 2.0 _
fibrisolvens because it produced butyrate, fermented xylan,
stained gram negative, and was a motile, rod-shaped bovine o 1.5-
ruminal bacterium. Although there is much variation among
Butyrivibrio strains, B. fibrisolvens is the only normal-flora 1.0-
ruminal bacterium with those characteristics (6-8, 22, 23). 0.5
Subsequent results (nutritional sources and requirements,
fermentation products) also supported this identification. A 0.c0 - ---------
minimal medium was constructed based on the medium of
Cotta and Hespell (13) and known requirements of B. -0.5-
-0.8 -0.6 -0.4 -0.2 -0.0 0.2 0.4 0.6 0.8 1.0
fibrisolvens (23). Biotin, folic acid, pyridoxamine, and/or
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log (EP g/L)
acetic acid (21, 23) but not additional vitamins, hemin, or
longer or branched-chain carboxylic acids (13, 21, 23) were FIG. 1. Rheology of strain nyx EP and xanthan gum, plotted as
required for growth. The resultant medium (medium A, log(cP) versus log(EP concentration) at three shear rates. Symbols:
Table 1) was used for all subsequent culture maintenance *, nyx EP, 1.22 s-1; O, xanthan gum, 1.22 s-1; *, nyx EP, 12.2 s-1;
and preparation of inocula. A, xanthan gum, 12.2 s'-; x, nyx EP, 122 sl; x, xanthan gum, 122
s-
Carbohydrate and nitrogen sources. Strain nyx utilized the
following sugars for growth: fructose, glucose, galactose,
sucrose, cellobiose, maltose, lactose, corn starch, potato
starch, soluble starch, xylan, xylose, and arabinose. Man- shear rates for each concentration (Fig. 2). At lower concen-
nose, mannitol, rhamnose, and pectin were not used. Strain trations and shear rates, xanthan gum resulted in higher
nyx also grew when the ammonium sulfate in medium A was viscosity, while at high concentrations and shear rates, this
replaced with urea, gelatin, casein, or casein hydrolysate. difference was reversed, indicating higher pseudoplasticity
With any of these substrates, the major fermentation prod- for the nyx product. Overall, however, these differences
ucts were lactate and butyrate. Acetate was not produced. were small, and the rheology of the strain nyx EP was
Chemical characterization of EP. With medium A, the yield essentially similar to that of xanthan gum.
of purified EP from original glucose was 11%. Strain nyx EP Development of basal-level experimental conditions. Me-
did not contain measurable protein or free glucose. Elemen- dium B (Table 1) was modified from medium A with in-
tal analysis (Purdue University Chemistry Department) in- creased glucose, ammonium sulfate, and phosphate to more
dicated that there was 40% carbon, 6% hydrogen, a trace closely approximate industrial EP production media and
(0.3%) of nitrogen, and 53% oxygen. Based on a formula increase buffering (39). The increased phosphate level did
weight calculation of 162 g of carbohydrate per mol (one not change EP production (P > 0.2). Higher levels of
H20 is lost per hexose in the polysaccharide), the EP should phosphate precipitated and were not used. When tested at
have been 44% carbon, 6% hydrogen, 49% oxygen, and 0% various temperatures, growth and EP production did not
nitrogen. The nitrogen may have indicated ammonia or a change significantly between 35 and 40°C (P > 0.05), result-
small number of amines, and the relatively higher oxygen
level may have indicated the presence of uronic acids, but
overall this result confirmed that the material was a polysac- 4.0
charide. Additional analysis (courtesy of D. J. Cherney,
Purdue University Animal Science Department and Agron-
omy Department) by high-pressure liquid chromatography 3.5 o
(HPLC) (17) indicated that the EP consisted of 48% glucose,
48% galactose, 3.5% mannose, and less than 0.1% xylose. 3.0-
Additional substituents, such as uronic acids, were not
measured. a.
Rheological characterization. The rheological characteris- 0
tics of strain nyx polysaccharide were compared with those
of xanthan gum. Log transformations, based on the power 2.0-
law for pseudoplastic solutions (9, 43), should produce linear
plots. When the results were plotted as log(viscosity) versus
log(EP concentration) (Fig. 1) or log(viscosity) versus log
(shear rate) (Fig. 2), the plots were generally linear. Polyno-
mial regressions of data plotted as log(viscosity) versus 1.0
log(EP concentration) (Fig. 1) resulted in no significant effect -0.5 0.0 0.5 1.0 1.5 2o as5
due to type of polysaccharide (P > 0.05 at all three shear log (shear rate /sec)
rates, r2 = 0.95 to 0.98). However, the effect of polysaccha- FIG. 2. Rheology of strain nyx EP and xanthan gum, plotted as
ride type when plotted as log(viscosity) versus log(shear log(cP) versus log(shear rate) at three EP concentrations. Symbols:
rate) (Fig. 2) was statistically significant (P < 0.05, r2 = 0.98 *, nyx EP, 1 g/liter; O, xanthan gum, 1 g/liter; *, nyx EP, 2 g/liter;
to 0.99). Therefore, testing a range of concentrations at a few A, xanthan gum, 2 g/liter; x, nyx EP, 4 g/liter; [gx, xanthan gum, 4
shear rates (Fig. 1) was less sensitive than testing a range of g/liter.388 WACHENHEIM AND PATTERSON APPL. ENVIRON. MICROBIOL.
TABLE 2. Effects of nitrogen sources on final OD and EP yields
Concna Final EP Final culture 3.5 4.5
Nitroen
Nitrogen surce
source (g/liter) yield(g/liter) 0Fact -4.0
Ammonium sulfate 0.96 2.52 ± 1.17b 3.77 ± 0.92 3.0
----m 3.5
Urea 4.32 2.40 ± 0.08 3.85 ± 0.08
VFCH 4.8 3.44 ± 0.57 2.85 ± 0.70 ._' 2.5 3.0
c
20 2
a Amounts are given for the highest EP yield for each N source. Less of a
0 2.0 -2.5 '
each N source resulted in lower amounts of EP, while additional N did not a-
increase EP production.
b Value ± standard
t 1.5 2.0w
deviation.
-1.5
1.0-
-1.0
ing in 3.0 g of EP per liter and an OD of 3.0. Above and 0.5 70.5
below that temperature range, growth and EP production 8. _
decreased (P < 0.01). The effect of initial acetate concentra- 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0
tion was also tested. Without acetate, no growth occurred Casein Hydrolysate (g/L)
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within 48 h. Increments of acetate (0.0, 0.15, 1.5, 15, and 150 FIG. 3. Effect of initial casein hydrolysate concentration on
mM) resulted in log-linear effects of acetate concentration growth (U) and EP production (CI) by strain nyx.
for OD (r2 = 0.93; P < 0.0001) and EP production (r2 = 0.96;
P < 0.0001) between 0 and 15 mM acetate. Additional
acetate had no effect.
Effect of carbohydrate source. Glucose, sucrose, lactose, effects and was used at 0.00, 0.48, 1.6, 4.8, and 16 g/liter
and xylose were compared at 40 g/liter, with measurements (Fig. 3). After excluding 16 g/liter (no difference from 4.8
made at 24, 48, and 72 h to allow for adaptation and g/liter), the effects of VFCH on OD and EP production were
differences in growth rate. Final EP production depended on linear (r2 = 0.89 and P < 0.0001 for OD, and r2 = 0.96 and
the substrate carbohydrate as follows (EP produced, in P < 0.0001 for EP).
grams per liter): glucose (2.5) > sucrose (1.8) > xylose or Although the concentrations of nitrogen sources were
lactose (1.25). The OD ranged from approximately 2.0 with selected on the basis of the effects of the individual nitrogen
lactose to 3.5 with glucose or xylose. At 24 h, glucose sources on growth and not on the basis of equal nitrogen
resulted in the highest OD (3.0) and EP (2.4 g/liter) (P < content, limited comparisons were made among nitrogen
0.05). At 48 h, the OD was the same for glucose and xylose sources. The amount of nitrogen was calculated from for-
(P > 0.6) but less with sucrose or lactose (P < 0.05), while mula weights of urea and ammonium sulfate and Kjeldahl
glucose resulted in about twice as much EP as xylose (2.5 analysis of VFCH (12.8% nitrogen). At low nitrogen concen-
versus 1.25 g/liter). The results at 72 h were similar to those trations, ammonium sulfate yielded the highest cell density
at 48 h. per amount of nitrogen, followed by urea and VFCH (P <
Effect of glucose concentrations. Strain nyx was grown with 0.05). At high nitrogen concentrations, either urea or ammo-
0 to 50 g of glucose per liter. EP, OD, and residual glucose nium sulfate resulted in the highest growth, OD of 3.9 (P >
were measured. There was no growth with 0 g of glucose. 0.5), with VFCH giving an OD of 2.75 (P < 0.05). Ammo-
The OD was highest (4.0) with 20 g of glucose per liter (P < nium sulfate resulted in higher EP levels at lower nitrogen
0.05). Increasing the glucose concentration did not increase concentrations relative to urea or VFCH (P < 0.05), but at
EP yield above that for initial glucose at 20 g/liter (2.5 g of EP the highest concentrations, VFCH yielded the most EP, 3.5
per liter). Above this level, growth and EP production were g/liter (P < 0.05).
probably limited by the pH drop. EP yield, calculated from Kinetics of growth and product formation by strain nyx.
glucose consumption, was 20.1% and was unaffected by the During kinetic experiments, vials were sampled most fre-
glucose concentration (P > 0.05). Glucose was completely quently during rapid growth, so that most of the data were
consumed only at 10 g of glucose per liter. collected during the periods of greatest change. The growth
Effects of nitrogen sources and concentrations. Ammonium rate and final OD increased because the vials were shaken
sulfate, urea, and vitamin-free casein hydrolysate (VFCH) during sampling. Fermentation products and residual glu-
were evaluated as nitrogen sources for EP production by cose were measured in addition to EP and OD. Early-
strain nyx (Table 2). Ammonium sulfate was tested at 0.00, exponential-phase culture results were tested for linearity
0.24, 0.48, 0.96, and 1.92 g/liter. After excluding 1.92 g/liter after log conversion.
(no difference from 0.96 g/liter, and therefore ammonium With ammonium sulfate as the nitrogen source (Fig. 4), the
sulfate was no longer limiting), the effects of ammonium specific growth rate was 0.43 h-1 (r2 = 0.97). EP production
sulfate were linear for OD (r2 = 0.82, P < 0.001) but not for followed first-order kinetics (r2 = 0.95). Glucose utilization
EP production. Urea was tested at 0.00, 0.86, 2.16, 4.32, and and production of butyrate were first order (r2 = 0.79 and
8.64 g/liter. After excluding 8.64 g/liter (no difference from 0.84, respectively; Fig. 5), while acetate concentrations did
4.32 g/liter), the effects of urea were significant but not linear not change with time (P > 0.10). Lactate followed the same
for OD (r2 = 0.49, P < 0.001). Increasing urea above 0.86 production pattern as butyrate.
g/liter did not affect EP production (P > 0.5). The concen- The results for VFCH were similar to those for ammonium
trations used in the experimental treatments were based on sulfate. The specific growth rate was 0.44 h-1 (r2 = 0.97); EP
early growth observations. With urea as the nitrogen source, production, butyrate production, and glucose utilization
the ranges of urea concentrations provided the expected were first order (r2 = 0.91, 0.76, and 0.78, respectively). No
results for OD but were apparently too high to demonstrate statistical differences were seen in comparing ammonium
statistical effects for EP production. VFCH was tested to sulfate and VFCH for specific growth rate (P > 0.8), EP
provide an array of amino acids while avoiding vitamin production (P > 0.9), glucose utilization (P > 0.7), butyrateVOL. 58, 1992 ANAEROBIC POLYSACCHARIDE BY BUTYRIVIBRIO FIBRISOLVENS 389
D U- 5._-0 from others working with other microorganisms. For exam-
4.5- - 4.5 ple, the highest yield of EP by strain nyx occurred with 30 to
40 g of glucose per liter; similar levels of optimal carbohy-
4.0- -4.0 drates benefited EP production by Xanthomonas campes-
3.5- -3.5 tris, with the highest yields of xanthan gum at glucose or
.0 3.0- -3.0
sucrose levels of 30 to 50 g/liter (39). The highest yield of
a) purified EP from glucose (20.1%) by strain nyx was higher
2.5- w E -2.5 than that reported for crude EP preparations from other
a. 2.0- i-2.0
Butyrivibrio strains (up to 16.3% yield) with 1% glucose (18).
0 Strain nyx produced a high amount of EP with ammonium
1.5- ; / -1.5 sulfate at 1 to 2 g/liter. The requirement for relatively higher
1.0- / 2 -1.0
amounts of VFCH, based on equimolar nitrogen content,
may be due to the inability of strain nyx to use some of the
0.5; t w ' -0.5 amino acids in VFCH. Ammonium sulfate and VFCH re-
0.01 i0.0 sulted in the same amounts of growth and EP production
0 5
..
10 15 20 25 30 35
40 i5 50- when the nitrogen sources were in excess. Therefore, com-
Hours of Growth plex and simple N sources gave similar results for strain nyx.
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FIG. 4. Growth (-) and EP production (O) by strain nyx in In contrast, the highest EP yields by X. campestris occurred
medium B. with amino acids as the N source, approximately doubling
the EP yields over those obtained with ammonium sulfate
(39).
production (P > 0.1), or lactate production (P > 0.7). The amounts of nitrogen sources relative to carbohydrate
Approximate final values for either set of cultures were: OD supplies have often been significant for EP production by
= 5.0 with EP at 2.5 g/liter, 60 mM lactate, and 25 mM microorganisms. Limiting the nitrogen source while provid-
butyrate. Approximately 15% of glucose utilization was ing excess carbon source frequently results in higher EP
accounted for by purified EP production, on a mole-per-mole production (15, 26, 37), but nitrogen deficiency decreased EP
basis. yield for Porphyridium sp. strain UTEX637 (2) and Halo-
ferax mediterranei (1); EP production by Pseudomonas sp.
strain NCIB11264 increased with increasing concentrations
DISCUSSION of ammonium chloride, up to a saturation level which
Based on the chemical and rheological tests that were resulted in a slight decrease in EP production (44). For strain
nyx, limiting amounts of nitrogen resulted in low amounts of
performed on the strain nyx EP, the material was clearly
EP. Once nitrogen was not limiting, additional nitrogen did
determined to be polysaccharide. Saline solutions of nyx EP not affect EP production.
were rheologically similar to those of xanthan gum. The
chemical composition of strain nyx EP was similar to that of Strain nyx produced EP at a nearly constant specific rate
the EP from several other strains of B. fibrisolvens, as while growth was active. EP production ceased when growth
described by Stack (40), in that strain nyx EP contained ceased. This kinetic pattern supported the assessment that
equal glucose and growth and EP production were linked for strain nyx. For
approximately parts galactose, plus a
other microorganisms, EP can be produced during either
small amount of mannose and a trace of xylose. exponential-growth or stationary phase or both, as indicated
Strain nyx produced EP from a variety of carbohydrate in Table 3.
substrates. Xylose supported a high final OD6. but a lower Many factors influence the applicability of a biological
final EP concentration than the hexoses. Although growth on process to commercial fermentation. Among these factors
xylose might result in a qualitatively different EP, Stack
are yield of product from substrate, concentration of final
reported that this did not occur for other strains of B. product, rate of production, and costs of operation and
fibrisolvens (40). These results can be compared with those
substrates. The highest final concentration of strain nyx EP
(3.5 g/liter) was produced within 24 h, comparable to EPs of
120.0 70.0 other, relatively fast-producing microorganisms. Most mi-
croorganisms with significantly higher yields produced EP
much more slowly (Table 3). The main exception is X.
100.0_ -60.0 campestris B-1459, which has undergone decades of strain
development. Comparing the specific growth rate for strain
gEE 80.0 2 -s50.0 nyx with the specific growth rate for X. campestris, strain
/S -40.0 n nyx grows faster (,. = 0.43 h 1) than X. campestris (p = 0.15
D 60.0, to 0.19 h-1) (32). The potential for enhancement of strain
CD
0
nyx
-~~~~~~~~~~~~~~~30.02
a
by strain development is unknown.
0
There was little rheological difference between xanthan
C.
(.0 40.0
< gum and strain nyx EP, so similar applications are possible.
The fermentation broth and polysaccharide that were pro-
duced by strain nyx were colorless, versus the yellow color
of xanthan gum (data not shown). Therefore, the nyx EP
does not require decolorization. The use of anaerobic bac-
teria, such as strain nyx, for commercial EP production is
Hours of Growth
possible, but additional information, expanding on the work
FIG. 5. Glucose utilization (A), lactate production (*), and bu- that was presented here, would be useful. For example, the
tyrate production (x) by strain nyx in medium B. effects of growth conditions, nutrients, and limiting condi-390 WACHENHEIM AND PATTERSON APPL. ENVIRON. MICROBIOL.
TABLE 3. Characteristics of EP production by various microorganisms
Time (h) to EP yield Growth phase for Ref
Microorganism EP maximum' (g/liter) highest EP production eference
Butyrivibriofibrisolvens nyx 24 3.5 Exponential This studyb
Pseudomonas aeruginosa 25 9.0 Exponential 26
Pseudomonas sp. strain NCIB11264 50 5.5 Stationary 44
Haloferax mediterranei 50 2.8 Exponential 1
Klebsiella aerogenes 72 0.9 Stationary 16
Xanthomonas campestris 96 33.0 Late exponential and 37, 39
stationary
Porphyridium sp. strain UTEX 637 360 2.9 Stationary 2
Aureobasidium pullulans 168 11.0 Exponential 33
Rhizobium meliloti 168 2.0 Exponential 15
Zoogloea ramigera 240 14.0 Stationary 28
a
Estimated from tables and figures in the indicated references, based on hours needed to reach maximum EP concentrations.
b Values are for cultures grown with VFCH as the N source.
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tions need more detailed consideration. The mineral nutri- 12. Costilow, R. 1981. Biophysical factors in growth, p. 66-111. In
tion of strain nyx was not explored in this investigation. P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester,
Because of the low pH of the growth medium at the end of W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.), Manual of
the fermentation (pH 5), it is possible that the culture was methods for general bacteriology. American Society for Micro-
biology, Washington, D.C.
ultimately limited by fermentation acids or acidity. There- 13. Cotta, M. A., and R. B. Hespell. 1986. Proteolytic activity of the
fore, the effects of pH control should also be examined. ruminal bacterium Butyrivibrio fibrisolvens. Appl. Environ.
Overall, therefore, while additional, directed research is Microbiol. 52:51-58.
needed, this project supported the hypothesis that a biolog- 14. Cottrell, I. W., and K. S. Kang. 1978. Xanthan gum, a unique
ical solution (anaerobic bacteria) to the engineering chal- bacteriai polysaccharide for food applications. Dev. Ind. Micro-
lenge of vigorously aerating growth media for EP production biol. 19:117-131.
is reasonable and deserves serious consideration. 15. Dudman, W. F. 1964. Growth and extracellular polysaccharide
production by Rhizobium meliloti. J. Bacteriol. 88:640-645.
ACKNOWLEDGMENTS 16. Duguid, J. P., and J. F. Wilkinson. 1953. The influence of
cultural conditions on polysaccharide production by Aerobacter
We thank D. J. R. Cherney for chromatographic analysis, P. A. aerogenes. J. Gen. Microbiol. 9:174-189.
Jaynes for Kjeldahl analysis of casein hydrolysate and help with 17. Garleb, K. A., L. D. Bourquin, and G. C. Fahey, Jr. 1989.
other procedures, and A. M. Wilson for reagents and equipment. Neutral monosaccharide composition of various fibrous sub-
This work was supported in part by a David Ross Fellowship and strates: a comparison of hydrolytic procedures and use of
assistantships from the Purdue Departments of Animal Science and anion-exchange high-performance liquid chromatography with
Biochemistry. pulsed amperometric detection of monosaccharides. J. Agric.
Food Chem. 37:1287-1293.
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