Bacterial cellulose production by fed-batch fermentation in molasses medium

Batch and fed-batch fermentations for bacterial cellulose (BC) production using molasses as a carbon source by Acetobacter xylinum BPR2001 were carried out in a jar fermentor. For improvement of BC production, molasses was subjected to H2SO4-heat treatment. The maximum BC concentration by this treated molasses increased 76%, and the specific growth rate increased 2-fold compared with that by untreated molasses. In batch fermentation, when the initial sugar concentrations of H2SO4-heat-treated molasses were varied from 20 to 70 g/L, the highest value of maximum BC concentration of 5.3 g/L was observed at 20 g/L. BC production in intermittent fed-batch (IFB) fermentation was conducted referring to the data in batch fermentation, and the highest BC production of 7.82 g/L was obtained when 0.2 L of molasses medium was added five times. When continuous fed-batch (CFB) fermentations were conducted, maximum BC concentration was obtained with a feeding rate of 6.3 g-sugar/h, which was derived from the optimal IFB experiment.     

Production of bacterial cellulose by Acetobacter xylinum BPR2001 using molasses medium in a jar fermentor

Bacterial cellulose (BC) production by Acetobacter xylinum subsp. sucrofermentans BPR2001 using molasses medium was carried out in a jar fermentor. When molasses was subjected to H₂SO₄-heat treatment, the maximum BC concentration increased to 76% more than that achieved using untreated molasses, and the specific growth rate increased 2-fold. When the initial sugar concentrations in the H₂SO₄-heat treated molasses were varied from 23 g/l to 72 g/l, BC concentration, production rate, and yield were maximum at sugar concentrations of 23 g/l and 37 g/l, and production of by-products, such as polysaccharides and CO₂, was lower than at sugar concentrations of 48 g/l and 72 g/l, indicating that maintaining a lower molasses concentration is essential for efficient BC production in jar fermentors, this being due mainly to the complex nature of molasses. Molasses has a clear advantage over pure sugars as a carbon source from an economic viewpoint.     

Statistical optimization of culture conditions for bacterial cellulose production using Box-Behnken design

Culture conditions in a jar fermentor for bacterial cellulose (BC) production from A. xylinum BPR2001 were optimized by statistical analysis using Box-Behnken design. Response surface methodology was used to predict the levels of the factors, fructose (X1), corn steep liquor (CSL) (X2), dissolved oxygen (DO) (X3), and agar concentration (X4). Total 27 experimental runs by combination of each factor were carried out in a 10-L jar fermentor, and a three-dimensional response surface was generated to determine the effect of the factors and to find out the optimum concentration of each factor for maximum BC production and BC yield. The fructose and agar concentration highly influenced the BC production and BC yield. However, the optimum conditions according to changes in CSL and DO concentrations were predicted at almost central values of tested ranges. The predicted results showed that BC production was 14.3 g/L under the condition of 4.99% fructose, 2.85% CSL, 28.33% DO, and 0.38% agar concentration. On the other hand, BC yield was predicted in 0.34 g/g under the condition of 3.63% fructose, 2.90% CSL, 31.14% DO, and 0.42% agar concentration. Under optimized culture conditions, improvement of BC production and BC yield were experimentally confirmed, which increased 76% and 57%, respectively, compared to BC production and BC yield before optimizing the culture conditions.     

Production of Fructose and Ethanol from Cane Molasses Using Saccharomyces cerevisiae ATCC 36858

The purpose of this research was to study the possibility of the production of ethanol and enriched fructose syrups from sugar cane molasses using the yeast Saccharomyces cerevisiae ATCC 36858. In batch experiments with a total sugar concentration of between 96.7 g/l and 323.5 g/l, the fructose yield was above 90% of the theoretical value. The ethanol yield and volumetric productivity were in the range of 66% and 77% of the theoretical value, and between 0.53 g ethanol/l × h and 3.15 g ethanol/l × h, respectively. The fructose fraction in the carbohydrates content of the produced syrups was more than 95% when the total initial sugar concentration in the medium was below 273.8 g/l. Some oligosaccharides and glycerol were also produced in all tested media. The maximum amount of produced oligosaccharides including raffinose accounted for 13.4 g/l in the cane molasses medium with 323.5 g/l sugars in the initial phase of the fermentation process. The oligosaccharides produced and raffinose were completely consumed by the end of the fermentation process when the total initial sugar concentration was less than 191.3 g/l. The glycerol concentration was below 9.9 g/l. These findings are useful in the production of ethanol and high fructose syrups using sugar cane molasses.     

Controlling substrate concentration in fed-batch candida magnoliae culture increases mannitol production

Candida magnoliae HH-01, a yeast strain that is currently used for the industrial production of mannitol, has the highest mannitol production ever reported for a mannitol-producing microorganism. However, when the fructose concentration exceeds 150 g/L, the volumetric mannitol production rate decreases because of a lag in mannitol production, and the yield decreases as a result of the formation of side products. In fed-batch culture, the volumetric production rate and mannitol yield from fructose vary substantially with the fructose concentration and are maximal at a controlled fructose concentration of 50 g/L. In continuous feeding experiments, the maximum mannitol yield was 85% (g/g) at a glucose/fructose feeding ratio of 1/20. A high glucose concentration in the production phase resulted in the formation of ethanol followed by a decrease in yield and productivity. NAD(P)H-dependent mannitol dehydrogenase was purified to homogeneity from C. magnoliae. In vitro, mannitol dehydrogenase was inhibited by increasing ethanol concentration. Mannitol product was also found to be inhibitory with a K(i) of 183 mM. Under optimum conditions, a final mannitol production of 213 g/L was obtained from 250 g fructose/L after 110 h.     

Production of arachidonic acid byMortierella alpina ATCC 32222

Summary WhenMortierella alpina ATCC 32222 was incubated in a glucose salts medium at 25°C the biomass (17.5 g/l) contained 9.62% arachidonic acid which amounted to 54% (w/w) of total biomass lipids. When the glucose concentration in the medium was varied from 0 to 150 g/l, the percentage of arachidonic acid in biomass and in lipids was highest at a glucose concentration of 30 g/l, but highest yield of arachidonic acid per litre of culture broth was observed at a glucose concentration of 100 g/l. While production of biomass reached a plateau of 17 g/l after a 3-day incubation at 25°C, the percentage of arachidonic acid in lipids and biomass increased dramatically from 3 to 6 days with a concurrent arachidonic acid yield increase from 0.89 to 1.63 g/l. Optimum initial culture pH for arachidonic acid production was in the range 6.0–6.7. By increasing the concentration of the glucose salts medium three-fold, yields of biomass and arachidonic acid were increased to 35.8 g/l and 3.73 g/l, respectively.     

A continuous lactic acid production system using an immobilized packed bed of Lactobacillus helveticus

A 5 l packed bed bioreactor was used to study the effect of initial lactose concentration and hydraulic retention time (HRT) on cell growth, lactose utilization and lactic acid production. Up to 95% of the initial lactose concentration was utilized at longer HRTs (30-36 h). The study showed that lactic acid production increased with increases in HRT (12-36 h) and initial lactose concentrations. The highest lactic acid production rate (3.90 g l(-1) h(-1)) was obtained with an initial lactose concentration of 100 g/l and an HRT of 18 h, whereas the lowest lactic acid production rate (1.35 g l(-1) h(-1)) was obtained with an initial lactose concentration of 50 g/l and an HRT of 36 h. This suggested that optimal lactic acid production can be achieved at an HRT of 18 h and initial lactose concentration of 100 g/l.     

Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O(2) tension maximizes bacterial cellulose pellicle production

Vitreoscilla hemoglobin (VHb) was constitutively expressed in Acetobacter xylinum to enhance bacterial cellulose (BC) production. A pronounced enhancement of BC production in static culture was observed. Reducing O(2) tension in gaseous phase of the culture by tightly sealing the culture tube could also enhance BC production by 70%. O(2) tension in gaseous phase reduced from 21 to 15% in the sealed and static culture of VHb-expressing A. xylinum after 7 days cultivation, while 7.36g/l of BC with yield of 0.44 were obtained. BC pellicle production by VHb-expressing A. xylinum was successfully scaled-up in a sealed 4l disposable zip lock plastic bag with BC yield of 0.38 and concentration of 6.73g/l.     

The effect of temperature,pH, and initial glucose concentration on the kinetics of ethanol production by Zymomonas mobilis in batch fermentation

Summary Zymomonas mobilis, strain ATCC 10988, was used to evaluate the effects of pH (5.0 to 8.0), temperature (30°C to 40°C), and initial glucose concentration (75 g/l to 150 g/l) on the kinetics of ethanol production from glucose using batch fermentation. Specific ethanol production rate was maximum and nearly constant over a pH range of 6.0 to 7.5. End-of-batch ethanol yield and specific growth rate were insensitive to pH in the range of 5.0 to 7.5. End-of-batch ethanol yield was maximum and nearly constant between 30°C and 37°C but decreased by 24% between 37°C and 40°C. All other kinetic parameters are greatest at 34°C. End-of-batch ethanol yield is maximum at an initial glucose concentration of 100 g/l. Specific growth rate reaches a maximum at 75 g/l, but specific ethanol production rate decreases throughout the range. The optimum initial glucose concentration of 100 g/l gives the highest ethanol yield at a specific ethanol production rate less than 10% below the maximum observed.     

Ethanol fermentation using a glucose negative mutant ofZymomonas mobilis

Summary The fermentation of an equimolar mixture of glucose and fructose into ethanol and sorbitol by a glucose negative mutant ofZymomonas mobilis was monitored. The results were analyzed using a recently described method based on polynomial fitting and calculation of intantaneous and overall parameters. These parameters described well the physiology of this mixed-substrate mixed-product fermentation. Growth of the mutant was greatly inhibited on this medium. Fructose was quantitatively converted into sorbitol while glucose was oxidized into gluconic acid .This latter product was utilized as substrate for cell growth and ethanol production.Nomenclature X biomass concentration, g/l - S total sugar concentration, g/l - Glu glucose concentration, g/l - Fru fructose concentration, g/l - Sor sorbitol concentration, g/l - P ethanol concentration, g/l - t fermentation time, h - specific growth rate, h^-1 - q_s specific sugar uptake rate, g/g.h - q_G specific glucose uptake rate, g/g.h - q_F specific fructose uptake rate, g/g.h - q_P specific ethanol productivity, g/g.h - q_Sor specific sorbitol productivity, g/g.h - Y_X/S biomass yield on total sugar, g/g - Y_P/S ethanol yield on total sugar, g/g - Y_Sor/S sorbitol yield on total sugar, g/g - y_Sor/f sorbitol yield on fructose, g/g - Y_P/G ethanol yield on glucose, g/g     

Influence of Culture Parameters on Biological Hydrogen Production by Clostridium saccharoperbutylacetonicum ATCC 27021

Summary Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H2/mol sugar) as much hydrogen as monosaccharides (1.29 mol-H2/mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H2/mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H2/mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.     

Repeated batch production of ethanol from Jerusalem artichoke tubers using recycled immobilized cells of Kluyveromyces fragilis

Summary Recycled immobilized cells of Kluyveromyces fragilis ATCC 28244 were used for repeated batch production of ethanol from the inulin sugars derived from Jerusalem artichoke tubers. Using 10% initial sugar concentration, a maximum ethanol concentration of 48 g/l was achieved in 7 h when the immobilized cell concentration in the Ca alginate beads was 72 g dry wt. immobilized cell/l bead volume. The maximum ethanol production rate was 13.5 g ethanol/l bioreactor volume/h. The same Ca alginate beads containing the cells were used repeatedly for 11 batch runs starting with fresh medium at the beginning of each run. The ethanol yield was found to be almost constant at 96% of the theoretical for all 11 batch runs, while the maximum ethanol production rate during the last batch run was found to be 70% of the original ethanol rate obtained in the first batch run.     

Mass production of poly-β-hydroxybutyric acid by fed-batch culture with controlled carbon/nitrogen feeding

Summary The effect of the ratio of methanol to ammonia, in the feeding solution the C/N ratio, on microbial PHB production was investigated. A constant C/N ratio regulated both the PHB content and the specific rate of PHB production. The results indicated that to produce the maximum PHB effectively in a short time the C/N ratio should be controlled automatically according to the increase in PHB content. Variation of the PHB content was estimated by tracing the timecourse of CO₂ concentration in exhaust gas. When the cell concentration reached 70 g/l, C/N ratio was gradually increased from the initial C/N ratio of 10 (mol methanol/mol ammonia). At 121 h, concentration of PHB reached 136 g/l, which was the maximum level so far obtained. The reaction time was considerably shortened compared with our previous study (175 h). Furthermore, PHB concentration reached 149 g/l at 170 h and total cell concentration became 233 g/l. PHB yield from methanol was 0.20 (g PHB/g methanol), which was also superior to the previous result of 0.18.     

Production of bacterial cellulose by Acetobacter xylinumwith an air-lift reactor

Bacterial cellulose was produced by Acetobacter xylinum subsp. surcrofermentans BPR2001 in a 50 liter air-lift reactor using fructose as the main carbon source. When air was supplied, the production of the cellulose was only 2.3 g/l in 80 h but when O -fortified air was supplied, the cellulose concentration increased to 5.63 g/l in 28 h and the productivity of the cellulose in an air-lift reactor with O -fortified air supply was comparable to that in a mechanically agitated jar fermenter.     

Metabolic flux analysis of Gluconacetobacter xylinus for bacterial cellulose production

Metabolic flux analysis was used to reveal the metabolic distributions in Gluconacetobacter xylinus (CGMCC no. 2955) cultured on different carbon sources. Compared with other sources, glucose, fructose, and glycerol could achieve much higher bacterial cellulose (BC) yields from G. xylinus (CGMCC no. 2955). The glycerol led to the highest BC production with a metabolic yield of 14.7 g/mol C, which was approximately 1.69-fold and 2.38-fold greater than that produced using fructose and glucose medium, respectively. The highest BC productivity from G. xylinus CGMCC 2955 was 5.97 g BC/L (dry weight) when using glycerol as the sole carbon source. Metabolic flux analysis for the central carbon metabolism revealed that about 47.96 % of glycerol was transformed into BC, while only 19.05 % of glucose and 24.78 % of fructose were transformed into BC. Instead, when glucose was used as the sole carbon source, 40.03 % of glucose was turned into the by-product gluconic acid. Compared with BC from glucose and fructose, BC from the glycerol medium showed the highest tensile strength at 83.5 MPa, with thinner fibers and lower porosity. As a main byproduct of biodiesel production, glycerol holds great potential to produce BC with superior mechanical and microstructural characteristics.     

Evaluation of an electrochemical bioreactor system in the biotransformation of 6-bromo-2-tetralone to 6-bromo-2-tetralol

Biotransformation of 6-bromo-2-tetralone (Br-beta-tetralone) to 6-bromo-2-tetralol (Br-beta-tetralol) by yeast cells of Trichosporon capitatum (ATCC 74312) and its partially purified Br-beta-tetralone reductase was evaluated in an electrochemical bioreactor. The biotransformation rates and final product formation were significantly affected by substrate concentration, biomass and electric potential. At 2 g/l of substrate, the initial reaction rate and final product were increased by 35% and 15%, respectively, with -1.5 V of electric potential compared to without electric potential. Additional substrate (2 g/l) provided by pulse feeding to the reaction mixture at different intervals resulted in 2.1 g/l Br-beta-tetralol compared to a total of 1.2 g/l without feeding. However, the increased production was not proportionate to the amount of additionally fed substrate. Increased substrate availability by the addition of 5% (v/v) ethanol resulted in the highest reaction rate and product formation, but addition of ethanol at a concentration higher than 5% decreased the reaction rate. At low biomass, the initial reaction rates were enhanced significantly when electric potential was high, but a higher biomass was necessary to obtain a similar reaction rate when electric potential was reduced. The highest initial reaction rate (59.2 mg/l per min) was achieved with a two-fold biomass concentration of 15.6 g of dry cell weight/l, substrate at 4 g/l and electric potential at -6 V. The conversion of Br-beta-tetralone to Br-beta-tetralol with partially purified Br-beta-tetralone reductase was slow in the presence of electric potential.     

Effects of environmental conditions on production of xylitol byCandida boidinii

Candida boidinii NRRL Y-17213 produced more xylitol thanC. magnolia (NRRL Y-4226 and NRRL Y-7621),Debaryomyces hansenii (C-98 M-21, C-56 M-9 and NRRL Y-7425), orPichia (Hansenula) anomala (NRRL Y-366). WithC. boidinii, highest xylitol productivity was at pH 7 but highest yield was at pH 8, using 5 g urea and 5 g Casamino acids/I. Decreasing the aeration rate decreased xylose consumption and cell growth but increased the xylitol yield. When an initial cell density of 5.1 g/l was used instead of 1.3 g/l, xylitol yield and the specific xylitol production rate doubled. Substrate concentration had the greatest effect on xylitol production; increasing xylose concentration 7.5-fold (to 150 g/l) gave a 71-fold increase in xylitol production (53 g/l) and a 10-fold increase in xylitol/ethanol ratio. The highest xylitol yield (0.47 g/g), corresponding to 52% of the theoretical yield, was obtained with 150 g xylose/l after 14 days. Xylose at 200 g/l inhibited xylitol production.E. Vandeska and S. Kuzmanova were and S. Amartey and T. Jeffries are with the Forest Products Laboratory, Institute for Microbial and Biochemical Technology, 1 Gifford Pinchot Drive, Madison, WI 53703, USA. E. Vandeska and S. Kuzmanova are now with the Faculty of Technology and Metallurgy, Rudjer Boskovic 16, 91000 Skopje, Macedonia     

Kinetics of ethanol production by immobilized Kluyveromyces marxianus cells at varying sugar concentrations of Jerusalem artichoke juice

Summary Kinetics of ethanol fermentation at varying sugar concentrations of Jerusalem artichoke tuber extract has been studied using Kluyveromyces marxianus cells immobilized in calcium alginate gel beads. A maximum ethanol concentration of 111 g/l was achieved at an initial sugar concentration of 260 g/l in 20 hours, when the immobilized cell concentration in the calcium alginate beads was 53.3 g dry wt./l bead volume. Ethanol yield remained almost unaffected by initial sugar concentration up to 250 g/l and was found to be about 88% of the theoretical. Maximum rate of ethanol production decreased from 22.5 g ethanol/l/h to 10.5 g ethanol/l/h while the maximum rate of total sugars utilization decreased from 74.9 g sugars/l/h to 28.5 g sugars/l/h as the initial substrate concentration was increased from 100 to 300 g/l. The concentration of free cells in the fermentation broth was low.     

Study of the production of fructose and ethanol from sucrose media by Saccharomyces cerevisiae

The production of ethanol and enriched fructose syrups from a synthetic medium with various sucrose concentrations using the mutant Saccharomyces cerevisiae ATCC 36858 was investigated. In batch tests, fructose yields were above 90% of theoretical values for the sucrose concentrations between 35 g/l and 257 g/l. The specific growth rates and biomass yields were from 0.218 to 0.128 h(-1) and from 0.160 to 0.075 g biomass/g of glucose and fructose consumed, respectively. Ethanol yields were in the range of 72 to 85% of theoretical value when sucrose concentrations were above 81 g/l. The volumetric ethanol productivity was 2.23 g ethanol/(l h) in a medium containing 216 g/l sucrose. Fructo-oligosaccharides and glycerol were also produced in the process. A maximum fructo-oligosaccharides concentration (up to 9 g/l) was attained in the 257 g/l sucrose medium in the first 7 h of the fermentation. These sugars started to be consumed when the concentrations of sucrose in the media were less than 30% of its initial values. The fructo-oligosaccharides mixture was composed of 6-kestose (61.5%), neokestose (29.7%) and 1-kestose (8.8%). The concentration of glycerol produced in the process was less than 9 g/l. These results will be useful in the production of enriched fructose syrups and ethanol using sucrose-based raw materials.     

Ethanol production from fructose in continuous culture by free and flocculent cells of Zymomonas mobilis

Summary Zymomonas mobilis strain ZM4 was used for ethanol production from fructose (100 g/l) in continuous culture with a mineral (containing Ca pantothenate) or a rich (containing yeast extract) mediium. With both media high conversion yields were observed but the ethanol productivity was limited by the low biomass content of the fermentor. A new flocculent strain of Z.mobilis (ZM4F) was cultivated in a CSTR with an internal settler and showed a maximal productivity of 93 g/l.h (fructose conversion of 80%). When the fructose conversion was 96% an ethanol productivity of 85.6 g/l.h with an ethanol yield of 0.49 g/g (96% of theoretical) was observed.