Effect of cell recycle on continuous butanol-acetone fermentation with Clostridium acetobutylicum under phosphate limitation

Summary An overflow filtration unit for cell recycle with Clostridium acetobutylicum was developed. A cellulose-triacetate ultrafiltration membrane with a cut-off volume of 20 000 MW was found to work best. C. acetobutylicum was grown in continuous culture under phosphate limitation (0.74 mM) at a pH value of 4.4 with cell recycle, the cell dry weight in the culture vessel reached 13.1 g/l at a dilution rate of D=0.10 h^-1 and 37°C. 377 mM of glucose were fermented to 190 mM butanol, 116.2 mM acetone and 25.8 mM ethanol. Total acids were 47.6 mM. The butanol productivity was 1.41 g/l/h. At a dilution rate of 0.40 h^-1 the butanol productivity was increased to 4.1 g/l/h but glucose consumption was decreased to 285 mM and butanol, acetone and ethanol production to 138.2, 97.5, 16.5 mM, respectively.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Effect of temperature and long-term operation on passively immobilizedZymomonas mobilis for continuous ethanol production

Summary A fibrous support was used forZ. mobilis immobilization. The system showed a broad optimum temperature range (25–35°C) for highest ethanol productivity, ethanol yield and glucose conversion during continuous fermentation of a 100 g/L glucose medium. Ethanol production and glucose conversion kept steady during two months of continuous operation at D=1h^–1.     

Bacterial conversion of molasses to acetone and butanol

Summary In cooperation with the company Copersucar (Brazil), several variants of a fermentation system for the continuous production of butanol and acetone from high-test or invert molasses were developed. These fermentation systems involve a relatively economic batch fermentation requiring little investment, using a continuous culture as the inoculation culture, as well as a modern two-stage continuous culture with cell recycling. For example, 13.3 g·1^–1 of solvent (acetone and butanol) are produced with a productivity of 3.3 g·1^–1 ·h^–1 by two-stage continuous molasses fermentation with cell recycling in the second stage. High-test molasses is converted completely into the products. Butanol and acetone production from molasses is economic in Brazil and the construction of a production plant is planned.Offprint requests to: A. S. Afschar     

Fermentation of glycerol to 1,3-propanediol in continuous cultures of Citrobacter freundii

The conversion of glycerol to 1,3-propanediol by Citrobacter freundii DSM 30040 was optimized in single- and two-stage continuous cultures. The productivity of 1,3-propanediol formation was highest under glycerol limitation and increased with the dilution rate (D) to a maximum of 3.7 g·l^–1·h^–1. Glycerol dehydratase seemed to be the rate-limiting step in 1,3-propanediol formation. Conditions for the two-stage fermentation process were as follows: first stage, glycerol limitation (250mM), pH 7.2, D=0.1 h^–, 31° C; second stage, additional glycerol, pH 6.6, D=0.05 h^–1, 28° C. Under these conditions 875mM glycerol were consumed, the final 1,3-propanediol concentration was 545mM, and the overall productivity 1.38 g·1^–1·h^–1. Correspondence to: G. Gottschalk     

Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H₂ and CO₂ as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l^–1 and 60 g l^–1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l^–1 and produced 17.6 g total solvents l^–1 (yield 0.39 g g^–1, productivity 0.29 g l^–1 h^–1). Using the integrated fermentation-gas stripping product-recovery system with CO₂ and H₂ as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l^–1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g^–1 and 1.16 g l^–1 h^–1, respectively.     

Production of butanol by Clostridium puniceum in batch and continuous culture

Summary The pink-pigmented, amylolytic and pectinolytic bacterium Clostridium puniceum in anaerobic batch culture at pH 5.5 and 25–30°C produced butan-1-ol as the major product of fermentation of glucose or starch. The alcohol was formed throughout the exponential phase of growth and surprisingly little acetone was simultaneously produced. Furthermore, acetic and butyric acids were only accumulated in low concentrations, and under optimal conditions were completely re-utilised before the fermentation ceased. Thus, in a minimal medium containing 4% w/v glucose as sole source of carbon and energy, after 65 h at 25°C, pH 5.5 all of the glucose had been consumed to yield (g product/100 g glucose utilised) butanol 32, acetone 3 and ethanol 2. Butanol was again the major product of glucose fermentation during phosphate-limited chemostat culture wherein, although the organism eventually lost its capacity to sporulate and to synthesize granulose, production of butanol continued for at least 100 volume changes. Under no growth condition was the organism capable of producing more than 13.3 g l^-1 of butanol. At pH 5.5, growth on pectin was slow and yielded a markedly lesser biomass concentration than when growth was on glucose or starch; acetic acid was the major fermentation product with lower concentrations of methanol, acetone, butanol and butyric acid. At pH 7, growth on all substrates produced virtually no solvents but high concentrations of both acetic and butyric acids.     

Production of xanthan by in-flow cultures of Xanthomonas campestris

The kinetics of xanthan formation in Xanthomonas campestris continuous and fed-batch fermentations was studied along with metabolic changes due to growth rate variation. A maximum growth rate within the range 0.11–0.12 h^–1 was obtained from the continuous culture data in defined medium, producing xanthan at rates up to 0.36 g l^–1 h^–1 corresponding to a maximum 67% glucose conversion at a dilution rate (D) of 0.05 h^–1. Comparatively, fed-batch cultivation was more efficient, producing maximum xanthan at 0.75 g l^–1 h^–1 and 63% glucose conversion at 0.1 h^–1. When reaching D=0.062 h^–1 in continuous cultures, a change was observed and the values of the specific rate of substrate consumption shifted, initiating an uncoupled growth region expressing a lack of balance of the catabolic and anabolic reactions. The deviation was not accompanied by a change in specific xanthan production indicating that xanthan metabolism was not affected by D. For fed-batch-grown X. campestris cells within the range D=0.03–0.1 h^–1, both metabolic parameters changed linearly with the growth rate showing a wide region coupled to growth. Outside that range, glucose accumulated and the specific xanthan production dropped, suggesting substrate inhibition. Correspondence to: J. C. Roseiro     

Improving performance of a gas stripping-based recovery system to remove butanol from Clostridium beijerinckii fermentation

The effect of factors such as gas recycle rate, bubble size, presence of acetone, and ethanol in the solution/broth were investigated in order to remove butanol from model solution or fermentation broth (also called acetone butanol ethanol or ABE or solvents). Butanol (8 g L^–1, model solution, Fig. 2) stripping rate was found to be proportional to the gas recycle rate. In the bubble size range attempted (<0.5 and 0.5–5.0 mm), the bubble size did not have any effect on butanol removal rate (Fig. 3, model solution). In Clostridium beijerinckii fermentation, ABE productivity was reduced from 0.47 g L^–1 h^–1 to 0.25 g L^–1 h^–1 when smaller (<0.5 mm) bubble size was used to remove ABE (Fig. 4, results reported as butanol/ABE concentration). The productivity was reduced as a result of addition of an excessive amount of antifoam used to inhibit the production of foam caused by the smaller bubbles. This suggested that the fermentation was negatively affected by antifoam.Mention of trade names of commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

Continuous solvent production by Clostridium beijerinckii BA101 immobilized by adsorption onto brick

The performance of a continuous bioreactor containing Clostridium beijerinckii BA101 adsorbed onto clay brick was examined for the fermentation of acetone, butanol, and ethanol (ABE). Dilution rates from 0.3 to 2.5 h^–1 were investigated with the highest solvent productivity of 15.8 g l^–1 h^–1 being obtained at 2.0 h^–1. The solvent yield at this dilution rate was found to be 0.38 g g^–1 and total solvent concentration was 7.9 g l^–1. The solvent yield was maximum at 0.45 at a dilution rate of 0.3 h^–1. The maximum solvent productivity obtained was found to be 2.5 times greater than most other immobilized continuous and cell recycle systems previously reported for ABE fermentation. A higher dilution rate (above 2.0 h^–1) resulted in acid production rather than solvent production. This reactor was found to be stable for over 550 h. Scanning electron micrographs (SEM) demonstrated that a large amount of C. beijerinckii cells were adsorbed onto the brick support.     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

The isolation and characterisation of a salicylate-hydroxylase-producing strain of Pseudomonas putida

Summary A salicylate-hydroxylase-producing strain of Pseudomonas putida with an unusual capability to grow at toxic levels of salicylate up to 10 g l^–1 has been isolated. It grew well under continuous culture conditions, with optimum growth at pH 6.5 and a temperature of 25° C. The use of an ammonium salt as a nitrogen source, instead of nitrate, resulted in a 30–40% increase in its biomass yield coefficient. Optimum growth under continuous culture conditions was achieved using 4 g l^–1 salicylate at 25° C, pH 6.5 and 0.2 h^–1 dilution rate. High salicylate hydroxylase enzyme activity [236 units (U) l^–1] and productivity (424.8 U h^–1) were obtained at a dilution rate of 0.45 h^–1 using a mineral medium containing 4 g l^–1 of salicylate. Operating under continuous culture conditions with oxygen limitation and a slight accumulation of residual salicylate (0.2 g l^–1) resulted in a decrease in culture performance and enzyme productivity. Correspondence to: R. Marchant     

The production of hemicellulose-degrading enzymes byBacillus macerans in anaerobic culture

Summary The cell-associated and exocellular hemicellulolytic polysaccharide depolymerase and glycoside hydrolase activity ofBacillus macerans NCDO 1764 was monitored over a range of anaerobic growth conditions in batch and continuous culture. The enzymes were detectable throughout the complete growth cycle in batch culture reaching and maintaining maximum levels in the stationary phase. In continuous culture enzyme activity was largely independent of growth rate (D=0.025–0.1 h^-1) although the activity was reduced at higher dilution rates (0.125–0.15 h^-1). Although activity was detectable over a wide pH range (pH 5.5–7.5) it was pH dependent, and maximum activities of both the cell-associated and exocellular enzymes were measured in cultures maintained at pH 6.5–7.0±0.1.The principal metabolites formed anaerobically from xylose byB. macerans in batch and continuous culture were acetic acid, formic acid and ethanol which represented 95–99% of the products formed. Smaller amounts of acetone,d,l-lactic acid and succinic acid were formed together with traces of butyric acid (<5 nmol/ml) and isovaleric acid (<25 nmol/ml). The proportions of the metabolites produced varied with growth conditions and were influenced by the pH of the culture and the rate and stage of growth of the microorganism.     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

The influence of H2, CO2 and dilution rate on the continuous fermentation of acetone-butanol

Summary The effect of product gases, H₂ and CO₂, on solvent production was studied using a continuous culture of alginate-immobilized Clostridium acetobutylicum. Initially, in order to find the optimum dilution rate for aceton--butanol production in this system, fermentations were carried out at various dilution rates. With 10% H₂ and 10% CO₂ in the sparging gas, a dilution rate of 0.07 h^–1 was found to maximize volumetric productivity (0.58 g·l^–1·h^–1), while the maximum specific productivity of 0.27 g^–·h^–1 occured at 0.12 h^–1. Continuous cultures with vigorous sparging of N₂ produced only acids. It was concluded that in the case of continuous fermentation H₂ is essential for good solvent production, although good solvent production is possible in an H₂-absent environment in the case of batch fermentations. When the fermentation was carried out at atmospheric pressure under H₂-enriched conditions, the presence of CO₂ in the sparging gas did not slow down glucose metabolism; rather it changed the direction of the phosphoroclastic reaction and as a result increased the butanol/acetone ratio.     

Growth kinetics ofSaccharomyces cerevisiae in batch and fedbatch cultivation using sugarcane molasses and glucose syrup from cassava starch

Growth kinetics ofSaccharomyces cerevisiae in glucose syrup from cassava starch and sugarcane molasses were studied using batch and fed-batch cultivation. The optimum temperature and pH required for growth were 30°C and pH 5.5, respectively. In batch culture the productivity and overall cell yield were 0.31 g L^–1 h^–1 and 0.23 g cells g^–1 sugar, respectively, on glucose syrup and 0.22 g L^–1 h^–1 and 0.18 g cells g^–1 sugar, respectively, on molasses. In fed-batch cultivation, a productivity of 3.12 g L^–1 h^–1 and an overall cell yield of 0.52 g cells g^–1 sugar in glucose syrup cultivation and a productivity of 2.33 g L^–1 h^–1 and an overall cell yield of 0.46 g cells g^–1 sugar were achieved in molasses cultivation by controlling the reducing sugar concentration at its optimum level obtained from the fermentation model. By using an on-line ethanol sensor combined with a porous Teflon^® tubing method in automating the feeding of substrate in the fed-batch culture, a productivity of 2.15 g L^–1 h^–1 with a yield of 0.47 g cells g^–1 sugar was achieved using glucose syrup as substrate when ethanol concentration was kept at a constant level by automatic control.     

Interactions in a mixed culture composed of Cellulomonas flavigena and Xanthomonas sp. growing in continuous culture on sugar cane bagasse

A mixed culture formed by Cellulomonas flavigen and Xanthomonas sp. was able to grow in continuous culture in a medium with sugar cane bagasses as carbon source without additional growth factors. The effect of temperature, pH and dilution rate (D) upon the microbial population ratio and biomass composition was studied. Both microorganisms co-existed between 30 and 40° C, at pH 5.8–7.0 and D = 0.04–0.11 h^–1. Correspondence to: T. Ponce-Noyola     

The influence of cyclic glucose feeding on a continuous bakers' yeast culture

Conclusions Except for the pronounced adaptation-hysteresis effect, the pulse experiments exhibited the expected trend: deviation from the steady feed reference curve was greatest at lowest dilution rates. Under conditions of lowest glucose level the effect of pulsing would be expected to cause the largest fluctuations in glucose, causing a certain fraction of the cells to ferment. Generally over the entire dilution rate range the biomass production was decreased and the ethanol was increased by pulsing the feed stream. There is, however, some evidence that pulse feeding can trigger quite unexpected results. Point (6) at D=0.3 h^–1 in Fig. 1 exhibit a biomass productivity which was about 20% greater than the continuous feeding reference value (DX=3.6 kg m^–3 h^–1 as compared with 3.0 kg m^–3 h^–1). Such performance would be of significant commercial value, but the poor reproducibility due to adaptation, as seen here, certainly would preclude its application.The results obtained should also be applicable to fed batch operation at the corresponding glucose level. Further experiments including the variation of the glucose feeding period would be necessary to obtain a conclusive picture. The observed phenomena are likely to occur in other fermentations and could eventually explain some of the problems existing with scale up of fermentation processes.Symbols D dilution rate h^–1 - P product (ethanol) concentration kg m^–3 - Q_O2 specific oxygen uptake rate mol kg^–1 s^–1 - Q_CO2 specific CO₂ production rate mol kg^–1 s^–1 - S substrate (glucose) concentration kg m^–3 - X biomass concentration kg m^–3 - Y_P/S yield of ethanol from glucose kg kg^–1 - Y_X/S yield of biomass from glucose kg kg^–1     

Effects of culture conditions on the co-fermentation of a glucose and xylose mixture to ethanol by a mutant of Saccharomyces diastaticus associated with Pichia stipitis

Substrates that contain hexose as well as pentose sugars can form an interesting substrate for the production of ethanol. Pichia stipitis and a respiratory-deficient mutant of Saccharomyces diastaticus were used to convert such a substrate into ethanol under continuous culture conditions. With a sugar mixture (glucose 70%/xylose 30%) at 50 g/l, the xylose was entirely consumed when the dilution rate (D) did not exceed 0.006 h^–1 whereas the glucose was entirely consumed whatever the D. The study of influence of initial substrate concentration (S₀) was performed at D = 0.015 h^–1. Under these conditions the substrate was entirely consumed when its initial concentration did not exceed 20 g/l. With S₀ = 80 g/l the residual xylose concentration reached 20.5 g/l. At low D or at low S₀, P. stipitis was the dominant species in the fermentor. Increasing the D or S₀ resulted in the wash-out of P. stipitis mainly because of its low ethanol tolerance. Correspondence to: J. P. Delgenes     

2-Propanol degradation by Sulfolobus solfataricus

Sulfolobus solfataricus used 2-propanol and 2-propanone (acetone) when grown in static cultures at 78 °C with or without glucose at 10 g l^–1. The presence of 3.92 g 2-propanol l^–1 in both cases inhibited growth. However, acetone accumulation following 2-propanol depletion suggested that 2-propanol was co-metabolized via the acetone metabolic pathway. Glucose at 10 g l^–1 increased 2-propanol and acetone utilization from 0.93 g l^–1 to 1.77 g l^–1 and from 0.11 g l^–1 to 1.62 g l^–1, respectively. Without glucose, immobilized S. solfataricus cells increased the 2-propanol removal rate to 0.035 g l^–1 h^–1, compared to 0.0012 g l^–1 h^–1 by its suspended counterpart. The results suggest the establishment of an immobilized reactor configuration is preferential for the treatment of high temperature solvent waste streams by this acidothermophilic Crenarchaeon.