High-productivity continuous biofilm reactor for butanol production

Corn steep liquor (CSL), a byproduct of the corn wet-milling process, was used in an immobilized cell continuous biofilm reactor to replace the expensive P2 medium ingredients. The use of CSL resulted in the production of 6.29 g/L of total acetone-butanol-ethanol (ABE) as compared with 6.86 g/L in a control experiment. These studies were performed at a dilution rate of 0.32 h⁻¹. The productivities in the control and CSL experiment were 2.19 and 2.01 g/(L·h), respectively. Although the use of CSL resulted in a 10% decrease in productivity, it is viewed that its application would be economical compared to P2 medium. Hence, CSL may be used to replace the P2 medium. It was also demonstrated that inclusion of butyrate into the feed was beneficial to the butanol fermentation. A control experiment produced 4.77 g/L of total ABE, and the experiment with supplemented sodium butyrate produced 5.70 g/L of total ABE. The butanol concentration increased from 3.14 to 4.04 g/L. Inclusion of acetate in the feed medium of the immobilized cell biofilm reactor was not found to be beneficial for the ABE fermentation, as reported for the batch ABE fermentation. Names are necessary to report factually on available data. However, the USDA neither guarantees nor warrants the standard of the product, and the use of the names by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Production of acetone butanol ethanol from degermed corn using Clostridium beijerinckii BA101

In this article we report on acetone butanol ethanol (ABE) fermentation characteristics of degermed corn when using Clostridium beijerinckii BA101. Recent economic studies suggested that recovery of germ from corn and hence corn oil would help to make the ABE fermentation process more economical. C. beijerinckii BA101 ferments corn mash efficiently to produce ABE under appropriate nutritional and environmental conditions. Corn mash contains germ/corn oil that is, possibly, ancillary to the production of butanol during the ABE fermentation process. Since the presence of corn oil is not a critical factor in solvent fermentation, it can be removed and this will allow for byproduct credit. Batch fermentation of degermed corn resulted in 8.93 g/L of total ABE production as compared with 24.80 g/L of total ABE when supplemented with P2 medium nutrients. During the course of the germ separation process, corn steeping is required prior to grinding and removing the germ. It is likely that some nutrients from the corn are leached out during the steeping process. This may reduce the rate of fermentation and impact the final concentration of butanol/ABE that can be achieved. Fermentation of degermed corn with corn steep liquor resulted in the production of 19.28 g/L of ABE.     

Continuous production of butanol by clostridium acetobutylicum immobilized in a fibrous bed bioreactor

We explored the influence of dilution rate and pH in continuous cultures of Clostridium acetobutylicum. A 200-mL fibrous bed bioreactor was used to produce high cell density and butyrate concentrations at pH 5.4 and 35°C. By feeding glucose and butyrate as a cosubstrate, the fermentation was maintained in the solventogenesis phase, and the optimal butanol productivity of 4.6g/(L h) and a yield of 0.42 g/g were obtained at a dilution rate of 0.9h⁻¹ and pH 4.3. Compared to the conventional acetone-butanol-ethanol fermentation, the new fermentation process greatly improved butanol yield, making butanol production from corn an attractive alternative to ethanol fermentation.     

Butanol production by Clostridium beijerinckii BA101 in an immobilized cell biofilm reactor

Acetone butanol ethanol was produced in a continuous immobilized cell (biofilm) plug-flow reactor inoculated with Clostridium beijerinckii BA101. To achieve high reactor productivity, C. beijerinckii BA101 cells were immobilized by adsorption onto clay brick. The continuous plug-flow reactor offers high productivities owing to reduced butanol inhibition and increased cell concentration. Although high productivity was achieved, it was at the expense of low sugar utilization (30.3%). To increase sugar utilization, the reactor effluent was recycled. However, this approach is complicated by butanol toxicity. The effluent was recycled after removal of butanol by pervaporation to reduce butanol toxicity in the reactor. Recycling of butanolfree effluent resulted in a sugar utilization of 100.7% in addition to high productivity of 10.2g/(L·h) at a dilution rate of 1.5 h⁻¹. A dilution rate of 2.0h⁻¹ resulted in a reactor productivity of 16.2g/(L·h) and sugar utilization of 101.4%. It is anticipated that this reactor-recovery system would be economical for butanol production when using C. beijerinckii BA101.     

Continuous production of butanol with immobilized cells ofClostridium acetobutylicum

Spores ofClostridium acetobutylicum were immobilized in calcium alginate. An active gel preparation was obtained after outgrowth of the spores to vegetative cells within the gel matrix. A 100 mL column containing the immobilized cells was used for continuous production. At steady-state conditions the productivity of butanol was 67 g/L reactor volume/day.     

Butanol production using Clostridium beijerinckii BA101 hyper-butanol producing mutant strain and recovery by pervaporation

Clostridium beijerinckii BA101 (mutant strain) and C. beijerinckii 8052 (wild type) were compared for substrate and butanol inhibition. The wild-type strain is more strongly inhibited by added butanol than is the mutant strain. Acetone and butanol were removed from a fed-batch reactor inoculated with C. beijcrinckii BA101 by pervaporation using a silicone membrane. In the batch reactor, C. beijerinckii BA101 produced 25.3 g/L of total solvents, whereas in the fermentation-recovery experiment it produced 165.1 g/L of total solvents. Solvent productivity increased from 0.35 (batch reactor) to 0.98 g/L·h (fed-batch reactor). The fed-batch reactor wasfed with 500 g/L of glucose-based P2 medium. Acetone selectivities ranged from 2 to 10 whereas butanol selectivities ranged from 7 to 19. Total flux varied from 26 to 31 g/m²·h.     

Continuous fermentation for the production of citric acid from glucose

Citric acid is finding new areas of use each year and the demand for the acid is constantly increasing. Being a bulk chemical, the continuous production of citric acid would be advantageous. The paper presents the results from ammonia limited batch and continuous fermentations using the yeast strainSaccharomycopsis (Candida) lipolytica (NRRL Y-7576). Mathematical models were developed for growth and glucose utilization in batch and continuous culture. Cell and acid yields appeared to be almost the same in batch and continuous culture. The specific production rates were found to be constant, equal to 0.053 g/g h, in the batch fermentations but varied in the continuous experiments from 0 to 0.11 g/g h depending on the fermentation conditions. Continuous production in a single stage CSTR was studied for over 1,000 hours without shutdown.     

Continuous production of mixed alcohols and acids from carbon monoxide

Continuous, steady-state fermentations using carbon monoxide gas as the sole carbon and energy source have been achieved with the CO strain ofButyribacterium methylotrophicum. Fermentation pH was found to regulate carbon monoxide metabolism over the pH range of 6.8 to 5.0. Cell growth diminished at low pH, with washout occurring at pH 5.0. As observed previously in batch culture, lower pH values favored production of butyrate over acetate. The mechanism responsible for this trend is currently being investigated by quantification of key intracellular enzyme activities. At low pH values, direct, steady-state fermentation of carbon monoxide to alcohols has been verified. Of major significance is the production of butanol from carbon monoxide in pure culture. This newly identified pathway provides a potential mechanism for direct bioconversion of synthesis gas to butanol.     

Continuous potable alcohol production by immobilizedSaccharomyces cerevisiae on mineral kissiris

A biocatalyst prepared by the immobilization of Saccharomyces cerevisiae on the surface of the mineral kissiris was used in the present study for continuous potable-alcohol production. An ethanol productivity (calculated on the basis of liquid volume) of 10.5 g/L/h was obtained at a 0.7/h dilution rate, 121 g/L sucrose content, and 29.6% conversion employing molasse as feed material. Glucose, raisin extracts, and molasse were successively used as feed materials without stopping the operation of the reactor for 6 mo. The ethanol productivity and yield remained constant during the operational-stability study of the reactor, carried out for 44 d. Biomass productivity, yield, and free-cell concentration in glucose, raisin extracts, and molasse were examined. Finally, a system with two continuous reactors joined successively was also studied in the present investigation.     

Continuous production of lactic acid in a cell recycle reactor

The production of lactic acid from glucose has been demonstrated using a CSTR (continuous stirred-tank reactor) with cell recycle. Studies were conducted withLactobacillus delbrueckii at a fermentation temperature of 42°C and a pH of 6.25. A cell density of 140 g dry weight/L and a volumetric productivity of 150 g/L.h, with complete glucose consumption, were obtained. It was not possible to obtain a lactic acid concentration above 60 g/L because of product inhibition. A cell purge was not necessary to maintain high viability bacteria culture or to obtain a steady state. At steady state the net cell growth appeared to be negligible. The specific glucose consumption for cell maintenance was 0.33 g glucose/g cells-h.     

Extraction of butanol isomers bym-cresol

Equilibrium distribution ratios were measured for the four butanol isomers between aqueous solutions and solutions of m-cresol in n-octane at various concentrations at 25°C. At any fixed extractant concentration, the order of the distribution ratios among the butanol isomers, from highest to lowest, is n>i>s>t. A comparison of the activity coefficients of the isomers in the aqueous and organic phases, the latter calculated from the measured distribution data, suggests that the dominant isomeric effects reside in the aqueous phase. The mass-action-law interpretation of the distribution data indicates a one-to-one stoichiometry of the butanol:cresol adduct for all of the isomers. The apparent stability of the adduct, with heterogeneous formation constant between 1 and 3 kJ-mol^–1, decreases with increasing branching of the butanol.deceased     

Continuous xylose fermentation byCandida shehatae in a two-stage reactor

Recent work has identified ethanol toxicity as a major factor preventing continuous production of ethanol at the concentrations obtainable in batch culture. In this paper we investigate the use of a continuous two-stage bioreactor design to circumvent toxic effects of ethanol. Biomass is produced via continuous culture in the first stage reactor in which ethanol concentrations are either absent or maintained at low levels. The freshly grown cells are fed into the second bioreactor in which high ethanol concentrations are produced. The steady influx of fresh cells and continuous removal of spent cells helps minimize the loss of fermentative activity that results from anaerobiosis and exposure to high ethanol concentrations. A final ethanol concentration of 37 g L⁻¹ and overall yield of .32 g g⁻¹ were obtained with the two-stage reactor as compared to corresponding values of 38 g L⁻¹ and .32 g g⁻¹ obtained in batch. The volumetric rate in the two-state process was .96 g L⁻¹ as compared to .46 g L⁻¹ h⁻¹ in batch. Maintained in cooperation with the University of Wisconsin-Madison. The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the US Department of Agriculture of any product or service to the exclusion of others which may be suitable.     

Production of acetone-butanol-ethanol from corn mash and molasses in batch fermentation

The production of solvents from corn mash and molasses in batch fermentation usingClostridium acetobutylicum P 262 was examined. The content of saccharose of beet molasses used in experiments is determined by using the gravimetric method (52.45% saccharose). The quantities of molasses that are used in the nutrient medium are calculated after doing the above determination. The samples of fermentation liquid are taken within a certain time, the determination of saccharose is done by using the same method, and all the saccharose is converted by the microorganism to organic end products. The quantitative and qualitative determination of acetone-butanol has been made by using gas chromatography. On the other hand, using the three isolation way, three different cultures are obtained, and with microscopic observations, the cultures obtained are of the C.acetobutylicum genus. According to the literature values, the concentration of maximum mixed solvent formed during fermentation is about 2%. This is seen in this experiment. There is only a slight difference from this value. This difference is caused by another organic product that is formed during fermentation.     

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10⁻⁴ cm/s.     

Continuous production of extracellular l-glutaminase by ca-alginate-immobilized marine Beauveria bassiana BTMF S-10 in packed-bed reactor

l-Glutamine amidohydrolase (l-glutaminase, EC 3.5.1.2) is a therapeutically and industrially important enzyme. Because it is a potent antileukemic agent and a flavor-enhancing agent used in the food industry, many researchers have focused their attention on l-glutaminase. In this article, we report the continuous production of extracellular l-glutaminase by the marine fungus Beauveria bassiana BTMF S-10 in a packed-bed reactor. Parameters influencing bead production and performance under batch mode were optimized in the order-support (Na-alginate) concentration, concentration of CaCl₂ for bead preparation, curing time of beads, spore inoculum concentration, activation time, initial pH of enzyme production medium, temperature of incubation, and retention time. Parameters optimized under batch mode for l-glutaminase production were incorporated into the continuous production studies. Beads with 12×10⁸ spores/g of beads were activated in a solution of 1% glutamine in seawater for 15 h, and the activated beads were packed into a packed-bed reactor. Enzyme production medium (pH 9.0) was pumped through the bed, and the effluent was collected from the top of the column. The effect of flow rate of the medium, substrate concentration, aeration, and bed height on continuous production of l-glutaminase was studied. Production was monitored for 5 h in each case, and the volumetric productivity was calculated. Under the optimized conditions for continuous production, the reactor gave a volumetric productivity of 4.048 U/(mL·h), which indicates that continuous production of the enzyme by Ca-alginate-immobilized spores is well suited for B. bassiana and results in a higher yield of enzyme within a shorter time. The results indicate the scope of utilizing immobilized B. bassiana for continuous commercial production of l-glutaminase.     

Process improvement in acetone-butanol production from hardwood by simultaneous saccharification and extractive fermentation

The acetone-butanol production by simultaneous saccharification and extractive fermentation (SSEF) was investigated. In the SSEF employing cellulase enzymes andClostridium acetobutylicum, both glucan and xylan fractions of pretreated aspen are concurrently converted into acetone and butanol. Continuous removal of the fermentation products from the bioreactor by extraction was an important factor that allowed long-term fed-batch operation. The use of membrane extraction prevented the problems of phase separation and extractant loss. Increase in substrate feeding as well as reduction of nutrient supply was found to be beneficial in suppressing the acid production, thereby improving the solvent yield. Because of prolonged low growth conditions prevalent in the fed-batch operation, the butanol-to-acetone ratio in the product was significantly higher at 2.6–2.8 compared to the typical value of two.     

Effect of the oxygen transfer coefficient on xylitol production from sugarcane bagasse hydrolysate by continuous stirred-tank reactor fermentation

The effect of the oxygen transfer coefficient on the production of xylitol by biocon version of xylose present in sugarcane bagasse hemicellulosic hydrolysate using the yeast Candiada guilliermondii was investigated. Continuous cultivation was carried out in a 1.25-L fermentor at 30°C, pH 5.5, 300 rpm, and a dilution rate of 0.03/h, using oxygen transfer coefficients of 10,20, and 30/h. The results showed that the microbial xylitol production (11 g/L) increased by 108% with the decrease in the oxygen volumetric transfer coefficient from 30 to 20/h. The maximum values of xylitol productivity (0.7g/[L…h]) and yield (0.58 g/g) were obtained at k _L a 20/h.     

Continuous production of lactic acid from glucose and lactose in a cell-recycle reactor

Growth and lactic acid production ofLactobacillus delbreuckii were compared using glucose and lactose as carbon sources. A continuous-flow stirred-tank fermenter was coupled with a cross-flow filtration unit to permit operation at high-cell concentrations. At steady state, yeast extract requirements for lactic-acid production were lower when glucose was used as a substrate than with lactose fermentation. Once steady state was obtained, with glucose feed, it was possible to lower the yeast extract concentration without affecting biomass concentration and lactic acid production. The lacticacid concentration that inhibited cell growth and lactic acid production was found to depend on the choice of a carbon substrate.     

Ethanol production by yeast cells immobilized in open-pore agar

An open-pore agar matrix has been shown to be suitable for the entrapment of microbial whole cells required for use in reactions that involve cell growth and gas evolution. Beads of porous agar with entrapped yeast cells have been used for the continuous fermentation of sugar cane molasses to ethanol, without apparent bead rupture, even after periods of 3 mo of use. The agar gel does not erode during prolonged operation, unlike porous gelatin cross-linked with glutaraldehyde.     

Ethanol production from corn starch in a fluidized-bed bioreactor

The production of ethanol from industrial dry-milled corn starch was studied in a laboratory-scale fluidized-bed bioreactor using immobilized biocatalysts. Saccharification and fermentation were carried out either simultaneously or separately. Simultaneous saccharification and fermentation (SSF) experiments were performed using small, uniform κ-carrageenan beads (1.5–2.5 mm in diameter) of co-immobilized glucoamylase and Zymomonas mobilis. Dextrin feeds obtained by the hydrolysis of 15% drymilled corn starch were pumped through the bioreactor at residence times of 1.5–4h. Single-pass conversion of dextrins ranged from 54–89%, and ethanol concentrations of 23–36 g/L were obtained at volumetric productivities of 9–15 g/L-h. Very low levels of glucose were observed in the reactor, indicating that saccharification was the rate-limiting step. In separate hydrolysis and fermentation (SHF) experiments, dextrin feed solutions of 150–160 g/L were first pumped through an immobilized-glucoamylase packed column. At 55°C and a residence time of 1 h, greater than 95% conversion was obtained, giving product streams of 162–172 g glucose/L. These streams were then pumped through the fluidized-bed bioreactor containing immobilized Z. mobilis. At a residence time of 2 h, 94% conversion and ethanol concentration of 70 g/L were achieved, resulting in an overall process productivity of 23 g/L-h. Atresidence times of 1.5 and 1 h, conversions of 75 and 76%, ethanol concentrations of 49 and 47 g/L, and overall process productivities of 19 and 25 g/L-h, respectively, were achieved.