Acetic acid production from lactose by an anaerobic thermophilic coculture immobilized in a fibrous-bed bioreactor

An anaerobic thermophilic coculture consisting of a heterofermentative bacterium (Clostridium thermolacticum) and a homoacetogen (Moorella thermoautotrophica) was developed for acetic acid production from lactose and milk permeate. The fermentation kinetics with free cells in conventional fermentors and immobilized cells in a recycle batch fibrous-bed bioreactor were studied. The optimal conditions for the cocultured fermentation were found to be 58 degrees C and pH 6.4. In the free-cell fermentation, C. thermolacticum converted lactose to acetate, ethanol, lactate, H(2) and CO(2), and the homoacetogen then converted lactate, H(2), and CO(2) to acetate. The overall acetate yield from lactose ranged from 0.46 to 0.65 g/g lactose fermented, depending on the fermentation conditions. In contrast, no ethanol was produced in the immobilized-cell fermentation, and the overall acetate yield from lactose increased to 0.8-0.96 g/g lactose fermented. The fibrous-bed bioreactor also gave a higher final acetate concentration (up to 25. 5 g/L) and reactor productivity (0.18-0.54 g/L/h) as compared to those from the free-cell fermentation (final acetate concentration, 15 g/L; productivity, 0.06-0.08 g/L/h). The superior performance of the fibrous-bed bioreactor was attributed to the high cell density (20 g/L) immobilized in the fibrous-bed and adaptation of C. thermolacticum cells to tolerate a higher acetate concentration. The effects of yeast extract and trypticase as nutrient supplements on the fermentation were also studied. For the free-cell fermentation, nutrient supplementation was necessary for the bacteria to grow in milk permeate. For the immobilized-cell fermentation, plain milk permeate gave a high acetate yield (0.96 g/g), although the reactor productivity was lower than those with nutrient supplementation. Balanced growth and fermentation activities between the two bacteria in the coculture are important to the quantitative conversion of lactose to acetic acid. Lactate and hydrogen produced by C. thermolacticum must be timely converted to acetic acid by the homoacetogen to avoid inhibition by these metabolites.     

Proteomic analysis of calcium alginate-immobilized Saccharomyces cerevisiae under high-gravity fermentation conditions

Saccharomyces cerevisiae KAY446 cells immobilized in calcium alginate gel, and supplemented with additional amino acids, were successfully used in enhancing ethanol production. This combination succeeded in improving the ethanol yield and reducing the fermentation time. The ethanol yield under these conditions was 0.40 g of ethanol/g of glucose, with a final ethanol concentration of 118 g/L after 72 h. This is compared to yields with immobilized cells alone of 0.35 g of ethanol/g of glucose and freely suspended cells with no amino acid supplementation of 0.30 g of ethanol/g of glucose, under the same VHG conditions. The maximum specific ethanol production rates were 0.98, 0.73, and 0.61 g (g dry weight) (-1) h (-1) for immobilized cells under VHG conditions with and without amino acid supplementation and free cells, respectively. A proteomic analysis showed significant stimulation of many pathways during fermentation under these conditions, including the Ras/cAMP, glycolysis, starch, and sucrose pathways, amino acids biosynthesis, and aminoacyl-tRNA synthetases. The upregulation of ribosomal, heat-shock proteins and proteins involved in cell viability confirmed that protein biosynthesis was accelerated and revealed likely mechanisms for improving cellular viability.     

Feasibility of ethanol production from coffee husks

The objective of this work was to evaluate the feasibility of ethanol production by fermentation of coffee husks by Saccharomyces cerevisiae. Batch fermentation studies were performed employing whole and ground coffee husks, and aqueous extract from ground coffee husks. It was observed that fermentation yield decreased with an increase in yeast concentration. The best results were obtained for the following conditions: whole coffee husks, 3 g yeast/l substrate, temperature of 30°C. Under these conditions ethanol production was 8.49 ± 0.29 g/100 g dry basis (13.6 ± 0.5 g ethanol/l), a satisfactory value in comparison to literature data for other residues such as corn stalks, barley straw and hydrolyzed wheat stillage (5–11 g ethanol/l). Such results indicate that coffee husks present excellent potential for residue-based ethanol production.     

Ethanol production from concentrated whey permeate using a fed-batch culture ofKluyveromyces fragilis

Summary Fed-batch fermentation of non-supplemented concentrated whey permeate resulted in high ethanol productivity for feeds of lactose for which batch fermentation had a poor performance. At an initial lactose concentration of 100 g/L and a constant lactose feeding rate of 18 g/h we have obtained: ethanol concentration 64 g/L, ethanol productivity 3.3 g/Lh, lactose consumption 100%, ethanol yield 0.47 g/g, and biomass yield 0.058 g/g.Nomenclature St total lactose fed per medium volume in the bioreactor, g/L - Si initial lactose concentration, g/L - F lactpse feeding rate, g/h - P final ethanol concentration, g/L - Y_p/s ethanol yield, g ethanol/g lactose - Y_x/s biomass yield, g biomass/g lactose - X_S lactose consumption, % - Qp overall ethanol volumetric productivity, g/Lh - m maximum specific growth rate, h - qsm maximum specific lactose consumption rate, g/gh - qpm maximum specific ethanol production rate, g/gh     

Alumina-doped alginate gel as a cell carrier for ethanol production in a packed-bed bioreactor

Alumina-doped alginate gel (AEC) was developed as a new type of cell carrier to be used in ethanol fermentation. The presence of the alumina particles in alginate gel not only improved the porous structure of the carrier, but also provided many advantageous characteristics including good mechanical strength, high stability, and high immobilization yield. The attachment of alumina particles and yeast cells by electrostatic attraction was shown to promote cell growth and increase ethanol productivity. The AEC carrier was found to be more effective for the immobilization of Saccharomyces cerevisiae M30 than the conventional Ca-alginate bead. Ethanol productivities of 1.4 and 7.9 ∼ 12.6 g/(L/h) were obtained using the AEC cultures in batch and continuous modes of operation, respectively, with an ethanol yield of 43.9 ∼ 46.7% and an immobilized yield of 81.4 ∼ 84.5%. Ethanol fermentation in a continuous packed-bed reactor using the AEC carrier was stable for > 30 days.     

Bacterial cellulose–alginate composite sponge as a yeast cell carrier for ethanol production

A bacterial cellulose–alginate (BCA) sponge, fabricated by a freeze-drying process, was successfully used as a yeast cell carrier for ethanol fermentation. The BCA sponge exhibited several advantageous properties, such as high porosity, appropriate pore size, strong hydrophilicity and high mechanical, chemical and thermal stabilities. BCA has an asymmetric structure, with a thin, dense outer layer covering an interior of interconnected macropores that are distributed throughout the sponge, which is effective for yeast immobilization. At 48 h of the fermentation, the maximum ethanol concentration produced by the immobilized culture (IC) in the BCA carrier was about 100 g/L, which was approximately 13% and 45% higher than that from the suspended culture (SC) and from IC in Ca-alginate matrix, respectively. Repeated-batch ethanol productions using IC in BCA carriers were also more stable than those using SC or IC in Ca-alginate matrix. The results of a 15 cycle repeated batch operation demonstrated that the system with IC in BCA exhibited superior long-term stability for ethanol fermentation with the average ethanol productivity at 1.9 g/L h and the immobilized yield at 86%. The improved ethanol fermentation performance was mainly due to the water uptake ability and properly interconnected pore structure, which help to overcome limiting mass transfer.     

Immobilized <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> cells in carboxymethyl cellulose for production of ethanol from cheese whey: experimental and kinetic studies

Cheese whey fermentation to ethanol using immobilized Kluyveromyces marxianus cells was investigated in batch and continuous operation. In batch fermentation, the yeast cells were immobilized in carboxymethyl cellulose (CMC) polymer and also synthesized graft copolymer of CMC with N-vinyl-2-pyrrolidone, denoted as CMC-g-PVP, and the efficiency of the two developed cell entrapped beads for lactose fermentation to ethanol was examined. The yeast cells immobilized in CMC-g-PVP performed slightly better than CMC with ethanol production yields of 0.52 and 0.49 g ethanol/g lactose, respectively. The effect of supplementation of cheese whey with lactose (42, 70, 100 and 150 g/l) on fermentative performance of K. marxianus immobilized in CMC beads was considered and the results were used for kinetic studies. The first order reaction model was suitable to describe the kinetics of substrate utilization and modified Gompertz model was quite successful to predict the ethanol production. For continuous ethanol fermentation, a packed-bed immobilized cell reactor (ICR) was operated at several hydraulic retention times; HRTs of 11, 15 and 30 h. At the HRT of 30 h, the ethanol production yield using CMC beads was 0.49 g/g which implies that 91.07 % of the theoretical yield was achieved.     

Rapid ethanol fermentation of cellulose hydrolysate. I. Batch versus continuous systems

Rapid fermentation of bagasse hydrolysate to ethanol under anaerobic conditions by a strain of Saccharomyces cerevisiae has been studied in batch and continuous cultures at pH 4.0 and 30°C temperature with cell recycle. By using a 23.6 g/liter cell concentration, a concentation of 9.7% (w/v)ethanol was developed in a period of 6 hr. The rate of fermentation was found to increase with supplementation of yeast vitamins in the hydrolysate. In continuous culture employing cell recycle and a 0.127 v/v/m air flow rate, a cell mass concentration of 48.5 g/liter has been achieved. The maximum fermentor productivity of ethanol obtained under these conditions was 32.0 g/liter/hr, which is nearly 7.5 times higher than the normal continuous process without cell recycle and air sparging. The ethanol productivity was found to decrease linearly with ethanol concentration. Conversion of glucose in the hydrolysate to ethanol was achieved with a yield of 95 to 97% of theoretical.     

Matrices (re)loaded: Durability,viability, and fermentative capacity of yeast encapsulated in beads of different composition during long-term fed-batch culture

Encapsulated microbes have been used for decades to produce commodities ranging from methyl ketone to beer. Encapsulated cells undergo limited replication, which enables them to more efficiently convert substrate to product than planktonic cells and which contributes to their stress resistance. To determine how encapsulated yeast supports long-term, repeated fed-batch ethanologenic fermentation, and whether different matrices influence that process, fermentation and indicators of matrix durability and cell viability were monitored in high-dextrose, fed-batch culture over 7 weeks. At most timepoints, ethanol yield (g/g) in encapsulated cultures exceeded that in planktonic cultures. And frequently, ethanol yield differed among the four matrices tested: sodium alginate crosslinked with Ca²⁺ and chitosan, sodium alginate crosslinked with Ca²⁺, Protanal alginate crosslinked with Ca²⁺ and chitosan, Protanal alginate crosslinked with Ca²⁺, with the last of these consistently demonstrating the highest values. Young's modulus and viscosity were higher for matrices crosslinked with chitosan over the first week; thereafter values for both parameters declined and were indistinguishable among treatments. Encapsulated cells exhibited greater heat shock tolerance at 50°C than planktonic cells in either stationary or exponential phase, with similar thermotolerance observed across all four matrix types. Altogether, these data demonstrate the feasibility of re-using encapsulated yeast to convert dextrose to ethanol over at least 7 weeks.     

Optimization of batch alcoholic fermentation of glucose syrup substrate

The quantitative effects of substrate concentration, yeast concentration, and nutrient supplementation on ethanol content, fermentation time, and ethanol productivity were investigated in a Box–Wilson central composite design experiment, consisting of five levels of each variable, High substrate concentration, up to 30° Brix, resulted in higher ethanol content (i.e., up to 15.7% w/v or 19.6% v/v) but longer fermentation time and hence lower ethanol productivity. Increasing yeast concentration, on the other hand, resulted in shorter fermentation time and higher productivity. The highest ethanol productivity of about 21 g EiOH/L h was obtained at low substrate concentration (i.e., 12° Brix), low alcohol content (i.e., 6% by weight), high yeast concentration (i.e., 4.4%), and high supplementation of yeast extract (i.e., 2.8). Productivity of this magnitude is substantially higher that that of the traditional batch fermentation of fed-batch fermentation. It is comparable to the results of continuous fermentation but lower than those of vacuum fermentation but lower than those of vacuum fermentation. Optimal conditions for maximal ethanol productivity can be established by a multiple regression analysis technique and by plotting the contours of constant response to conform to the constraints of individual operations.     

Bioethanol Production from Horticultural Waste Using Crude Fungal Enzyme Mixtures Produced by Solid State Fermentation

It was desired to study efficient and simplified methods to convert organosolv-pretreated horticultural waste (HW) to ethanol fuel using cellulase produced under solid-state fermentation (SSF). The unprocessed cellulase crude (72.2 %) showed better reducing sugar yield using filter paper than the commercial enzyme blend (68.7 %). Enzymatic hydrolysis of organosolv-pretreated HW using the crude cellulase with 20 % solid content, enzyme loading of 15 FPU/g HW at 50 °C, and pH 5.5 resulted in a HW hydrolysate containing 25.06 g/L glucose after 72 h. Fermentation of the hydrolysate medium produced 12.39 g/L ethanol with 0.49 g/g yield from glucose and 0.062 g/g yield from HW at 8 h using Saccharomyces cerevisiae. This study proved that crude cellulase complex produced under SSF and organosolv pretreatment can efficiently convert woody biomass to ethanol without any commercial cellulase usage.     

Studies on immobilized Saccharomyces cerevisiae. I. Analysis of continuous rapid ethanol fermentation in immobilized cell reactor

Rapid fermentation of cane molasses into ethanol has been studied in batch, continuous (free-cell and cell-immobilized systems) by a strain of Saccharomyces cerevisiae at temperature 30 degrees C and pH 5.0. The maximum productivity of ethanol obtained in immobilized system was 28.6 g L(-1) h(-1). The cells were immobilized by natural mode on a carrier of natural origin and retention of 0.132 g cells/g carrier was achieved. The immobilized-cell column was operated continuously at steady state over a period of 35 days. Based on the parameter data monitored from the system, mathematical analysis has been made and rate equations proposed, and the values of specific productivity of ethanol and specific growth rate for immobilized cells computed. It has been established that immobilized cells exhibit higher specific rate of ethanol formation compared to free cells but the specific growth rate appears to be comparatively low. The yield of ethanol in the immobilized-cell system is also higher than in the free-cell system.     

A comparative study of glucose conversion to ethanol by Zymomonas mobilis and a recombinant Escherichia coli

Summary Zymomonas mobilis and recombinant Escherichia coli B (pLOI297) were compared in side-by-side batch fermentations using a synthetic cellulose hydrolysate (glucose/salts) medium with pH control at 6.0 and an inoculation cell density of 35–50 mg dry wt. cells/L. At a nominal glucose concentration of 6%, both cultures achieved near maximal theoretical ethanol yields; however, the Z. mobilis fermentation was complete at 13h compared to 33h for the E.coli fermentation. With approx.12% glucose, the Z. mobilis fermentation was complete in 20h with a process yield of 0.49 g ethanol/g added glucose compared to the E. coli fermentation which remained 20% incomplete after 6 days resulting in a process yield of only 0.32 g/g. Nutrient supplementation (10g tryptone/L) resulted in complete fermentation of 12% glucose (pH 6.3) by the recombinant E. coli in 4 days, with a yield of 0.48 g/g.     

Strain improvement of thermotolerant Saccharomyces cerevisiae VS strain for better utilization of lignocellulosic substrates

AIM: Pentose-utilizing yeast development by protoplast fusion and sequential mutations and ethanol fermentation using lignocellulosic substrate. METHODS AND RESULTS: Protoplasts of thermotolerant Saccharomyces cerevisiae and mesophilic, xylose-utilizing Candida shehatae were fused by electrofusion. The fusants were selected based on their growth at 42 degrees C and ability to utilize xylose. The selected best fusant was mutated sequentially and 3 mutant fusants obtained were tested for their stability. The mutant fusant CP11 was found to be stable and used for lignocellulosic fermentation. The Prosopis juliflora wood material was hydrolysed with 1% sulphuric acid initially for 18 h at room temperature and then for 20 min at 121 degrees C. The acid hydrolysate was separated and used for detoxification by ethyl acetate and overliming. The hard cellulosic fraction was hydrolysed with Aspergillus niger crude cellulase enzyme for 18 h at 50 degrees C. The substrate (15% w/v) yielded 84 g l(-1) sugars, representing 80% (w/w) hydrolysis of carbohydrate content present in the lignocellulosic material. The acid and enzyme hydrolysates were then equally mixed and used for fermentation with the developed fusant yeast (CP11). The fusant yeast gave an ethanol yield of 0.459 +/- 0.012 g g(-1), productivity of 0.67 +/- 0.015 g l(-1) h(-1) and fermentation efficiency of 90%. CONCLUSIONS: Protoplast fusion followed by sequential mutations method gave a stable and good performing fusant with maximum utilization of reducing sugars in the media. SIGNIFICANCE AND IMPACT OF THE STUDY: This new method could be applied to develop fusants for better biotechnological applications.     

Direct fermentation of fodder maize, chicory fructans and perennial ryegrass to hydrogen using mixed microflora

This study examined the feasibility of producing hydrogen by direct fermentation of fodder maize, chicory fructooligosaccharides and perennial ryegrass (Lolium perenne) in batch culture (pH 5.2-5.3, 35 degrees C, heat-treated anaerobically digested sludge inoculum). Gas was produced from each substrate and contained up to 50-80% hydrogen during the peak periods of gas production with the remainder carbon dioxide. Hydrogen yields obtained were 62.4+/-6.1mL/g dry matter added for fodder maize, 218+/-28mL/g chicory fructooligosaccharides added, 75.6+/-8.8mL H(2)/g dry matter added for wilted perennial ryegrass and 21.8+/-8mL H(2)/g dry matter added for fresh perennial ryegrass. Butyrate, acetate and ethanol were the main soluble fermentation products. Hydrogen yields of 392-501m(3)/hectare of perennial ryegrass per year and 1060-1309m(3)/hectare of fodder maize per year can be obtained based on the UK annual yield per hectare of these crops. These results significantly extend the range of substrates that can be used for hydrogen production without pre-treatment.     

Improved ethanol production of a newly isolated thermotolerant Saccharomyces cerevisiae strain after high-energy-pulse-electron beam

Aims: To isolate thermotolerant Saccharomyces cerevisiae with high‐energy‐pulse‐electron (HEPE) beam, to optimize the mutation strain fermentation conditions for ethanol production and to conduct a preliminary investigation into the thermotolerant mechanisms. Methods and Results: After HEPE beam radiation, the thermotolerant S. cerevisiae strain Y43 was obtained at 45°C. Moreover, the fermentation conditions of mutant Y43 were optimized by L3³ orthogonal experiment. The optimal glucose content and initial pH for fermentation were 20% g l^?1 and 4·5, respectively; peptone content was the most neglected important factor. Under this condition, ethanol production of Y43 was 83·1 g l^?1 after fermentation for 48 h at 43°C, and ethanol yield was 0·42 g g^?1, which was about 81·5% of the theoretical yield. The results also showed that the trehalose content and the expression of the genes MSN2, SSA3 and TPS1 in Y43 were higher than those in the original strain (YE0) under the same stress conditions. Conclusions: A genetically stable mutant strain with high ethanol yield under heat stress was obtained using HEPE. This mutant may be a suitable candidate for the industrial‐scale ethanol production. Significance and Impact of the Study: High‐energy‐pulse‐electron radiation is a new efficient technology in breeding micro‐organisms. The mutant obtained in this work has the advantages in industrial ethanol production under thermostress.     

Cellulosic ethanol production on temperature-shift simultaneous saccharification and fermentation using the thermostable yeast Kluyveromyces marxianus CHY1612

In cellulosic ethanol production, use of simultaneous saccharification and fermentation (SSF) has been suggested as the favorable strategy to reduce process costs. Although SSF has many advantages, a significant discrepancy still exists between the appropriate temperature for saccharification (45-50 °C) and fermentation (30-35 °C). In the present study, the potential of temperature-shift as a tool for SSF optimization for bioethanol production from cellulosic biomass was examined. Cellulosic ethanol production of the temperature-shift SSF (TS-SSF) from 16 w/v% biomass increased from 22.2 g/L to 34.3 g/L following a temperature shift from 45 to 35 °C compared with the constant temperature of 45 °C. The glucose conversion yield and ethanol production yield in the TS-SSF were 89.3% and 90.6%, respectively. At higher biomass loading (18 w/v%), ethanol production increased to 40.2 g/L with temperature-shift time within 24 h. These results demonstrated that the temperature-shift process enhances the saccharification ratio and the ethanol production yield in SSF, and the temperature-shift time for TS-SSF process can be changed according to the fermentation condition within 24 h.     

Effect of substrate concentration on ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary A cellulose hydrolysate from Aspen wood, containing mainly glucose, was fermented into ethanol by a thermotolerant strain MSN77 of Zymomonas mobilis. The effect of the hydrolysate concentration on fermentation parameters was investigated. Growth parameters (specific growth rate and biomass yield) were inhibited at high hydrolysate concentrations. Catabolic parameters (specific glucose uptake rate, specific ethanol productivity and ethanol yield) were not affected. These effects could be explained by the increase in medium osmolality. The results are similar to those described for molasses based media. Strain MSN77 could efficiently ferment glucose from Aspen wood up to a concentration of 60 g/l. At higher concentration, growth was inhibited.Nomenclature S glucose concentration (g/l) - X biomass concentration (g/l) - P ethanol concentration (g/l) - C conversion of glucose (%) - t fermentation time (h) - q_S specific glucose uptake rate (g/g.h) - q_p specific ethanol productivity (g/g.h) - YINX/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

AIMS: To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. METHODS AND RESULTS: The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP(+)-dependent activity in both strains with mutated XDH, reaching 0.782 and 0.698 U mg(-1). In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. CONCLUSIONS: Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important.     

Ethanol production from cotton-based waste textiles

Ethanol production from cotton linter and waste of blue jeans textiles was investigated. In the best case, alkali pretreatment followed by enzymatic hydrolysis resulted in almost complete conversion of the cotton and jeans to glucose, which was then fermented by Saccharomyces cerevisiae to ethanol. If no pretreatment applied, hydrolyses of the textiles by cellulase and beta-glucosidase for 24 h followed by simultaneous saccharification and fermentation (SSF) in 4 days, resulted in 0.140-0.145 g ethanol/g textiles, which was 25-26% of the corresponding theoretical yield. A pretreatment with concentrated phosphoric acid prior to the hydrolysis improved ethanol production from the textiles up to 66% of the theoretical yield. However, the best results obtained from alkali pretreatment of the materials by NaOH. The alkaline pretreatment of cotton fibers were carried out with 0-20% NaOH at 0 degrees C, 23 degrees C and 100 degrees C, followed by enzymatic hydrolysis up to 4 days. In general, higher concentration of NaOH resulted in a better yield of the hydrolysis, whereas temperature had a reverse effect and better results were obtained at lower temperature. The best conditions for the alkali pretreatment of the cotton were obtained in this study at 12% NaOH and 0 degrees C and 3 h. In this condition, the materials with 3% solid content were enzymatically hydrolyzed at 85.1% of the theoretical yield in 24 h and 99.1% in 4 days. The alkali pretreatment of the waste textiles at these conditions and subsequent SSF resulted in 0.48 g ethanol/g pretreated textiles used.