Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses

Due to the environmental concerns and the increasing price of oil, bioethanol was already produced in large amount in Brazil and China from sugarcane juice and molasses. In order to make this process competitive, we have investigated the suitability of immobilized Saccharomyces cerevisiae strain AS2.1190 on sugarcane pieces for production of ethanol. Electron microscopy clearly showed that cell immobilization resulted in firm adsorption of the yeast cells within subsurface cavities, capillary flow through the vessels of the vascular bundle structure, and attachment of the yeast to the surface of the sugarcane pieces. Repeated batch fermentations using sugarcane supported-biocatalyst were successfully carried out for at least ten times without any significant loss in ethanol production from sugarcane juice and molasses. The number of cells attached to the support increased during the fermentation process, and fewer yeast cells leaked into fermentation broth. Ethanol concentrations (about 89.73–77.13 g/l in average value), and ethanol productivities (about 59.53–62.79 g/l d in average value) were high and stable, and residual sugar concentrations were low in all fermentations (0.34–3.60 g/l) with conversions ranging from 97.67–99.80%, showing efficiency (90.11–94.28%) and operational stability of the biocatalyst for ethanol fermentation. The results of this study concerning the use of sugarcane as yeast supports could be promising for industrial fermentations. L. Liang and Y. Zhang have contributed equally to this work.     

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS₃) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L⁻¹ at 30°C. The maximum amount of ethanol produced by immobilized VS₃ using 150 g L⁻¹ glucose was only 44 g L⁻¹ after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L⁻¹. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L⁻¹ to 25 g L⁻¹ after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L⁻¹. The maximum amount of ethanol produced by immobilized VS₃ cells using 150, 200 and 250 g glucose L⁻¹ was 72.5, 93 and 87 g ethanol L⁻¹ at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226. Received 16 September 1999/ Accepted in revised form 22 December 1999     

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae

Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase approximately 10-fold. The performance of the two reactors was compared and a respective rate model was proposed. The present research has shown that high sugar concentration (150 g/l) in the ICR column was successfully converted to ethanol. The achieved results in ICR with high substrate concentration are promising for scale up operation. The proposed model can be used to design a lager scale ICR column for production of high ethanol concentration.     

Production of pullulanase by free and immobilized cells of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NRC22 in batch and continuous cultures

The production of extracellular pullulanase by Bacillus licheniformis NRC22 was investigated using different fermentation modes. In batch culture maximal enzyme activity of 18 U/ml was obtained after 24 h of growth. In continuous fermentation by the free cells, maximal reactor productivity (4.15 KU/l/h) with enzyme concentration of 14.8 U/ml and specific productivity of 334.9 U/g wet cells/h was attained at a dilution rate of 0.28/h, over a period of 25 days. B. licheniformis NRC22 cells were immobilized on Ca-alginate. The immobilization conditions with respect to matrix concentration and cell load was optimized for maximal enzyme production. In repeated batch operation, the activity of the immobilized cells was stable during the 10 cycles and the activity remained between 9.8 and 7.7 U/ml. Continuous production of pullulanase by the immobilized cells was investigated in a packed–bed reactor. Maximal reactor productivity (7.0 KU/h) with enzyme concentration of 16.8 U/ml and specific productivity of 131.64 U/g wet cells/h was attained at dilution rate of 0.42/h. The enzyme activity in the effluent started to decline gradually to the level of 8.7 U/ml after 25 days of the operation.     

Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain FBR5

Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H₂SO₄) pretreated (160 °C, 10 min) and enzymatically saccharified (pH 5.0, 45 °C, 72 h) WS (86 g/l) was 50.0 ± 1.4 g/l. The hydrolyzate contained 1,184 ± 19 mg furfural and 161 ± 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 °C. However, it produced 21.9 ± 0.3 g ethanol from non-abated WSH (total sugars, 44.1 ± 0.4 g/l) in 90 h including the lag time of 24 h at controlled pH 7.0 and 35 °C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 °C for 15 h. The bacterium produced 21.6 ± 0.5 g ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 ± 0.4 g) at pH 6.0. It produced 24.9 ± 0.3 g ethanol in 96 h and 26.7 ± 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF, respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement and increase in ethanol yield.     

Production of 2,3-butanediol by newly isolated Enterobacter cloacae

Enterobacter cloacae NRRL B-23289 was isolated from local decaying wood/corn soil samples while screening for microorganisms for conversion of l-arabinose to fuel ethanol. The major product of fermentation by the bacterium was meso-2,3-butanediol (2,3-BD). In a typical fermentation, a BD yield of 0.4 g/g arabinose was obtained with a corresponding productivity of 0.63 g/l per hour at an initial arabinose concentration of 50 g/l. The effects of initial arabinose concentration, temperature, pH, agitation, various monosaccharides, and multiple sugar mixtures on 2,3-BD production were investigated. BD productivity, yield, and byproduct formation were influenced significantly within these parameters. The bacterium utilized sugars from acid plus enzyme saccharified corn fiber and produced BD (0.35 g/g available sugars). It also produced BD from dilute acid pretreated corn fiber by simultaneous saccharification and fermentation (0.34 g/g theoretical sugars). Received: 17 December 1998 / Revision received: 9 March 1999 / Accepted: 20 March 1999     

Effects of ethanol concentration and stripping temperature on continuous fermentation rate

The operation of a pilot plant consisting of a 14-l fermentor, 10-cm packed column and condenser for continuous fermentation and stripping of ethanol was stable for more than 100 days. The feed consisted of a non-sterile solution of 560 g/l glucose with 100 g/l corn steep water. Fouling of the packing in the column with attached growth of yeast cells was controlled by in situ washing at intervals of 3–6 days. A computer simulation of the pilot plant was developed and used to analyze the data. The productivity of the continuous fermentor varied from 14 g ethanol to 17 g ethanol l⁻¹ h⁻¹. The yield was equal to the maximum theoretically possible: 0.51 g ethanol/g glucose consumed. Results are fit to linear models for the effects of ethanol concentration on specific growth rate and cell yield, and for the effect of stripping temperature on specific growth rate. Received: 16 October 1996 / Received revision: 3 January 1997 / Accepted: 24 January 1997     

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.     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

Ethanol production from starch by immobilized Aspergillus awamori and Saccharomyces pastorianus using cellulose carriers

A simultaneous saccharification and fermentation (SSF) process was investigated to produce ethanol using two kinds of cellulose carriers that were respectively suitable for immobilization of Aspergillus awamori and Saccharomyces pastorianus. The maximum ethanol concentration attained by the batch operation was 25.5 g l⁻¹. Under suitable conditions, both cellulose carriers with immobilized cells could be reused efficiently for three cycles. The total amount of ethanol production was 66.0 g (per 1 l working volume) after the repeated operation. Ethanol productivity mainly depends on a saccharification process. There is a limit in durability in the repeated batch operation, and it is important to maintain high activity of the fungus in order to produce ethanol efficiently. Journal of Industrial Microbiology & Biotechnology (2001) 27, 52–57. Received 11 December 2000/ Accepted in revised form 02 June 2001     

Comparative study of d-xylose conversion to ethanol by immobilized growing or non-growing cells of the yeast Pachysolen tannophilus

Summary The direct conversion of d-xylose to ethanol was investigated using immobilized growing and non-growing cells of the yeast Pachysolen tannophilus. Both preparations produced ethanol from d-xylose, however the d-xylose conversion to ethanol was much better with immobilized growing cells. Ethanol concentration up to 22.9 g/l and ethanol yield of 0.351 g/g of d-xylose were obtained in batch fermentation by immobilized growing cells whereas only 17.0 g/l and 0.308 g/g of d-xylose were obtained by immobilized non-growing cells. With continuous systems, immobilized growing cells were necessary for the long-term operation, since a steady state ethanol concentration of 17.7 g/l was maintained for only one week by immobilized non-growing cell reactor. With simultaneous control of aeration rate and concentrations of nitrogen sources in feed medium, immobilized growing cells of P. tannophilus showed excellent performance. At a residence time of 25 h, the immobilized cell reactor produced 26.9 g/l of ethanol from 65 g/l of d-xylose in feed medium.     

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.     

Improved bioethanol production through simultaneous saccharification and fermentation of lignocellulosic agricultural wastes by <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> 6556

The combined effect of simultaneous saccharification and fermentation and separate hydrolysis and fermentation (SHF) for ethanol production by Kluyveromyces marxianus 6556 was studied using two lignocellulosic feedstocks viz., corncob and soybean cake. The ethanologenic efficiency of K. marxianus 6556 was observed as 28% (theoretical yield) in a fermentation medium containing glucose, but, there was no ethanol production by cells grown on xylose. A maximum sugar release of 888 mg/g corncob and 552 mg/g soybean cake was achieved through acid hydrolysis pretreatment. Furthermore, corncob and soybean cake treated with commercial cellulase (100 IU for 48 h) from Trichoderma reesei yielded reducing sugars of 205 and 100 mg/g, respectively. Simultaneous saccharification and fermentation resulted in highest ethanol production of 5.68 g/l on corncob and 2.14 g/l on soybean cake after 48 h of incubation. On the contrary, the presence of inhibitors decreased the overall ethanol yield in the hydrolysates obtained through SHF of corncob and soybean cake.     

Repeated batch fermentation from raw starch using a maltose transporter and amylase expressing diploid yeast strain

We successfully demonstrated batch ethanol fermentation repeated ten times from raw starch with high ethanol productivity. We constructed a yeast diploid strain coexpressing the maltose transporter AGT1, α-amylase, and glucoamylase. The introduction of AGT1 allows maltose and maltotriose fermentation as well as the improvement of amylase activities. We also found that α-amylase activity during fermentation was retained by the addition of 10 mM calcium ion and that the highest α-amylase activity was 9.26 U/ml during repeated fermentation. The highest ethanol productivity was 2.22 g/l/h at the fourth batch, and after ten cycles, ethanol productivity of more than 1.43 g/l/h was retained, as was α-amylase activity at 6.43 U/ml.     

Optimization of temperature,sugar concentration,and inoculum size to maximize ethanol production without significant decrease in yeast cell viability

Aiming to obtain rapid fermentations with high ethanol yields and a retention of high final viabilities (responses), a 2³ full-factorial central composite design combined with response surface methodology was employed using inoculum size, sucrose concentration, and temperature as independent variables. From this statistical treatment, two well-fitted regression equations having coefficients significant at the 5% level were obtained to predict the viability and ethanol production responses. Three-dimensional response surfaces showed that increasing temperatures had greater negative effects on viability than on ethanol production. Increasing sucrose concentrations improved both ethanol production and viability. The interactions between the inoculum size and the sucrose concentrations had no significant effect on viability. Thus, the lowering of the process temperature is recommended in order to minimize cell mortality and maintain high levels of ethanol production when the temperature is on the increase in the industrial reactor. Optimized conditions (200 g/l initial sucrose, 40 g/l of dry cell mass, 30 °C) were experimentally confirmed and the optimal responses are 80.8 ± 2.0 g/l of maximal ethanol plus a viability retention of 99.0 ± 3.0% for a 4-h fermentation period. During consecutive fermentations with cell reuse, the yeast cell viability has to be kept at a high level in order to prevent the collapse of the process.     

Critical factors affecting ethanol production by immobilized Pichia stipitis using corn cob hemicellulosic hydrolysate

Fermentation of xylose from hydrolysate of acid-treated corn cob by Pichia stipitis is inhibited by acetic acid and lignin derivatives. In the present study, we have designed and implemented an immobilized cell culture for xylose to ethanol conversion from acid-treated corn cob hydrolysate without the removal of fermentation inhibitors. In this study, cultivations of suspended and immobilized Pichia were compared in terms of ethanol yield and productivity to investigate whether the cell immobilization could improve resistance to inhibitors. Cell immobilization clearly favored the fermentative metabolism in nondetoxified corn cob hydrolysate leading to an improvement of twofold ethanol productivity as compared to that achieved with suspension culture. Calcium alginate as an immobilization matrix was selected to immobilize Pichia cells. Concentrations of sodium alginate, calcium chloride, and fermentor agitation speed were optimized for ethanol production using statistical method. Statistical analysis showed that agitation speed had maximum influence on ethanol production by immobilized Pichia cells. In comparison to suspension culture, immobilization had a positive impact on the fermentative metabolism of Pichia, improving the ethanol yield from 0.40 to 0.43?g/g and productivity from 0.31 to 0.51?g/L/h for acid-treated corn cob hydrolysate.     

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.     

Candida shehatae and Saccharomyces cerevisiae work synergistically to improve ethanol fermentation from sugarcane bagasse and rice straw hydrolysate in immobilized cell bioreactor

Sugarcane bagasse (SCB) and rice straw (RS), abundant lignocellulosic agro‐industrial residues in South‐East Asia, are potent feedstocks for bioethanol production as they contain significant amount of glucose and xylose monomers after fractionation and subsequent enzymatic hydrolysis. To simultaneously convert glucose and xylose to ethanol, it requires co‐cultivation of Saccharomyces cerevisiae and Candida shehatae which are hexose and pentose‐fermenting yeasts, respectively. Xylose‐fermenting strain grows slower than glucose‐fermenting one, therefore low efficiency of xylose‐to‐ethanol conversion was found. To enhance the efficiency of ethanol fermentation, the present work proposed to improve xylose assimilation by using co‐immobilization of two strains in a packed bed bioreactor and to increase oxygenation of the medium by applying a recycled batch system when the recycle stream was intervened by a mixing system in a naturally aerated vessel. Initially, conversion of glucose and xylose to ethanol using pure culture was investigated. Subsequently, influence of different immobilization techniques was investigated. Cells entrapment in Ca‐alginate beads provided considerably high ethanol yield over cells immobilized on delignified cellulose, and thus it was selected to use as inoculum in an immobilized cell bioreactor (ICB). The results showed that continuous ethanol production yielded 0.38 and 0.40 g/g corresponding to 74.5% and 78.4% theoretical yields from SCB and RS hydrolysate, respectively. However, recycled batch system produced significantly improved ethanol yield to 0.49 g/g and 0.50 g/g corresponding to 96.1% and 98.0% theoretical yields for SCB and RS hydrolysate, respectively. In this study, higher ethanol concentration and less unfermented sugar concentration was successfully achieved in the ICB with recycled batch system when using SCB and RS hydrolysate as the substrate.     

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.     

Short communication: Ethanol production from cellulose at 45°C using a batch-fed system containing alginate-immobilized Kluyveromyces marxianus IMB3

The thermotolerant yeast, Kluyveromyces marxianus IMB3, was grown in batch culture at 45°C on cellulose-containing media, supplemented with exogenous cellulase activity. At various stages during fermentation, both substrate and enzyme were added in batch mode and fermentation was continued for 220 h. Ethanol production increased to 20 g/l at 200 h, representing 45% of the maximum theoretical yield. In subsequent experiments, the organism was immobilized in calcium alginate beads and these were used in a similar, batch-fed system at 45°C. Again, fermentation was continued for 220 h and ethanol production increased to its maximum, of 28 g/l, within 100 h and this represented in excess of 60% of the maximum theoretical yield.