Improved Ethanol and Reduced Xylitol Production from Glucose and Xylose Mixtures by the Mutant Strain of <Emphasis Type="Italic">Candida shehatae</Emphasis> ATCC 22984

A mutant Cs3512, which showed better fermentation of xylose and the mixtures of xylose and glucose, was obtained through mutation of Candida shehatae ATCC 22984 and screening with a medium containing antimycin A and TTC (2,3,5-triphenyltetrazolium chloride). Cs3512 produced 44.4 g/l of ethanol from 121.3 g/l of xylose, which was 13% higher than that by ATCC 22984. At the same time, xylitol production was reduced by 38% to 10.2 g/l from 16.3 g/l by ATCC 22984. Cs3512 also showed 8% increase in ethanol yield from 0.39 to 0.42 g/g comparing to ATCC 22984 when fermenting the sugar mixture composed of 52.9 g/l glucose and 21.2 g/l xylose. When Cs3512 was used in the simultaneous saccharification and fermentation of lime pretreated rice straw via CaCCO (calcium capturing by carbonation) process, it produced ethanol at 77% of the theoretical yield. The results imply that Cs3512 is a potential non-recombinant yeast strain for ethanol production from lignocellulosic biomass.     

Continuous Ethanol Production from Cassava Through Simultaneous Saccharification and Fermentation by Self-Flocculating Yeast Saccharomyces Cerevisiae CHFY0321

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h⁻¹. Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h⁻¹. At dilution rates in the range of 0.014 h⁻¹ to 0.042 h⁻¹, high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h⁻¹, the ethanol volumetric productivity and ethanol yield were 3.58 g/L∙h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.     

Constitutive Expression of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Lipase LIP2 in <Emphasis Type="Italic">Pichia pastoris</Emphasis> Using GAP as Promoter

A gene encoding Yarrowia lipolytica lipase LIP2 (YlLIP2) was cloned into a constitutive expression vector pGAPZαA and electrotransformed into the Pichia pastoris X-33 strain. The high-yield clones obtained by high copy and enzyme activity screening were chosen as the host strains for shaking flask and fermentor culture. The results showed that glucose was the optimum carbon source for YlLIP2 production, and the maximum hydrolytic activity of recombinant YlLIP2 reached 1,315 U/ml under the flask culture at 28 °C, pH 7.0, for 48 h. The fed-batch fermentation was carried out in 3- and 10-l bioreactors by continuously feeding glucose into the growing medium for achieving high cell density and YlLIP2 yields. The maximum hydrolytic activity of YlLIP2 and cell density obtained in the 3-l bioreactor were 10,300 U/ml and 116 g dry cell weight (DCW)/l, respectively. The peak hydrolytic activity of YlLIP2 and cell density were further improved in the 10-l fermentor where the values respectively attained were 13,500 U/ml and 120 g DCW/l. The total protein concentration in the supernatant reached 3.3 g/l and the cell viability remained approximately 99% after 80 h of culture. Furthermore, the recombinant YlLIP2 produced in P. pastoris pGAP and pAOX1 systems have similar content of sugar (about 12%) and biochemical characteristics. The above results suggest that the GAP promoter-derived expression system of P. pastoris is effective for the expression of YlLIP2 by high cell density culture and is probably an alternative to the conventional AOX1 promoter expression system in large-scale production of industrial lipases.     

Fermentation Characteristics of <Emphasis Type="Italic">Mortierella alpina</Emphasis> in Response to Different Nitrogen Sources

The fermentation characteristics of Mortierella alpina were investigated in response to various nitrogen sources. Influences on nitrogen source and glucose uptake rate, mycelial morphology of M. alpina, and pH of medium in relation to different nitrogen sources were discussed. Effects of different nitrogen sources on cell growth, fatty acid composition, arachidonic acid (ARA), and total lipid concentration were also evaluated. It revealed that the maximum nitrogen source uptake ratio was obtained when corn steep liquor was used as nitrogen source. When yeast extract was used as the sole nitrogen source, glucose was completely exhausted at the end of fermentation. The maximum dry cell weight obtained from medium with yeast extract as nitrogen source had the highest total lipid concentration. Sodium nitrate was the favorable nitrogen source for ARA accumulation, and the highest ARA percentage in total fatty acids was obtained, 35.9%. Urea was identified as the favorable nitrogen source for ARA production, the highest ARA concentration obtained from urea was 5.8 g/l. Compared with inorganic nitrogen sources, organic nitrogen compounds are favorable for both cell growth and total lipids accumulation.     

Production and Characterization of Cellulose by <Emphasis Type="Italic">Acetobacter</Emphasis> sp. V6 Using a Cost-Effective Molasses–Corn Steep Liquor Medium

In order to reduce of the manufacturing cost of bacterial cellulose (BC), BC production by Acetobacter sp. V6 was investigated in shaking culture using molasses and corn steep liquor (CSL) as the sole carbon and nitrogen sources, respectively. The highest BC production was obtained with Ca₃(PO₄)₂-treated molasses. Maximum BC yield (2.21 ± 0.04 g/l) was obtained at 5% (w/v) total sugar in molasses. In improved medium containing molasses and CSL, BC production was observed in the medium after 1 day of incubation and increased rapidly thereafter with maximum yield (3.12 ± 0.03 g/l) at 8 days. This value was approximately twofold higher than the yield in the complex medium. Physical properties of BC from the complex and molasses media were studied using Fourier-transform infrared (FT-IR) spectroscopy and X-ray diffractometer. By FT-IR, all the BC were found to be of cellulose type І, the same as typical native cellulose. The relative crystallinity of BC produced in the complex and molasses media were 83.02 and 67.27%, respectively. These results suggest that molasses and CSL can be useful low-cost substrates for BC production by Acetobacter sp. V6 without supplementation with expensive nitrogen complexes such as yeast extract and polypeptone, leading to the reduction in the production costs.     

Ethanol Production from H<Subscript>2</Subscript>SO<Subscript>3</Subscript>-Steam-Pretreated Fresh Sweet Sorghum Stem by Simultaneous Saccharification and Fermentation

The present work presents an alternative approach to ethanol production from sweet sorghum: without detoxification, acid-impregnated fresh sweet sorghum stem which contains soluble (glucose and sucrose) and insoluble carbohydrates (cellulose and hemicellulose) was steam pretreated under mild temperature of 100 °C. Simultaneous saccharification and fermentation experiments were performed on the pretreated slurries using Saccharomyces cerevisiae. Experimentally, ground fresh sweet sorghum stem was combined with H₂SO₃ at dosages of 0.25, 0.50, and 0.75 g/g dry matter (DM) and steam pretreated by varying the residence time (60, 120, or 240 min). According to enzymatic hydrolysis results and ethanol yields, H₂SO₃ was a powerful and mild acid for improving enzymatic digestibility of sorghum stem. At a solid loading of 10% (w/v) and acid dosage of 0.25 g/g DM H₂SO₃ at 100 °C for 120 min, 44.5 g/L ethanol was obtained after 48 ± 4 h of simultaneous saccharification and fermentation. This corresponded to an overall ethanol yield of 110% of the theoretical one, based on the soluble carbohydrates in the fresh sweet sorghum stem. The concentrations of hydroxymethylfurfural and furfural of the sulfurous acid pretreated samples were below 0.4 g/L. Ethanol would not inhibit the cellulase activity, at least under the concentration of 34 g/L.     

Characterization of Cellulolytic Extract from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> PF-2 and Its Application in Biomass Saccharification

The aim of this work was to evaluate the biochemical features of the white-rot fungi Pycnoporus sanguineus cellulolytic complex and its utilization to sugarcane bagasse hydrolysis. When cultivated under submerged fermentation using corn cobs as carbon source, P. sanguineus produced high FPase, endoglucanase, β-glucosidase, xylanase, mannanase, α-galactosidase, α-arabinofuranosidase, and polygalacturonase activities. Cellulase activities were characterized in relation to pH and temperature. β-Glucosidase and FPase activities were higher at 55 °C, pH 4.5, and endoglucanase activity was higher at 60 °C, in a pH range of 3.5–4.0. All cellulase activities were highly stable at 40 and 50 °C through 48 h of pre-incubation. Crude enzymatic extract from P. sanguineus was applied in a saccharification experiment using acid-treated and alkali-treated sugarcane bagasse as substrate, and the hydrolysis yields were compared to that obtained by a commercial cellulase preparation. Reducing sugar yields of 60.4% and 64.0% were reached when alkali-treated bagasse was hydrolyzed by P. sanguineus extract and commercial cellulase, respectively. Considering the glucose production, it was observed that P. sanguineus extract and commercial cellulase ensured yields of 22.6% and 36.5%, respectively. The saccharification of acid-treated bagasse was lower than that of alkali-treated bagasse regardless of the cellulolytic extract. The present work showed that P. sanguineus has a great potential as an enzyme producer for biomass saccharification.     

Effect of Concentration and Substrate Flow Rate on Isomaltulose Production from Sucrose by <Emphasis Type="Italic">Erwinia</Emphasis> sp. Cells Immobilized in Calcium-Alginate Using Packed Bed Reactor

Isomaltulose was obtained from sucrose solution by immobilized cells of Erwinia sp. D12 using a batch and a continuous process. Parameters for sucrose conversion into isomaltulose were evaluated using both experimental design and response surface methodology. Erwinia sp. D12 cells were immobilized in different alginates, and the influence of substrate flow rate and concentration parameters to produce isomaltulose from sucrose were observed. Response surface methodology demonstrated that packed bed columns containing cells immobilized in low-viscosity sodium alginate (250 cP) presented a mean isomaltulose conversion rate of 47%. In a continuous process, both sucrose substrate concentration and substrate flow rate parameters had a significant effect (p < 0.05) and influenced the conversion of sucrose into isomaltulose. Higher conversion rates of sucrose into isomaltulose, from 53–75% were obtained using 75 g of immobilized cells at a substrate flow rate of 0.6 mL/min.     

Water Hyacinth as Carbon Source for the Production of Cellulase by <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Water hyacinth (Eichhornia crassipes), an aquatic weed common to the subtropic/tropical regions, was utilized as an inexpensive lignocellulosic substrate for production of cellulase by Trichoderma reesei. The effects of process parameters like substrate pretreatment, substrate concentration, initial medium pH, mode of inoculation, and incubation temperature on cellulase production were investigated. Under optimal conditions, a maximal cellulase activity of 0.22 ± 0.04 IU/ml (approximately 73.3 IU/g cellulose) was recorded at the end of 15-day incubation period. Specific activity of the enzyme was 6.25 IU/mg protein. Hydrolysis of 1% substrate (water hyacinth) using crude enzyme dosage of 1.2 IU/g water hyacinth showed 28.7% saccharification in 1 h. The observations in present study indicate that saccharification of cellulose from water hyacinth was significantly higher by laboratory-produced cellulase than the commercial blend.     

Statistical Optimization for Succinic Acid Production from E. Coli in a Cost-Effective Medium

Response surface methodology (RSM) was employed for optimization of medium components and cultural parameters in cost effective cane molasses based medium for attaining high yield of succinic acid. The important factors obtained by “one-variable-at-a-time-approach” (cane molasses, corn steep liquor, sodium carbonate, and inoculum density) were further optimized by RSM. The optimum values of the parameters obtained through RSM (cane molasses 12.5%, corn steep liquor 7.5%, and sodium carbonate 25 mM) led to almost double yield of succinic acid (15.2 g/l in 36 h) as against “one-variable-at-a-time-approach” (7.1 g/l in 36 h) in 500-ml anaerobic bottles containing 300-ml cane molasses based medium. Subsequently, in 10-l bioreactor succinic acid production from Escherichia coli was further improved to 26.2 g/l in 30 h under conditions optimized through RSM. This fermentation-derived succinic acid will definitely help in replacing existing environmentally hazardous and cost-intensive chemical methods for the production of succinic acid.     

Kinetic Study on the Pretreatment and Enzymatic Saccharification of Rice Hull for the Production of Fermentable Sugars

The production of fermentable sugars from rice hull was studied by dilute acid pretreatment and enzymatic saccharification. Rice hull (15%, w/v) was pretreated by 1% (v/v) sulfuric acid at high temperature (120∼160 °C) for 15, 30, 45, and 60 min, respectively. The maximum sugar concentration from rice hull in the prehydrolysate was obtained at 140 °C for 30 min, but the enzymatic saccharification yield from the corresponding pretreated rice hull is not high. To another aspect, the maximum enzymatic saccharification yield was achieved at 160 °C for 60 min, while the recovery of fermentable sugars was the poorest. To take account of fermentable sugars from pretreatment and enzymatic saccharification, the maximum yield of sugars was obtained only when rice hull was treated at 140 °C for 30 min. Under this condition, 72.5% (w/w) of all sugars generated from the raw material can be recovered. The kinetic study on the enzymatic saccharification of dilute acid pretreated rice hull was also performed in this work by a modified Michaelis–Menten model and a diffusion-limited model. After calculation by a linear and a non-linear regression analysis, both models showed good relation with the experimental results.     

Continuous Production of Oxytetracycline in Fluidized Bed Bioreactor by Immobilized Cells of Streptomyces varsoviensis MTCC-1537: Effect of Dilution and Glucose Loading Rates

Oxytetracycline (OT) production using glutaraldehyde cross-linked calcium alginate immobilized cells of Streptomyces varsoviensis in continuous fluidized bed reactor (FBR) was investigated. Initially, batch experiments were carried in stirred tank reactor (STR) and FBR using calcium alginate immobilized cells. Higher OT production of 0.45 gm/L was achieved by FBR when compared with 0.33 g/L of OT in STR. All subsequent studies were carried out in continuous mode of operation in FBR. During 21 days of operation, effect of glucose concentration and different dilution rates were studied. A maximum of 0.75 g/L OT was achieved in the medium having 10 g/L of glucose concentration. The highest OT concentration of 0.92 g/L and the highest yield of OT with respect to biomass at 0.1713 g/g were obtained at the dilution rate of 0.25 day⁻¹.     

Ethanol production in immobilized-cell bioreactors from mixed sugar syrups and enzymatic hydrolysates of steam-exploded biomass

We investigated ethanol production from mixed sugar syrups. Hydrolysates were prepared from enzymatic saccharification of steam-pretreated aspen chips. Syrups containing 45 g/L of glucose and 12 g/L of xylose were detoxified through two ion-exchange resins and then fermented with Pichia stipitis and Saccharomyces cerevisiae immobilized in Ca-alginate gel beads. Combinations of different gel fractions in the fermentation volume, amount of yeast cells, and ratios of P. stipitis vs S. cerevisiae within each bead were compared. In the best conditions, by using a total beads volume corresponding to 25% of the working volume, we obtained a yield of 0.39 g_ethanol/g_{initial sugars}. This amount of gel entrapped an initial cell concentration of 6×10¹²cells/L with ratio of S. cerevisiae/P. stipitis of 0.25 g/g. Modified stirredtank reactors were obtained either by adding marbles or by inserting a perforated metal cylinder, which reduced considerably the rupture of beads while visibly improving oxygenation of the medium.     

Microbial Production of 1,3-Propanediol by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> XJPD-Li under Different Aeration Strategies

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae XJPD-Li under different aeration strategies were investigated. In batch fermentation, the results showed that the final concentration of 1,3-PD and yield on glycerol were 13.44 g/l and 0.73 mol/mol under the anaerobic condition (N₂, 0.4 vvm), 11.55 g/l and 0.62 mol/mol without aeration, and 8.73 g/l and 0.47 mol/mol under the aerobic condition (air, 0.4 vvm), respectively. Under the aerobic condition, the yield of 1,3-PD on glycerol was the lowest, while the biomass (optical density at 650 nm) was the highest among these three conditions. In the fed-batch culture, the final concentration and the yield of 1,3-PD was 60.82 g/l and 0.61 mol/mol under the anaerobic condition (N₂, 0.4 vvm), 56.43 g/l and 0.53 mol/mol without aeration, and 65.26 g/l and 0.56 mol/mol under the aerobic condition. All these three conditions had good productivities of 1,3-PD, which were 3.35 g/l·h under the anaerobic condition (N₂, 0.4 vvm), 3.13 g/l·h without aeration, and 3.16 g/l·h under the aerobic condition within the initial 12 h.     

Pretreatment of Corn Stover Silage with Fe(NO<Subscript>3</Subscript>)<Subscript>3</Subscript> for Fermentable Sugar Production

Corn stover silage is an attractive raw material for the production of biofuels and chemicals due to its high content of carbohydrates and easy degradability. The effects of Fe(NO₃)₃ pretreatment conditions on sugar yields were investigated for corn stover silage. In addition, a combined severity factor was used to evaluate the effect of pretreatment conditions on the concentration of total sugars and inhibitors. Optimum pretreatment condition was obtained at 150 °C for 10 min with 0.05 M Fe(NO₃)₃, at which the yields of soluble xylose and glucose in liquid achieved 91.80% of initial xylose, 96.74% of initial arabinose and 19.09% of initial glucose, respectively, meanwhile, 91.84% of initial xylose, 98.24% of initial arabinose, and 19.91% of initial glucose were removed. In addition, a severity analysis showed that the maximum sugar concentration of 33.48 g/l was achieved at combined severity parameter value of 0.62, while the inhibitor concentration was only 0.03 g/l. Fe(NO₃)₃ is an effective catalyst to enhance hemicellulose hydrolysis in corn stover silage, the yields of monomeric xylose in the liquid fraction reached as high as 91.06% of initial xylose and 96.22% of initial arabinose, respectively.     

Ethanol fermentation in a tower fermenter using self-aggregatingSaccharomyces uvarum

A self-aggregating strain ofSaccharomyces uvarum (U4) was used as a biocatalyst to carry out continuous ethanol fermentation in a tower fermentor equipped with a cell separator. Cell aggregates (2–3 mm) formed a stable packed bed in the fermentor, and the cell separator retained yeast cells effectively. Corn steep liquor was used as a nitrogen source for the fermentation of corn syrup and black strap molasses. An ethanol productivity of 54 g/L/h was reached using corn syrup at a dilution rate of 0.7/h, and sugar concentration in the feed was 15% (w/v). For molasses fermentation, an ethanol productivity of 22 g/L/h was obtained at a dilution rate of 0.7/h, and sugar concentration in the feed was 12.5% (w/v). Ethanol yields obtained from tower fermentation are higher than those obtained from flask fermentation (96% for corn syrup fermentation and 92% for molasses fermentation). No significant loss in fermentation activity was observed after 3 mo of operation.     

Ethanol production from jerusalem artichoke by inulinase and zymomonas mobilis

Ethanol production from Jerusalem artichoke was studied using inulinase and Z.mobilis by simultaneous saccharification and fermentation (SSF) process. The SSF process showed higher ethanol yield and productivity than the acid or enzymatic prehydrolyzed two-step process. The optimum temperature and inulinase concentration for SSF were 35°C and 0.25% (v/w, 4.4 units/g of sugar), respectively. In order to operate the SSF process in a continuous mode, inulinase and Z.mobilis cells were coimmobilized in alginate beads, using chitin as a matrix for enzyme immobilization. The maximum ethanol productivity of the continuous SSF process was 55.1 g/L/h, with 55% conversion yield. At the conversion yield of 90%, the productivity was 32.7 g/L/h. The continuous SSF system could be operated stably over 2 wk with an ethanol concentration of 48.6 g/L (95% of theoretical yield).     

Oil Palm Empty Fruit Bunch as Alternative Substrate for Acetone–Butanol–Ethanol Production by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> EB6

Acetone–butanol–ethanol (ABE) production from renewable resources has been widely reported. In this study, Clostridium butyricum EB6 was employed for ABE fermentation using fermentable sugar derived from treated oil palm empty fruit bunch (OPEFB). A higher amount of ABE (2.61 g/l) was produced in a fermentation using treated OPEFB as the substrate when compared to a glucose based medium that produced 0.24 g/l at pH 5.5. ABE production was increased to 3.47 g/l with a yield of 0.24 g/g at pH 6.0. The fermentation using limited nitrogen concentration of 3 g/l improved the ABE yield by 64%. The study showed that OPEFB has the potential to be applied for renewable ABE production by C. butyricum EB6.     

Feasibility of Hydrothermal Pretreatment on Maize Silage for Bioethanol Production

The potential of maize silage as a feedstock to produce bioethanol was evaluated in the present study. The hydrothermal pretreatment with five different pretreatment severity factors (PSF) was employed to pretreat the maize silage and compared in terms of sugar recovery, toxic test, and ethanol production by prehydrolysis and simultaneous saccharification and fermentation. After pretreatment, most of the cellulose remained in the residue, ranging between 85.87% by the highest PSF (185°C, 15 min) and 92.90% obtained at the lowest PSF (185°C, 3 min). A larger part of starch, varying from 71.64% by the highest PSF to 78.28% by the lowest, was liberated into liquor part, leaving 8.05–11.74% in the residues. Xylan recovery in the residues increased from 44.25% at the highest PSF to 82.95% at the lowest. The recovery of xylan in liquor changed from 20.13% to 50.33%. Toxic test indicated that all the liquors from the five conditions were not toxic to the Baker’s yeast. Pretreatment under 195°C for 7 min had the similar PSF with that of 185°C for 15 min, and both gave the higher ethanol concentration of 19.92 and 19.98 g/L, respectively. The ethanol concentration from untreated maize silage was only 7.67 g/L.     

Cultivation of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> in the Stirred Bioreactor with Different Feeding Strategies for CoQ<Subscript>10</Subscript> Production

The logistic growth model combined with the Luedeking-Piret equation was adopted in this study to model the batch production of CoQ₁₀ in the cultivation of Rhodobacter sphaeroides. The simulation results indicated that CoQ₁₀ production was a primary metabolite. As being a primary metabolite, a longer cell growing stage would tend to accumulate more biomass and lead to a higher CoQ₁₀ concentration being produced. In this context, a fed-batch operation by molasses feeding was performed to increase the biomass and subsequent CoQ₁₀ production. Three different molasses feeding strategies were operated in this study. Results suggested that the fed-batch operation with molasses controlled at 10 ± 1 g/l could increase the cell mass and CoQ₁₀ concentration to reach their maximum values of 18.6 g/l and 83.8 mg/l, respectively, nearly 2.2 times and 1.9 times their respective values obtained in the batch cultivation.