Simultaneous saccharification and fermentation of Kanlow switchgrass by thermotolerant Kluyveromyces marxianus IMB3: the effect of enzyme loading, temperature and higher solid loadings

Switchgrass (Panicum virgatum) was subjected to hydrothermolysis pretreatment and then used to study the effect of enzyme loading and temperature in a simultaneous saccharification and fermentation (SSF) with the thermotolerant yeast strain Kluyveromyces marxianus IMB3 at 8% solid loading. Various loadings of Accellerase 1500 between 0.1 and 1.1 mL g(-1) glucan were tested in SSF at 45 °C (activity of enzyme was 82.2 FPU mL(-1)). The optimum enzyme loading was 0.7 mL g(-1) glucan based on the six different enzyme loadings tested. SSFs were performed at 37, 41 and 45 °C with an enzyme loading of 0.7 mL g(-1) glucan. The highest ethanol concentration of 22.5 g L(-1) was obtained after 168 h with SSF at 45 °C, which was equivalent to 86% yield. Four different batch and fed-batch strategies were evaluated using a total solid loading of 12% (dry basis). About 32 g L(-1) ethanol was produced with the four strategies, which was equivalent to 82% yield.     

Productivity of laccase in solid substrate fermentation of selected agro-residues by Pycnoporus sanguineus

A comparative study on solid substrate fermentation (SSF) of sago 'hampas', oil palm frond parenchyma tissue (OPFPt) and rubberwood sawdust with Pycnoporus sanguineus for laccase production was carried out. Optimal mycelial growth of Pyc. sanguineus was observed on all the substrates studied over a 21 days time-course fermentation. Laccase productivity was highest during degradation of sago 'hampas' and OPFPt and a range from 7.5 to 7.6 U/g substrate on the 11th day of fermentation compared to degradation of rubberwood sawdust with a maximum laccase productivity of 5.7 U/g substrate on day 11 of SSF. Further optimization of laccase production was done by varying the inoculum age, density and nitrogen supplementation. SSF of OPFPt by Pyc. sanguineus gave maximum productivity of laccase of 46.5 U/g substrate on day 6 of fermentation with a 30% (w/w) of 4 weeks old inoculum and 0.92% nitrogen in the form of urea supplemented in the substrate. The extraction of laccase was also optimized in this study. Recovery of laccase was fourfold higher at 30.6 U/g substrate on day 10 of SSF using unadjusted tap water at pH 8.0 as extraction medium at 25+/-2 degrees C compared to laccase recovery of 7.46 U/g substrate using sodium acetate buffer at pH 4.8 at 4 degrees C. Further optimization showed that laccase recovery was increased by 50% with a value of 46.5 U/g substrate on day 10 of SSF when the extraction medium was tap water adjusted to pH 5.0 at 25+/-2 degrees C.     

Temperature cycling to improve the ethanol production with solid state simultaneous saccharification and fermentation

Simultaneous saccharification and fermentation (SSF) widely used in submerged state could be effective in solid state. Solid state SSF was first compared with solid state separate hydrolysis and fermentation on ethanol production. Ethanol yield using solid state separate hydrolysis and fermentation (SHF) in 5 days was only half of that in solid state SSF in 3 days. In solid state SSF, the ethanol concentration using temperature cycling (10 h at 37 degrees C followed by 15 min at 42 degrees C) was 2 times that using constant 37 degrees C within 72 h, reached 5.2%.     

Simultaneous saccharification and fermentation of steam-pretreated bagasse using Saccharomyces cerevisiae TMB3400 and Pichia stipitis CBS6054

Sugarcane bagasse--a residue from sugar and ethanol production from sugar cane--is a potential raw material for lignocellulosic ethanol production. This material is high in xylan content. A prerequisite for bioethanol production from bagasse is therefore that xylose is efficiently fermented to ethanol. In the current study, ethanolic fermentation of steam-pretreated sugarcane bagasse was assessed in a simultaneous saccharification and fermentation (SSF) set-up using either Saccharomyces cerevisiae TMB3400, a recombinant xylose utilizing yeast strain, or Pichia stipitis CBS6054, a naturally xylose utilizing yeast strain. Commercial cellulolytic enzymes were used and the content of water insoluble solids (WIS) was 5% or 7.5%. S. cerevisiae TMB3400 consumed all glucose and large fraction of the xylose in SSF. Almost complete xylose conversion could be achieved at 5% WIS and 32 degrees C. Fermentation did not occur with P. stipitis CBS6054 at pH 5.0. However, at pH 6.0, complete glucose conversion and high xylose conversion (>70%) was obtained. Microaeration was required for P. stipitis CBS6054. This was not necessary for S. cerevisiae TMB3400.     

Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus

Kluyveromyces marxianus DMKU 3-1042, isolated by an enrichment technique in a sugar cane juice medium supplemented with 4% (w/v) ethanol at 35 degrees C, produced high concentrations of ethanol at both 40 and 45 degrees C. Ethanol production by this strain in shaking flask cultivation in sugar cane juice media at 37 degrees C was highest in a medium containing 22% total sugars, 0.05% (NH(4))(2)SO(4), 0.05% KH(2)PO(4), and 0.15% MgSO(4).7H(2)O and having a pH of 5.0; the ethanol concentration reached 8.7% (w/v), productivity 1.45 g/l/h and yield 77.5% of theoretical yield. At 40 degrees C, a maximal ethanol concentration of 6.78% (w/v), a productivity of 1.13 and a yield 60.4% of theoretical yield were obtained from the same medium, except that the pH was adjusted to 5.5. In a study on ethanol production in a 5l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.2 vvm throughout the fermentation, K. marxianus DMKU 3-1042 yielded a final ethanol concentration of 6.43% (w/v), a productivity of 1.3g/l/h and a yield of 57.1% of theoretical yield.     

Recombinant Production of an Inulinase in a Saccharomyces cerevisiae gal80 Strain

The inulinase gene (INU1) from Kluyveromyces marxianus NCYC2887 strain was overexpressed by using GAL10 promotor in a △gal80 strain of Saccharomyces cerevisiae. The inulinase gene lacking the original signal sequence was fused in-frame to mating factor alpha signal sequence for secretory expression. Use of the △gal80 strain allowed the galactose-free induction of inulinase expression using a glucose-only medium. Shake flask cultivation in YPD medium produced 34.6 U/ml of the recombinant inulinase, which was approximately 13-fold higher than that produced by K. marxianus NCYC2887. It was found that the use of the △gal80 strain improved the expression of inulinase in the recombinant S. cerevisiae in both the aerobic and the anaerobic condition by about 2.9- and 1.7-fold, respectively. 5 L fed-batch fermentation using YPD medium was performed under aerobic condition with glucose feeding, which resulted in the inulinase production of 31.7 U/ml at OD600 of 67. Ethanol fermentation of dried powder of Jerusalem artichoke, an inulin-rich biomass, was also performed using the recombinant S. cerevisiae expressing INU1 and K. marxianus NCYC2887. Fermentation in a 5L scale fermentor was carried out at an aeration rate of 0.2 vvm, an agitation rate of 300 rpm, and the pH was controlled at 5.0. The temperature was maintained at 30degrees C and 37degrees C, respectively, for the recombinant S. cerevisiae and K. marxianus. The maximum productivities of ethanol were 59.0 and 53.5 g/L, respectively.     

Temperature cycling to improve the ethanol production with solid state simultaneous saccharification and fermentation

Simultaneous saccharification and fermentation (SSF) widely used in submerged state could be effective in solid state. Solid state SSF was first compared with solid-state separate hydrolysis and fermentation on ethanol production. Ethanol yield using solid-state separate hydrolysis and fermentation (SHF) in 5 days was only half of that in solid state SSF in 3 days. In solid state SSF, the ethanol concentration using temperature cycling (10 h at 37°C followed by 15 min at 42°C) reached 5.2% which was 2 times higher than that observed at 37°C within 72 h. The text was submitted by the authors in English.     

Continuous ethanol production by thermotolerant Kluyveromyces marxianus IMB3 immobilized on mineral Kissiris at 45°C

Thermotolerant Kluyveromyces marxianus var. marxianus IMB3 yeast strain was immobilized on Kissiris (mineral glass foam derived from lava) in column packed reactors, and used for ethanol production from glucose or molasses under continuous culture conditions at temperatures between 40 and 50°C. Both ethanol yield and fermentation efficiency were highest at 45°C and a dilution rate (D) of 0.15/h. Increasing sugar concentration led to an increase in ethanol yield of up to 68.6 and 55.9 g/l on approx. 200g glucose or molasses, respectively. Optimum fermentation efficiency (experimental yields over theoretical maximum yields) however was at about 15% sugar for both glucose and molasses. Slight aeration (25 ml of air/min) through the medium addition line was found advantageous due to its mixing effect and probable maintenance of activity.     

Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1

A newly isolated strain of Kluyveromyces marxianus YS-1 was used for the production of extra cellular inulinase in a medium containing inulin, meat extract, CaCl2 and sodium dodecyl sulphate (SDS). Fermentation medium pH 6.5, cultivation temperature 30 degrees C and 5% (v/v) inoculum of 12 h-old culture were optimal for enzyme production (30.8 IU/ml) with a fermentation time of 72 h at shake flask level. Raw inulin (2%, w/v) extracted from dahlia tubers by processing at 15 kg/cm2 for 10 min was optimum for bioreactor studies. Maximum enzyme production (55.4 IU/ml) was obtained at an agitation rate of 200 rpm and aeration of 0.75 vvm in a stirred tank reactor with a fermentation time of 60 h.     

黑曲霉固态发酵及酶解玉米皮

以玉米提取淀粉后的玉米皮渣为主要原料,采用黑曲霉固态发酵法产酶再酶解的二步法降解玉米皮中纤维素类物质。经Plackett-Burman法及响应面设计优化发酵条件得:温度30℃,接种量10%,初始水分体积分数60%,物料厚度2.47 cm,初始pH 5.79,发酵时间6 d;滤纸比酶活可达11.01 U/g,较原始酶活提高了40.61%;产酶结束后加入pH 4.8醋酸-醋酸钠缓冲液,置于50℃下酶解144 h,中性洗涤纤维与酸性洗涤纤维降解率分别为46.09%、48.82%,还原糖质量分数达到9.02%。     

Enzymatic properties of immobilized Alcaligenes faecalis cells with cell-associated beta-glucosidase activity

Enzymatic properties of Alcaligenes faecalis cells immobilized in polyacrylamide were characterized and compared with those reported for the extracted enzyme, and with those measured for free cells. Many of the properties reflected those of the extracted enzyme rather than those measured in the free whole cells prior to immobilization, suggesting cell disruption during immobilization. These properties included the pH activity profile, a slightly broader pH stability profile, and the activation energy. Electron micrographs showed evidence of cell debris among the polymer matrix. The immobilized cells were not viable, and did not consume glucose. Thermal stability was less after immobilization with a half-life of 16 h at 45 degrees C, and 3.5 h at 50 degrees C. The immobilized preparation was more stable when stored lyophilized rather than in buffer, losing 23 and 52% activity, respectively, after six months. The enzyme was irreversibly inhibited by both acetate and citrate buffers. If the immobilized enzyme is to be used in conjunction with cellulases from Trichoderma reesei for cellulase saccharification, the optimal conditions would be pH 5.5 and 45 degrees C in a buffer containing no carboxylic acid groups.     

Designing simultaneous saccharification and fermentation for improved xylose conversion by a recombinant strain of Saccharomyces cerevisiae

Wheat straw is an abundant agricultural residue which can be used as a raw material for bioethanol production. Due to the high xylan content in wheat straw, fermentation of both xylose and glucose is crucial to meet desired overall yields of ethanol. In the present work a recombinant xylose fermenting strain of Saccharomyces cerevisiae, TMB3400, cultivated aerobically on wheat straw hydrolysate, was used in simultaneous saccharification and fermentation (SSF) of steam pretreated wheat straw. The influence of fermentation strategy and temperature was studied in relation to xylose consumption, ethanol formation and by-product formation. In addition, model SSF experiments were made to further investigate the influence of temperature on xylose fermentation and by-product formation. In particular for SSF at the highest value of fibre content tested (9% water insoluble substance, WIS), it was found that a fed-batch strategy was clearly superior to the batch process in terms of ethanol yield, where the fed-batch gave 71% of the theoretical yield (based on all available sugars) in comparison to merely 59% for the batch. Higher ethanol yields, close to 80%, were obtained at a WIS-content of 7%. Xylose fermentation significantly contributed to the overall ethanol yields. The choice of temperature in the range 30-37 degrees C was found to be important, especially at higher contents of water insoluble solids (WIS). The optimum temperature was found to be 34 degrees C for the raw material and yeast strain studied. Model SSF experiments with defined medium showed strong temperature effects on the xylose uptake rate and xylitol yield.     

Hydrogen production from sewage sludge using mixed microflora inoculum: effect of pH and enzymatic pretreatment

Hydrogen was successfully produced by fermenting primary sewage sludge which had been both heat treated and digested with a commercially available enzyme preparation. When either heat treatment or enzymatic digestion were not used, no hydrogen was produced during fermentation. Heat treated mesophilic anaerobic sludge was used as an inoculum rather than a pure microbial culture. Fermentation was conducted at pH levels ranging from of 4.5 to 7.0. When fermentation took place at pH 5.5 a peak hydrogen production rate of 3.75 ml min(-1) was observed. At this pH the hydrogen yield was 0.37 mol H(2)mol(-1) carbohydrate, equivalent to 18.14L H(2)kg(-1) dry solids.     

Solid state fermentation of Achras zapota lignocellulose by Phanerochaete chrysosporium

The biological transformation of lignocellulose of Achras zapota by white rot fungi, Phanerochaete chrysosporium, in solid state fermentation (SSF) was studied for 28 days. The kinetic transformation of lignocellulose was monitored through the determination of acid soluble and acid insoluble lignin content, total organic carbon (TOC) and chemical oxygen demand (COD). The lignolytic enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP) were quantified on weekly intervals. The degradation of lignin and other structural moieties of A. zapota lignocellulose were confirmed by high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The degradation of lignin was increased after 7 days of fermentation with the release of water soluble and fermentable products. The LiP and MnP activities were increased in the first week of SSF and lignin degradation was also set to increase. This was accompanied with increase in COD by 94.6% and TOC by 80% and lignin content was decreased by 76%. The maximum activities of the enzymes LiP and MnP in extracellular fluid of SSF under nitrogen limitation, at pH 5.0, at temperature 37 degrees C and at 60% humidity were 2100 U/L and 1200 U/L.     

High alcohol production by solid substrate fermentation from starchy substrates using thermotolerant Saccharomyces cerevisiae

Solid Substrate Fermentation system (SSF) was used to produce ethanol from various starchy substrates like sweet sorghum, sweet potato, wheat flour, rice starch, soluble starch and potato starch using thermotolerant yeast isolate (VS₃) by simultaneous saccharification and fermentation process. Alcohol produced was estimated by gas chromatography after an incubation time of 96 hrs at 37v°C and 42v°C. More ethanol was produced from rice starch and sweet sorghum. The maximum amount of ethanol produced from these substrates using VS₃ was 10 g/100 g and 3.5 g/100 g substrate (rice starch) and 8.2 g and 7.5 g/100 g substrate (sweet sorghum) at 37v°C and 42v°C respectively.     

Sporulation of Penicillium camemberti in submerged batch culture.

Sporulation of Penicillium camemberti was studied in submerged batch fermentation. A defined medium was used with glucose and ammonium as C- and N-sources. Temperature was set to 25 degrees C at pH 5.6. Essential for submerged sporulation was the presence of calcium (14 mM) which was adsorbed to the cell walls in all sporulating strains and inhibited mycelial growth. Acetate led to highly branched conidiophores and was the second main factor for efficient sporulation. The chelating properties of citrate were necessary for keeping calcium and phosphate in solution. Fermentation conditions allowed high spore yields after 96 h (1.6 x 10(8) spores/ml). Cyclopiazonic acid, the mycotoxin common for P. camemberti was produced during fermentation. The levels observed (0.5-4 ppm at 96 h) were strain specific and not related to spore yield.     

Influence of pH regulation and nutrient content on cephalosporin C production in solid-state fermentation by Acremonium chrysogenum C10

Aims: To investigate the effect of pH regulation and nutrient concentration on cephalosporin C (CPC) production in solid‐state fermentation (SSF), using sugarcane bagasse as inert support, impregnated with liquid medium. Methods and Results: Solid‐state fermentation using different initial pH values, buffer and nutrient concentrations were performed. Results revealed pH as a key parameter in CPC SSF, as it hampered the antibiotic production not only above 7·8, but also under 6·4. Using initial pH lower than 6·8 and PB in the solid medium, it was possible to keep pH within the production range, increase the production period (from 1 to 3 days) and hence the CPC yield from 468 to 3200 μg gdm^?1 (g^?1 of dry matter). Conclusion: Parameters that help to keep pH in adequate values for CPC production in SSF, such as initial pH, buffering system and nutrient concentration, can greatly increase the production time and CPC yields in this fermentation technique. Significance and Impact of the Study: This is the first work on CPC production on impregnated support, and the only one revealing pH as a key parameter; it is also shown that high nutrient concentration can improve CPC yields in SSF as long as pH is kept under control.     

Bioconversion of corn straw by coupling ensiling and solid-state fermentation

A two-stage process that combined solid-state fermentation (SSF) and ensiling was used for bioconversion of corn straw, in order to increase nutritional value and palatability for animal feed. SSF of corn straw increased the level of protein from 6.7% to 14.7% and decreased the cellulose by 38.0% and hemicellulose by 21.2%. Cellulase and xylanase were produced during SSF. After SSF, the fermented substrate was directly ensiled by inoculating with lactic acid bacteria (LAB). In situ produced enzymes and bacterial inoculation resulted in a rapid drop in pH, a high level of lactic acid production, partial degradation of cell wall components and generation of reducing sugars (RSs). Efficiency of ensiling at 25 degrees C, 30 degrees C, 35 degrees C, 40 degrees C was evaluated. Temperature influenced the effect of ensiling; the higher the temperature, the shorter the ensiling period. The combined fermentation upgraded the nutritional value, enhanced the efficiency of ensiling and reduced bioprocessing costs.     

Kinetics and mathematical modeling of homoacetic fermentation of lactate by Clostridium formicoaceticum

Fermentation kinetics of Clostridium formicoaceticum grown on lactate at pH 7.0 and 35 degrees C was studied. Acetate was the only fermentation product and its production was growth associated. The growth of this bacterium was insensitive to the lactate concentrations studied, but was inhibited by acetic acid. A Monod-type expression with product inhibition similar to the noncompetitive inhibition of enzyme kinetics was used to model the batch fermentation. An integrated equation was developed and used to help estimating the kinetic parameters in the model. This mathematical model can be used to simulate the homoacetic fermentation of lactate by C. formicoaceticum at pH 7.0 and 35 degrees C.     

Synthesis and characterisation of a new pH-sensitive amphotericin B--poly(ethylene glycol)-b-poly(L-lysine) conjugate

This paper reports on the synthesis, characterisation, and efficiency of a new intravenous conjugate of amphotericin B (AMB). Twelve molecules of AMB were attached to block copolymer poly(ethylene glycol)-b-poly(L-lysine) via pH-sensitive imine linkages. In vitro drug release studies demonstrated the conjugate (M(w)=26,700) to be relatively stable in human plasma and in phosphate buffer (pH 7.4, 37 degrees C). Controlled release of AMB was observed in acidic phosphate buffer (pH 5.5, 37 degrees C) with the half-life of 2 min. The LD(50) value determined in vivo (mouse) is 45 mg/kg.