Impacts of main factors on bioethanol fermentation from stalk juice of sweet sorghum by immobilized Saccharomyces cerevisiae (CICC 1308)

In order to attain a higher ethanol yield and faster ethanol fermentation rate, orthogonal experiments of ethanol fermentation with immobilized yeast from stalk juice of sweet sorghum were carried out in the shaking flasks to investigate the effect of main factors, namely, fermentation temperature, agitation rate, particles stuffing rate and pH on ethanol yield and CO(2) weight loss rate. The range analysis and analysis of variance (ANOVA) were applied for the results of orthogonal experiments. Results showed that the optimal condition for bioethanol fermentation should be A(4)B(3)C(3)D(4), namely, fermentation temperature, agitation rate, particles stuffing rate and pH were 37 degrees C, 200rpm, 25% and 5.0, respectively. The verification experiments were carried out in shaking flasks and 5L bioreactor at the corresponding parameters. The results of verification experiments in the shaking flasks showed that ethanol yield and CO(2) weight loss rate were 98.07% and 1.020gh(-1), respectively. The results of ethanol fermentation in the 5L bioreactor showed that ethanol yield and fermentation time were 93.24% and 11h, respectively. As a result, it could be concluded that the determined optimal condition A(4)B(3)C(3)D(4) was suitable and reasonable for the ethanol fermentation by immobilized Saccharomyces cerevisiae. The conclusion in the research would be beneficial for application of ethanol fermentation by immobilized S. cerevisiae from stalk juice of sweet sorghum.     

Comparison of different pretreatments in ethanol fermentation using corn cob hemicellulosic hydrolysate with Pichia stipitis and Candida shehatae

A hemicellulosic hydrolysate was prepared with 0.3 M H₂SO₄ at 98 °C for 1 h. The total initial reducing sugar was maintained at 45 g l^–1 by synthetic xylose supplementation. The seven detoxification methods were employed including either the single addition of solid CaO (to pH 10 or 6) or its combinations with zeolite shaking. Over-liming gave the hydrolysate that was most completely fermented by Pichia stipitis and Candida shehatae at 30 °C, pH 6, among the tested methods.     

Xylitol production from corncob hydrolysate using polyurethane foam with immobilized Candida tropicalis

Polyurethane foam (PUF) was used as a carrier for Candida tropicalis (C. tropicalis) in the multi-batches fermentation of xylitol from xylose-containing corncob hemicellulose hydrolysate. After washing and sterilization, PUF (density of 320kgm(-3), specific surface area of 1.5-2.0×10(5)m(2)m(-3), average porosity of 95%, pore diameter of 0.03mm and cubic length of 5mm) was mixed with the culture medium at appropriate proportion followed by the inoculation. The fermentation parameters such as initial cell concentration, PUF dosage, pH value and temperature were controlled to study the effects on xylitol fermentation. In the 21-day durability tests, the optimal xylitol yield and volumetric productivity reached to 71.2% and 2.10gL(-1)h(-1) respectively. Moreover, the average xylitol yield and volumetric productivity were 66.3% and 1.90gL(-1)h(-1) for ten batchwise operations. The current research demonstrated that the PUF immobilization could serve as an efficient method for improving the cells vitality and enzyme reactivity in the continuous operation of fermentation.     

Coupling two sizes of CSTR-type bioreactors for sequential lactic acid and xylitol production from hemicellulosic hydrolysates of vineshoot trimmings

This study develops a system for the efficient valorisation of hemicellulosic hydrolysates of vineshoot trimmings. By connecting two reactors of 2L and 10L, operational conditions were set up for the sequential production of lactic acid and xylitol in continuous fermentation, considering the dependence of the main metabolites and fermentation parameters on the dilution rate. In the first bioreactor, Lactobacillus rhamnosus consumed all the glucose to produce lactic acid at 31.5°C, with 150rpm and 1L of working volume as the optimal conditions. The residual sugars were employed for the xylose to xylitol bioconversion by Debaryomyces hansenii in the second bioreactor at 30°C, 250rpm and an air-flow rate of 2Lmin(-1). Several steady states were reached at flow rates (F) in the range of 0.54-5.33mLmin(-1), leading to dilution rates (D) ranging from 0.032 to 0.320h(-1) in Bioreactor 1 and from 0.006 to 0.064h(-1) in Bioreactor 2. The maximum volumetric lactic acid productivity (Q(P LA)=2.908gL(-1)h(-1)) was achieved under D=0.266h(-1) (F=4.44mLmin(-1)); meanwhile, the maximum production of xylitol (5.1gL(-1)), volumetric xylitol productivity (Q(P xylitol)=0.218gL(-1)h(-1)), volumetric rate of xylose consumption (Q(S xylose)=0.398gL(-1)h(-1)) and product yield (0.55gg(-1)) were achieved at an intermediate dilution rate of 0.043h(-1) (F=3.55mLmin(-1)). Under these conditions, ethanol, which was the main by-product of the fermentation, was produced in higher amounts (1.9gL(-1)). Finally, lactic acid and xylitol were effectively recovered by conventional procedures.     

Toxin production by Clostridium botulinum type A under various fermentation conditions.

The time of appearance and the quantity of toxin produced by the Hall strain of Clostridium botulinum type A were examined under various conditions. A 70-liter fermentor and a complex medium consisting of 2% casein hydrolysate and 1% yeast extract plus an appropriate concentration of glucose were employed. Optimal conditions for toxin production were as follows: a nitrogen overlay at a rate of 5 liters/min, an agitation rate of 50 rpm, a temperature of 35 degrees C, and an initial glucose concentration of 1.0% with the pH uncontrolled. Under these conditions, the maximum toxin concentration (6.3 x 10(5) mouse median lethal doses/ml) was attained within 24 h. Cell lysis was apparently not required to obtain maximum toxin concentrations under the fermentation conditions described.     

Toxin production by Clostridium botulinum type A under various fermentation conditions.

The time of appearance and the quantity of toxin produced by the Hall strain of Clostridium botulinum type A were examined under various conditions. A 70-liter fermentor and a complex medium consisting of 2% casein hydrolysate and 1% yeast extract plus an appropriate concentration of glucose were employed. Optimal conditions for toxin production were as follows: a nitrogen overlay at a rate of 5 liters/min, an agitation rate of 50 rpm, a temperature of 35 degrees C, and an initial glucose concentration of 1.0% with the pH uncontrolled. Under these conditions, the maximum toxin concentration (6.3 x 10(5) mouse median lethal doses/ml) was attained within 24 h. Cell lysis was apparently not required to obtain maximum toxin concentrations under the fermentation conditions described.     

Succinic acid production from Bacteroides fragilis: process optimization and scale up in a bioreactor

We report the effect of different physiological and nutritional parameters on succinic acid production from Bacteroides fragilis. This strain initially produced 0.70gL(-1) of succinic acid in 60h. However, when process optimization was employed, 5.4gL(-1) of succinic acid was produced in medium consisting of glucose (1.5%); tryptone (2.5%); Na(2)CO(3) (1.5%), at pH 7.0, when inoculated with 4% inoculum and incubated at 37 degrees C, 100rpm for 48h. A marked enhancement in succinic acid production was observed when the optimized conditions were employed in a 10L bioreactor. A total of 12.5gL(-1) of succinic acid was produced in 30h. This is approximately 12-fold increase in succinic acid production when compared to the initial un-optimized medium production. This enhancement in succinic acid production may be due to the control of CO(2) supply and the impeller speed. This is also resulted in the reduction of the production time. The present study provides useful information to the industrialists seeking environmentally benign technology for the production of bulk biomolecules through manipulation of various chemical parameters.     

Continuous butanol fermentation and feed starch retrogradation: butanol fermentation sustainability using Clostridium beijerinckii BA101

Use of starch solution as feed for butanol bioconversion processes employing Clostridium beijerinckii BA101 may have added economic advantage over the use of glucose. Acetone butanol ethanol (ABE) was produced from 30 gL(-1) starch solution using a continuous process. The bioreactor was fed at a dilution rate of 0.02 h(-1) and starch solution/feed volume (3 L) was replaced every 72 h. The continuous reactor fed with cornstarch solution (feed temperature 19 degrees C) produced approximately 6.0 gL(-1) total ABE. Increasing the feed storage temperature to 37 degrees C improved ABE production to 7.2 gL(-1) suggesting that retrogradation was occurring more rapidly at 19 degrees C. In both these cases the fermentation drifted toward acid production after approximately 260 h, consistent with the retrogradation of starch overtime. The use of soluble starch, which is less prone to retrogradation, resulted in the production of 9.9 gL(-1) ABE at 37 degrees C feed storage temperature, as compared to 7.2 gL(-1) ABE when cornstarch was used. It should be noted that gelatinized starch retrogradation takes place after sterilization and prior to use of the feed medium, and does not occur during long-term storage of the raw corn material in the months leading up to processing. The degree of hydrolysis of gelatinized starch decreased from 68.8 to 56.2% in 3 days when stored at 37 degrees C. Soluble starch which does not retrograde demonstrated no change in the degree of hydrolysis.     

Influence of pH and temperature on soluble substrate generation with primary sludge fermentation

The study was undertaken to evaluate the effect of pH and temperature control on the generation of soluble fermentation products from primary sludge. The effect was tested by running parallel experiments under pH and temperature controlled and uncontrolled conditions. In fermentation experiments conducted at 20 degrees C without pH control, the average soluble COD release was 14 mg per liter of wastewater treated, representing a potential increase of 5% in the biodegradable COD content of the primary sedimentation effluent. The corresponding average VFA generation was 9.2mg COD l(-1). The nutrient release was practically negligible and stayed at 0.4 mg l(-1) for nitrogen and 0.1mg l(-1) for phosphorus. Acetic acid accounted more than 45% of the generated VFA in all experimental runs. The acetic acid content of the VFA decreased with increasing initial VSS concentrations and higher pH levels. VFA generation by fermentation was significantly affected with temperature and pH control. Temperature change between 10 and 24 degrees C induced a five-fold increase in VFA generation, from 610 mg l(-1) at 10 degrees C to 2950 mg l(-1) at 24 degrees C.     

Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis

The optimization of submerged culture conditions for mycelial growth and exopolysaccharide (EPS) production in an edible mushroom Tremella fuciformis was studied in shake flasks and bioreactors. The temperature of 28 degrees C and pH 8 in the beginning of fermentation in agitated flasks was the most efficient condition to obtain maximum mycelial biomass and EPS. The optimal medium constituents were as follows (gL(-1)): glucose 20, tryptone 2, KH(2)PO(4) 0.46, K(2)HPO(4) 1 and MgSO(4).7H(2)O 0.5. The fungus was cultivated under various agitation and aeration conditions in a 5L stirred-tank bioreactor. The maximum cell mass and EPS production were obtained at a relatively high agitation speed of 200 rpm and at an aeration rate of 2 vvm. The flow behavior of the fermentation broth was Newtonian and the maximum apparent viscosity (35 cP) was observed at a highly aerated condition (2 vvm). The EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor. The morphological study revealed that the fungus grows in mainly three different yeast-like forms: ovoid, elongated, and double yeast forms. The high population of the elongated yeast has a very close relationship to high EPS production. The EPS were protein-bound polysaccharides consisted of mainly mannose, xylose, and fucose. The molecular weights of EPS were determined to be (1.3-1.5)x10(6).     

假丝酵母发酵玉米芯半纤维素水解液生产木糖醇

采用一株驯化过的假丝酵母(Candida sp.)直接发酵经过简单脱毒处理的玉米芯半纤维素水解液生产木糖醇。确定了水解液的最适浓缩倍数在3.0～3.72的范围内。利用正交实验,确定了摇瓶分批发酵工艺条件的最适组合为：摇床转速180r/min,起始C/N为50,起始pH 5.5,接种量5% (体积比)。在此基础上,重点研究了在发酵罐中通气量对酵母发酵玉米芯水解液生产木糖醇的影响。结果表明采用先高后低的分段通气发酵在木糖醇得率方面明显优于恒定通气发酵;其中,在0～24h,3.75 L/min;24～108h,1.25 L/min的分段通气条件下(装液量为2.5L),木糖醇得率(木糖醇/木糖,g/g) 达到0.75 g/g。该结果将有助于建立一种高效的、大规模的利用玉米芯半纤维素水解液发酵生产木糖醇的工艺。     

Detoxification of dilute acid hydrolysates of lignocellulose with lime

The hydrolysis of hemicellulose to monomeric sugars by dilute acid hydrolysis is accompanied by the production of inhibitors that retard microbial fermentation. Treatment of hot hydrolysate with Ca(OH)(2) (overliming) is an effective method for detoxification. Using ethanologenic Escherichia coli LY01 as the biocatalyst, our results indicate that the optimal lime addition for detoxification varies and depends on the concentration of mineral acids and organic acids in each hydrolysate. This optimum was shown to be readily predicted on the basis of the titration of hydrolysate with 2 N NaOH at ambient temperature to either pH 7.0 or pH 11.0. The average composition of 15 hydrolysates prior to treatment was as follows (per L): 95.24 +/- 7.29 g sugar, 5.3 +/- 2.99 g acetic acid, 1.305 +/- 0.288 g total furans (furfural and hydroxymethylfurfural), and 2.86 +/- 0.34 g phenolic compounds. Optimal overliming resulted in a 51 +/- 9% reduction of total furans, a 41 +/- 6% reduction in phenolic compounds, and a 8.7 +/- 4.5% decline in sugar. Acetic acid levels were unchanged. Considering the similarity of microorganisms, it is possible that the titration method described here may also prove useful for detoxification and fermentation processes using other microbial biocatalysts.     

Isomaltulose production using free cells: optimisation of a culture medium containing agricultural wastes and conversion in repeated-batch processes

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-alpha-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by intramolecular transglucosylation. The experimental designs and response surface methodology (RSM) were applied for the optimisation of the nutrient concentrations in the culture medium for the production of glucosyltransferase by Erwinia sp. D12 in shaken flasks at 200 rpm and 30 degrees C. A statistical analysis of the results showed that, in the range studied, the factors had a significant effect (P < 0.05) on glucosyltransferase production and the highest enzyme activity (10.84 U/ml) was observed in culture medium containing sugar cane molasses (150 g l(-1)), corn steep liquor (20 g l(-1)), yeast extract Prodex Lac SD (15 g l(-1)) and K2HPO4 (0.5 g l(-1)) after 8 h at 30 degrees C. The production of cell biomass by the strain of Erwinia sp. D12 was carried out in a 6.6-l fermenter with a mixing rate of 200 rpm and an aeration rate of 1 vvm. Fermentation time, cellular growth, medium pH and glucosyltransferase production were observed. The greatest glucosyltransferase activity was 22.49 U/ml, obtained after 8 h of fermentation. The isomaltulose production from sucrose was performed using free Erwinia sp. D12 cells in a batch process using an orbital shaker. The influence of the parameters sucrose concentration, temperature, pH, and cell concentration on the conversion of sucrose into isomaltulose was studied. The free cells showed a high conversion rate of sucrose into isomaltulose using batch fermentation, obtaining an isomaltulose yield of 72.11% from sucrose solution 35% at 35 degrees C.     

Bioconversion of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to motor fuel ethanol by xylose-fermenting yeast

Water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate has been utilized as a substrate for ethanol production using Pichia stipitis NRRL Y-7124. Hydrolysate fermentability was considerable improved by boiling, and overliming up to pH 10.0 with solid Ca(OH)(2) in combination with sodium sulfite. The percent total sugar utilized and ethanol yield (Y(p/s)) for the untreated hydrolysate were 20.15+/-0.17% and 0.19+/-0.003 g(p) g(s)(-1), respectively, compared with 76.0+/-0.32% and 0.35 g(p) g(s)(-1), respectively for the treated material. The fermentation was very effective at an aeration rate of 0.02 v/v/m, temperature 30+/-0.2 degrees C and pH 6.0+/-0.2. However, the volumetric productivity (Q(p)) was still considerably less than observed in a simulated synthetic hydrolysate medium with a sugar composition similar to the hemicellulose acid hydrolysate. L-Arabinose was not fermented but assimilated. The presence of acetic acid in the hydrolysate decreased the ethanol yield and productivity considerably.     

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.     

Production of phytase (myo-inositolhexakisphosphate phosphohydrolase) by Aspergillus niger van Teighem in laboratory-scale fermenter

The growth and production pattern of phytase by a filamentous fungus, Aspergillus niger van Teighem, were studied in submerged culture at varying agitation rates and controlled and uncontrolled pH conditions. Allowing the initial culture to grow under neutral condition with subsequent decline in pH resulted in increased phytase productivity. A maximum of 141 nkat/mL phytase was obtained when the broth pH was maintained at pH 2.5 as compared to 17 nkat/mL units at controlled pH 5.5. The culture morphology and rheological properties of the fermentation broth significantly varied with the agitation rate. The volumetric oxygen transfer coefficient was determined at different phases of fungal growth during batch fermentation using static gassing out and dynamic gassing out methods. The oxygen transfer coefficient (k(L)a) of the fermenter was found to be 125 h(-)(1) at 500 rpm as compared to 38 h(-)(1) at 200 rpm. The oxygen transfer rates at different phases of growth were significantly affected by cell mass concentration and fungal morphology. During the course of fermentation there was a gradual decline of k(L)a from 97 h(-)(1) on day 2 to 63 h(-)(1) on day 6 of fermentation, after which no significant change was observed. The degree of agitation considerably influenced the culture morphology where shear thinning of filamentous fungus was observed with the increase in agitation.     

Utilization of meat industry by products: protein hydrolysate from sheep visceral mass

Protein hydrolysate was prepared from pre-treated sheep visceral mass (including stomach, large and small intestines) by enzymatic treatment at 43+/-1 degrees C (at the in situ pH 7.1+/-0.2 of the visceral mass) using fungal protease. The enzyme readily solubilized the proteins of the visceral mass as indicated by the degree of hydrolysis (34%) and nitrogen recovery (>64%). Hydrolysis with an enzyme level of 1% (w/w of total solids) at 43+/-1 degrees C with a pH around 7.0 for 45 min was found to be the optimum condition. The yield of protein hydrolysate was about 6% (w/w). The amino acid composition of the protein hydrolysate that was very hygroscopic, was comparable to that of casein.     

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.     

Production of intracellular milk-clotting enzyme in submerged cultures of Fusarium subglutinans.

Fusarium subglutinans (Wollenweber and Reinking) Nelson et al. was found to produce intracellular milk-clotting enzyme (MCE) with good milk-clotting activity (MCA). The crude activity of the produced enzyme was recorded as optimum at 55 degrees C and pH 4.5. The highest yield i.e. 78.43 SU/mg dry biomass was obtained after 4 days of rotary shaking at 30 degrees C when the fermentation medium containes wheat flour 2%, glucose 1% and (NH4)2SO4 0.1% with an initial pH value 6.0. Under these conditions, the maximum ratio of MCA to proteolytic activity (PA) amounting to 603.31 SU/PU mg(-1) was also achieved. Production of intracellular MCE by F. subglutinans was assumed to be active growth-associated type. This enzyme preparation was less active than the calf rennet, but was superior to those of Meito's and Pfizer's rennets.     

Effect of temperature on the microaerophilic metabolism of Pachysolen tannophilus

Xylitol production by Pachysolen tannophilus from detoxified hemicellulose hydrolysate was investigated under microaerophilic conditions at temperature ranging from 20 to 40 degrees C. A carbon balance previously proposed to study the influence of pH was used in this work to evaluate the amounts of carbon source (xylose) utilised in competitive metabolic ways: reductive production of xylitol, ethanol fermentation and respiration. At pH = 5.5 more than 83% of xylose was reduced to xylitol at 25 < T < 30 degrees C, whereas respiration became the main process at low temperature (71.1% at 20 degrees C). At high temperature, on the other hand, all three processes took place at comparable rate, consuming at 40 degrees C nearly the same percentage of carbon source (33-35%). Finally, the maximum values of volumetric productivity calculated at variable temperature were used to estimate the main thermodynamic parameters of both xylitol production (Deltah* = 105.4 kJ mol(-1); Deltas* = -13.2 J mol(-1) K(-1)) and thermal deactivation (Deltah*(D) = 210.5 kJ mol(-1); Deltas*(D) = 3.63 J mol(-1) K(-1)).