Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol

Wheat straw consists of 48.57 ± 0.30% cellulose and 27.70 ± 0.12% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for production of ethanol. Dilute acid pretreatment at varied temperature and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from wheat straw (7.83%, w/v, DS) by dilute H₂SO₄ (0.75%, v/v) pretreatment and enzymatic saccharification (45 °C, pH 5.0, 72 h) using cellulase, β-glucosidase, xylanase and esterase was 565 ± 10 mg/g. Under this condition, no measurable quantities of furfural and hydroxymethyl furfural were produced. The yield of ethanol (per litre) from acid pretreated enzyme saccharified wheat straw (78.3 g) hydrolyzate by recombinant Escherichia coli strain FBR5 was 19 ± 1 g with a yield of 0.24 g/g DS. Detoxification of the acid and enzyme treated wheat straw hydrolyzate by overliming reduced the fermentation time from 118 to 39 h in the case of separate hydrolysis and fermentation (35 °C, pH 6.5), and increased the ethanol yield from 13 ± 2 to 17 ± 0 g/l and decreased the fermentation time from 136 to 112 h in the case of simultaneous saccharification and fermentation (35 °C, pH 6.0).     

Increased levansucrase production by a genetically modified Acetobacter diazotrophicus strain in shaking batch cultures

Acetobacter diazotrophicus levansucrase (LsdA) is a potential new candidate enzyme for kestose production from sucrose. Culture conditions for maximal LsdA yield were investigated. Variations in the medium pH had the most significant influence on LsdA production. The highest yield (32 mg l⁻¹) was achieved at an initial pH of 8·0, although optimal growth occurred under acidic conditions. The introduction of extrachromosomal copies of the levansucrase gene increased the enzyme yield to 72 mg l⁻¹. In the genetically modified A. diazotrophicus strain, levansucrase represented more than 95% of total secreted proteins showing an overall activity of 189 units ml⁻¹.     

Purification and characterization of an endocellulase from the thermophilic fungus Chaetomium thermophilum CT2

Chaetomium thermophilum CT2 produced endocellulases at 50 °C, when grown on 2% microcrystalline cellulose, 1% soluble starch, and 0.4% yeast extract medium. A major endocellulase component was purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography and gel filtration on Sephacryl S-100. The molecular weight of the enzyme was estimated to be 67.8 kDa and the enzyme was found to be a glycoprotein containing 18.9% carbohydrate. The K_m of the purified enzyme for carboxymethyl cellulose, sodium salt (CMC), was 4.6 mg ml⁻¹. The enzyme displayed highest activity towards CMC and significantly lower activities towards phosphoric acid swollen cellulose and filter paper. The activity was enhanced in the presence of Na⁺, K⁺ and Ca²⁺ but inhibited by Hg²⁺, Zn²⁺, Ag⁺, Mn²⁺, Ba²⁺, Fe²⁺, Cu²⁺, Mg²⁺ and NH₄⁺. Optimum activity was at 60 °C and pH 4.0. The enzyme was stable over 60 min incubation at 60 °C and half-life at 70, 80 and 90 °C was approximately 45, 24 and 7 min, respectively.     

Effect of pH, temperature and culture medium composition on the production of an extracellular cysteine proteinase by Micrococcus sp. INIA 528

Growth and proteinase production by Micrococcus sp. INIA 528 in a batch-operated laboratory fermentor were investigated, with trypticase soy broth as the basal medium for studies on optimum temperature, pH and medium composition. Maximum growth was recorded at 34°C and pH 715, whereas optimum temperature and pH for proteinase production were 31°C and pH 6.25. Maximum rate of enzyme production occurred during the late log and early stationary phases of growth. Addition of 5.0 g 1^-1 yeast extract, 1.0 g 1^-1 glucose, 1.0 g 1^-1 MgSO₄ or 1.0 g 1^-1 K₂HPO₄ to basal medium resulted in a lower enzyme yield, but supplementation of basal medium with 2.5 g 1^-1 (NH₄)₂SO₄ increased enzyme production by 45%. A high initial biomass added to fresh broth supplemented with 2.5 g 1^-1 (NH₄)₂SO₄ only increased enzyme activity by 19%, compared to the maximum enzyme activity achieved with the standard inoculum.     

Production, purification and properties of endoglucanase from a newly isolated strain of Mucor circinelloides

A newly isolated strain of the fungus, Mucor circinelloides (NRRL 26519), when grown on lactose, cellobiose, or Sigmacell 50 produces complete cellulase (endoglucanase, cellobiohydrolase, and β-glucosidase) system. The extracellular endoglucanase (EG) was purified to homogeneity from the culture supernatant by ethanol precipitation (75%, v/v), CM Bio-Gel A column chromatography, and Bio-Gel A-0.5 m gel filtration. The purified EG (specific activity 43.33 U/mg protein) was a monomeric protein with a molecular weight of 27 000. The optimum temperature and pH for the action of the enzyme were at 55 °C and 4.0–6.0, respectively. The purified enzyme was fully stable at pH 4.0–7.0 and temperature up to 60 °C. It hydrolysed carboxymethyl cellulose and insoluble cellulose substrates (Avicel, Solka-floc, and Sigmacell 50) to soluble cellodextrins. No glucose, cellobiose, and short chain cellooligosaccarides were formed from these substrates. The purified EG could not degrade oat spelt xylan and larch wood xylan. It bound to Avicell, Solka-floc, and Sigmacell 50 at pH 5.0 and the bound enzyme was released by changing the pH to 8.0. The enzyme activity was enhanced by 27±5 and 44±14% by the addition of 5 mM MgCl₂ and 0.5 mM CoCl₂, respectively, to the reaction mixture. Comparative properties of this enzyme with other fungal EGs are presented.     

Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst

The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of β-cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase.

The enzymatic properties of immobilized CGTase were investigated and compared with those of the soluble enzyme. Soluble and immobilized CGTases showed similar optimum temperature (80–85 °C) and pH (5.5) values, but the pH profile of the immobilized CGTase was broader at higher pH values. The thermoinactivation of the CGTase coupled to Eupergit C was slower than the observed with the native enzyme. The half-life of the immobilized enzyme at 95 °C was five times higher than that of the soluble enzyme. The immobilized CGTase maintained 40% of its initial activity after 10 cycles of 24 h each. After immobilization, the selectivity of CGTase (determined by the ratio CDs/oligosaccharides) was notably shifted towards oligosaccharide production.     

Production of tannase from Aspergillus ruber under solid-state fermentation using jamun (Syzygium cumini) leaves

Tannase producing fungal strains were isolated from different locations including garbages, forests and orchards, etc. The strain giving maximum enzyme yield was identified to be Aspergillus ruber. Enzyme production was studied under solid state fermentation using different tannin rich substrates like ber leaves (Zyzyphus mauritiana), jamun leaves (Syzygium cumini), amla leaves (Phyllanthus emblica) and jawar leaves (Sorghum vulgaris). Jamun leaves were found to be the best substrate for enzyme production under solid-state fermentation (SSF). In SSF with jamun leaves, the maximum production of tannase was found to be at 30 °C after 96 h of incubation. Tap water was found to be the best moistening agent, with pH 5.5 in ratio of 1:2 (w/v) with substrate. Addition of carbon and nitrogen sources to the medium did not increase tannase production. Under optimum conditions as standardized here, the enzyme production was 69 U/g dry substrate. This is the first report on production of tannase by A. ruber, giving higher yield under SSF with agro-waste as the substrate.     

Influence of initial pH on hydrogen production from cheese whey

Batch experiments were conducted to investigate the effect of initial pH, between 5 and 10, on fermentative hydrogen production from crude cheese whey (87.5% (v/v) by Clostridium saccharoperbutylacetonicum). Hydrogen was produced over the range of pH studied. The hydrogen production rate and yield peaked at an initial pH 6 and then steadily decreased as the pH increased. The highest rate and yield were 28.3 ml h⁻¹ and 7.89 mmol g⁻¹ lactose, respectively. Sugar consumption was unaffected between pH 5 and 9 and remained at 97%. All final pHs were acidic and increased alongside the initial pH. There was no correlation between the initial pH and the fermentation time; the times were shorter (50–52 h) between pH 6 and 8, and longer (62–82 h) outside this range. A modified Gompertz equation adequately described fermentative hydrogen production from cheese whey. The respective maximum hydrogen production rate and hydrogen potential at an optimal pH of 6 were 47.07 ml h⁻¹ and 1432 ml. Lag phase times were much longer at acidic pHs than at alkaline pHs.     

Optimisation of growth medium for the production of cyclodextrin glucanotransferase from Bacillus stearothermophilus HR1 using response surface methodology

Cyclodextrin glucanotransferase (CGTase) activity was observed when the bacterium was grown in the medium at various initial pH values, containing carbon, nitrogen, phosphorus and mineral salt sources at 50 °C for 24 h in the shake flasks. The optimisation of this growth medium was carried out using response surface methodology. The design contains a total of 32 experimental trials involving 10 star points and 6 replicates at the centre points. The design was employed by selecting sago starch, peptone from casein, K₂HPO₄, CaCl₂ and initial pH as five independent variables in this study. The optimal calculated values of tested variables for maximal production of CGTase were found to be comprised of: sago starch, 16.02 g/l; peptone from casein, 20 g/l; K₂HPO₄, 1.4 g/l; CaCl₂, 0.2 g/l and initial pH, 7.54 with a predicted CGTase activity of 14.20 U/ml. These predicted optimal parameters were tested in the laboratory and the final CGTase activity obtained was very close to the predicted value at 14.80 U/ml.     

Influence of different carbon sources on bacterial cellulose production by Gluconacetobacter xylinus strain ATCC 53524

Aims:  To determine the effect of carbon sources on cellulose produced by Gluconacetobacter xylinus strain ATCC 53524, and to characterize the purity and structural features of the cellulose produced.
Methods and Results:  Modified Hestrin Schramm medium containing the carbon sources mannitol, glucose, glycerol, fructose, sucrose or galactose were inoculated with Ga . xylinus strain ATCC 53524. Plate counts indicated that all carbon sources supported growth of the strain. Sucrose and glycerol gave the highest cellulose yields of 3·83 and 3·75 g l⁻¹ respectively after 96 h fermentation, primarily due to a surge in cellulose production in the last 12 h. Mannitol, fructose or glucose resulted in consistent rates of cellulose production and yields of >2·5 g l⁻¹. Solid state ¹³C CP/MAS NMR revealed that irrespective of the carbon source, the cellulose produced by ATCC 53524 was pure and highly crystalline. Scanning electron micrographs illustrated the densely packed network of cellulose fibres within the pellicles and that the different carbon sources did not markedly alter the micro-architecture of the resulting cellulose pellicles.
Conclusions:  The production rate of bacterial cellulose by Ga . xylinus (ATCC 53524) was influenced by different carbon sources, but the product formed was indistinguishable in molecular and microscopic features.
Significance and Impact of the Study:  Our studies for the first time examined the influence of different carbon sources on the rate of cellulose production by Ga . xylinus ATCC 53524, and the molecular and microscopic features of the cellulose produced.     

Heat-induced electrical signals affect cytoplasmic and apoplastic pH as well as photosynthesis during propagation through the maize leaf

Combining measurements of electric potential and pH with such of chlorophyll fluorescence and leaf gas exchange showed heat stimulation to evoke an electrical signal (propagation speed: 3–5 mm s⁻¹) that travelled through the leaf while reducing the net CO₂ uptake rate and the photochemical quantum yield of both photosystems (PS). Two-dimensional imaging analysis of the chlorophyll fluorescence signal of PS II revealed that the yield reduction spread basipetally via the veins through the leaf at a speed of 1.6 ± 0.3 mm s⁻¹ while the propagation speed in the intervein region was c. 50 times slower. Propagation of the signal through the veins was confirmed because PS I, which is present in the bundle sheath cells around the leaf vessels, was affected first. Hence, spreading of the signal along the veins represents a path with higher travelling speed than within the intervein region of the leaf lamina. Upon the electrical signal, cytoplasmic pH decreased transiently from 7.0 to 6.4, while apoplastic pH increased transiently from 4.5 to 5.2. Moreover, photochemical quantum yield of isolated chloroplasts was strongly affected by pH changes in the surrounding medium, indicating a putative direct influence of electrical signalling via changes of cytosolic pH on leaf photosynthesis.     

Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN₃, ascorbic acid, β-mercaptoethanol and dithreitol. The K_m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.     

Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117

Aim:  To produce high laccase activities from the white-rot fungus Fomes fomentarius .
Methods and Results:  Different culturing methods, viz, cell immobilization on stainless steel sponges and plastic material and solid-state fermentation (SSF) using wheat bran as substrate were used for laccase production by the white-rot fungus F. fomentarius . The SSF study expresses the highest laccase activities, nearly to 6400 U l⁻¹ after 13 days of laboratory flasks cultivation. When the wheat bran medium was supplemented with 2 mmol l⁻¹ copper sulfate, laccase activity increased by threefold in comparison to control cultures, reaching 27 864 U l⁻¹. With the medium thus optimized, further experiments were performed in a 3 l fixed-bed bioreactor (working volume 1·5 l) leading to a laccase activity of about 6230 U l⁻¹ on day 13.
Conclusions:  The results obtained clearly showed the superiority of wheat bran for laccase production over stainless steel sponges and plastic material. Supplementing the wheat bran solid medium with 2 mmol l⁻¹ copper sulfate allowed obtaining high activities at flask scale. The system was scaled to fixed-bed laboratory reactor.
Significance and Impact of the Study:  The high enzyme production along with the low-cost of the substrate, showed the suitability of the system F. fomentarius – SSF for industrial purposes.     

Optimization of submerged culture conditions for mycelial polysaccharide production in Cordyceps pruinosa

Cordyceps pruinosa is an entomogenous fungus noteworthy for its various bioactivities. The influence of synthetic medium and cultural conditions on polysaccharides production was investigated in shake flask culture. In the present study, optimal medium and submerged culture conditions were investigated using an orthogonal layout. Media and cultural conditions including potato starch 2% (w/v), sucrose 2.5%, soybean 0.5%, beef extract 0.5%, yeast extract 0.1%, KCl 0.02%, K₂HPO₄ 0.1%, MgSO₄·7H₂O 0.05%, pH 7.0, inoculum size 5%, medium capacity 50 ml/250 ml flask, dispersant 15 beads, culture time 7 days were employed. In fermentation medium, sucrose, beef extract and yeast extract were replaced with molasses of sucrose, groundnut and Vitamin B complex, respectively. Under optimal culture conditions, the yield of polysaccharides production was 9.51 g l⁻¹ after 54 h of fermentation in a 25 l fermenter, which was approximately twice as high as that in shake flask cultures. In addition the entire period of fermentation was shorted to around 1/4 of flask culture time (9 days). Thus, it will meet closely the requirements of industrial fermentation scale of polysaccharides production in C. pruinosa.     

Purification and characterization of UDPG:ginsenoside Rd glucosyltransferase from suspended cells of Panax notoginseng

Heterogeneity of ginsenosides is an interesting and important issue because those structure-similar secondary metabolites have different or even totally opposite pharmacological activities. In this work, a new enzyme UDP-glucose:ginsenoside Rd glucosyltransferase (UGRdGT), which catalyzes the formation of ginsenoside Rb₁ from ginsenoside Rd [Biotechnol. Bioeng. 89: 444–52, 2005], was purified approximately 145-fold from suspended cells of Panax notoginseng with an overall yield of 0.2%. Purification to apparent homogeneity, as judged by SDS-PAGE, was successfully achieved by using sequential ammonium sulphate precipitation, anion-exchange chromatography and native PAGE. The enzyme had a molecular mass of 36 kDa, and its activity was optimal at pH 8.5 and 35 °C. The enzyme activity was enhanced by Mn²⁺, Ca²⁺ and Mg²⁺, but strongly inhibited by Zn²⁺, Hg²⁺, Co²⁺, Fe²⁺ and Cu²⁺. The apparent K_m value for UDP-glucose and ginsenoside Rd was 0.32 and 0.14 mM, respectively. The biotransformation yield from ginsenoside Rd to Rb₁ by UGRdGT in 50 mM Tris–HCl buffer at pH 8.5 and 35 °C was over 80%. This work provides a basis for further molecular study on the ginsenoside Rb₁ biosynthesis by P. notoginseng cells and it is also useful for potential application to in vitro biotransformation from ginsenoside Rd to Rb₁.     

Optimization of bio-indigo production by recombinant E. coli harboring fmo gene

A bacterial flavin-containing monooxygenase (FMO) gene was cloned from Methylophaga aminisulfidivorans MP^T, and a plasmid pBlue 2.0 was constructed to express the bacterial fmo gene in E. coli. To increase the production of bio-indigo, upstream sequence size of fmo gene was optimized and response surface methodology was used. The pBlue 1.7 plasmid (1686 bp) was prepared by the deletion of upstream sequence of pBlue 2.0. The recombinant E. coli harboring the pBlue 1.7 plasmid produced 662 mg l⁻¹ of bio-indigo in tryptophan medium after 24 h of cultivation in flask. The production of bio-indigo was optimized using a response surface methodology with a 2ⁿ central composite design. The optimal combination of media constituents for the maximum production of bio-indigo was determined as tryptophan 2.4 g l⁻¹, yeast extract 4.5 g l⁻¹ and sodium chloride 11.4 g l⁻¹. In addition, the optimum culture temperature and pH were 30 °C and pH 7.0, respectively. Under the optimized conditions mentioned above, the recombinant E. coli harboring pBlue 1.7 plasmid produced 920 mg of bio-indigo per liter in optimum tryptophan medium after 24 h of cultivation in fermentor. The combination of truncated insert sizes and culture optimization resulted in a 575% increase in the production of bio-indigo.     

Production of trehalose by permeabilized Micrococcus QS412 cells

Permeabilized Micrococcus QS412 cells were used to produce trehalose from starch through catalysis of maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase in the cells. The permeabilized cells could omit the enzyme purification and simplify the immobilization of intracellular enzymes. The reagent, reagent dosage and time of cell permeabilization treatment were determined. The maximum trehalose biosynthesis activity was obtained after the cells were treated with 5% (w/v) of toluene at 30 °C for 40 min. Reaction conditions of trehalose synthesis of permeabilized cells were optimized. The yield of trehalose was up to 188 mg/g wet permeabilized cells in pH 8.0, 100 mmol/l phosphate buffer at 30 °C after 12 h reaction. Batch reactions showed that the permeabilized cells could be reused for 16 cycles in the biosynthesis reaction. The total trehalose yield was up to 2.5 g/g wet permeabilized cells. Development of permeabilized cells provide a new cheaply alternative technology for trehalose production.     

Optimization of culture conditions for production of cis-epoxysuccinic acid hydrolase using response surface methodology

Response surface methodology, which allows for rapid identification of important factors and optimization of them to enhance enzyme production, was employed here to optimize culture conditions for the production of cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3. In the first step, a Plackett–Burman design was used to evaluate the effects of nine variables (yeast extract, cis-epoxysuccinic acid, KH₂PO₄, K₂HPO₄ · 3H₂O, MgSO₄ · 7H₂O, trace minerals solution, culture volume, initial pH and incubation time) on the enzyme production. Yeast extract, cis-epoxysuccinic acid and KH₂PO₄ had significant influences on cis-epoxysuccinic acid hydrolase production and their concentrations were further optimized using central composite design and response surface analysis. A combination of adjusting the concentration of yeast extract to 7.8 g/l, cis-epoxysuccinic acid to 9.8 g/l, and KH₂PO₄ to 1.12 g/l would favor maximum cis-epoxysuccinic acid hydrolase production. An enhancement of cis-epoxysuccinic acid hydrolase production from 5.6 U/ml to 9.27 U/ml was gained after optimization.     

Utilization of -xylose as carbon source for production of bacterial cellulose

Utilization of -xylose as carbon source for production of bacterial cellulose was studied. Seventeen strains of acetic acid bacteria were screened for their cellulose productivity in -glucose, -xylose, and -xylose/ -xylulose mixed media, respectively. -Xylose was not well metabolized by any bacterial strains that exhibited high cellulose production in -glucose medium. Consequently, bacterial cellulose production in -xylose medium was unsuccessful. -Xylose, however, became utilizable substrate for bacterial strains if xylose-isomerase was added to the medium. Acetobacter xylinus IFO 15606 was the best cellulose producer in -xylose/ -xylulose mixed medium, so cultural conditions were studied for enhanced cellulose production. With pH controlled, the strain could produce cellulose at a yield exceeding 0.3 g per 100 ml of -xylose/ -xylulose mixed medium, which was comparable to the yields in -glucose medium by excellent producers in the literature.