Improvement of wheat straw hydrolysis by cellulolytic blends of two Penicillium spp.

Co-culture of fungal strains Penicillium janthinellum EMS-UV-8 (E), Penicillium funiculosum strain P (P) and Aspergillus sp. strain G (G) and blending of their crude cellulase were evaluated for improvements in cellulase activities as well as for enhanced hydrolysis of dilute acid pretreated wheat straw (PWS). The blending of crude enzymes of P and E enhanced the hydrolysis of PWS more effectively due to synergism in cellulolytic enzyme activities. Here, three types of blends were made on the basis of equal FPUs, equal protein content or fixed volume containing different proportions of individual enzymes, the former blend hydrolyzed 42.6% of PWS due to the 98%,62%, 64% and 34% synergistic enhancement in endo-glucanase, cellulase (FPU), β-glucosidase and xylanase activities, respectively. Hydrolysis at 10% solid loading of PWS in roller bottle reactor with this blend further enhanced hydrolysis yield to 74% within 24 h, which was much better than the corresponding hydrolysis yields of individual (38.1% by E and 61.5% by P) or the commercial enzyme (62.3%). This study proved that synergistic blends of cellulases from two Penicillium spp. are cost-effective tools for efficient wheat straw hydrolysis for on-site biofuel production.     

Reutilization of carbon sources through sugar recovery from waste rice straw

Rice straw was utilized for the cultivation of Phanerochaete chrysoporium to produce cellobiose dehydrogenase. The composition of the rice straw after fermentation was found to be 28.77% glucan, 19.05% xylan and 54.81% other lignin containing sugars. The glucan and xylan content decreased due to the consumption of glucan and xylan by P. chrysoporium. After fermentation, the rice straw waste was subjected to chemical pretreatment to remove lignin. The effect of dilute acid pretreatment was not notable because of the glucose loss. However, when the rice straw after fermentation was treated with aqueous ammonia, the composition changed to 44.73% glucan, 25.43% xylan and 29.52% other lignin containing sugars. The aqueous ammonia pretreatment was optimized and an ammonia concentration, reaction time and temperature of 20%, 6 h and 60 °C, respectively, were determined to be the optimal pretreatment conditions After removal of lignin, the initial reaction rate was increased to 0.009583 g/L s, which was about 3 fold higher than the rice straw after fermentation. X-ray diffractometry was performed to investigate the crystallinity index, and the XRD results showed that biological treatment and the combination of both biological treatment and chemical pretreatment decreased the crystallinity index.     

Insights into biological delignification of rice straw by Trametes hirsuta and Myrothecium roridum and comparison of saccharification yields with dilute acid pretreatment

Rice straw is the most abundant agricultural residue on a global scale and is widely available as feedstock for cellulosic fuel production. However, it is highly recalcitrant to biochemical deconstruction and also generates inhibitors that affect enzymatic saccharification. Rice straw from eastern Arkansas was subjected to dilute acid pretreatment (160 °C, 48 min and 1.0% sulfuric acid) and solid-state fermentation with two lignocellulolytic fungi, Trametes hirsuta and Myrothecium roridum, and their saccharification efficacies were compared. T. hirsuta and M. roridum were tested separately; pretreatment of rice straw with either strain for seven days resulted in 19 and 70% enrichment of its holocellulose content, respectively. However, liquid chromatography analysis of the alkali extracts showed significant differences in cell wall degradation by T. hirsuta and M. roridum. T. hirsuta removed 15% more phenolic compounds and 38% more glucan than M. roridum, while M. roridum removed 77% more xylan than T. hirsuta. Fungal and dilute acid pretreated biomass was then hydrolyzed using Accellerase^® 1500, a saccharification cocktail. Saccharification efficiency of M. roridum was 37% higher than that of dilute acid pretreatment of rice straw, requiring 8% lower enzyme loading and 50% shorter enzymatic hydrolysis duration. Alkali extraction of fungal pretreated biomass also yielded 10–15 g kg⁻¹ of acid precipitable polymeric lignin (APPL), which is a valuable co-product for biorefineries. In comparison to dilute acid pretreatment, fungal pretreatment could be a cost-effective alternative for the degradation of recalcitrant biomass, such as rice straw.     

Enhanced enzymatic conversion with freeze pretreatment of rice straw

Production of bioethanol by the conversion of lignocellulosic waste has attracted much interest in recent years, because of its low cost and great potential availability. The pretreatment process is important for increasing the enzymatic digestibility of lignocellulosic materials. Enzymatic conversion with freeze pretreatment of rice straw was evaluated in this study. The freeze pretreatment was found to significantly increase the enzyme digestibility of rice straw from 48% to 84%. According to the results, enzymatic hydrolysis of unpretreated rice straw with 150 U cellulase and 100 U xylanase for 48 h yielded 226.77 g kg⁻¹ and 93.84 g kg⁻¹ substrate-reducing sugars respectively. However, the reducing sugar yields from freeze pretreatment under the same conditions were 417.27 g kg⁻¹ and 138.77 g kg⁻¹ substrate, respectively. In addition, hydrolyzates analysis showed that the highest glucose yield obtained during the enzymatic hydrolysis step in the present study was 371.91 g kg⁻¹ of dry rice straw, following pretreatment. Therefore, the enhanced enzymatic conversion with freeze pretreatment of rice straw was observed in this study. This indicated that freeze pretreatment was highly effective for enzymatic hydrolysis and low environmental impact.     

Two-stage co-hydrolysis of rice straw by Trichoderma reesei ZM4-F3 and Pseudomonas aeruginosa BSZ-07

Rhamnolipid biosurfactant was added to rice straw hydrolysis system to enhance the production of reducing sugars. Differing from the traditional method, on-site production of rhamnolipid made the rice straw decomposing fungus Trichoderma reesei ZM4-F3 and rhamnolipid producing bacteria Pseudomonas aeruginosa BSZ-07 work together. As the growth periods of these two strains are 96 h and 48 h, respectively, a new two-stage co-hydrolysis bioprocess was achieved. T. reesei ZM4-F3 was cultivated for rice straw hydrolysis in the first 48 h at suitable conditions. For the next 48 h, the results showed that the temperature of 34 °C and pH value of 5.5 were the optimum conditions, and the optimum adding inoculation concentration ratio of P. aeruginosa BSZ-07 to T. reesei ZM4-F3 was 4%. Under these conditions, the co-hydrolysis sample could improve the production of reducing sugars to 2.57 g l⁻¹, 15.20% higher than that of the control. The increased enzyme stability was indicated to be one of the mechanisms of the stimulatory effect of rhamnolipid on rice straw hydrolysis system. Compared with Tween-80 and sodium dodecylsulphate, rhamnolipid biosurfactant exhibited a better stimulatory effect on rice straw hydrolysis. Since the two-stage co-hydrolysis system could leave out the rhamnolipid purification process, thus reducing the biosurfactant production cost effectively, it seems to be a new prospective bioprocess for rice straw hydrolysis.     

Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production

A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which include the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). In the present study, cellulolytic enzymes for biomass hydrolysis were produced using solid state fermentation on wheat bran as substrate. Crude cellulase and a relatively glucose tolerant BGL were produced using fungi Trichoderma reesei RUT C30 and Aspergillus niger MTCC 7956, respectively. Saccharification of three different feed stock, i.e. sugar cane bagasse, rice straw and water hyacinth biomass was studied using the enzymes. Saccharification was performed with 50 FPU of cellulase and 10 U of β-glucosidase per gram of pretreated biomass. Highest yield of reducing sugars (26.3 g/L) was obtained from rice straw followed by sugar cane bagasse (17.79 g/L). The enzymatic hydrolysate of rice straw was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.093 g per gram of pretreated rice straw.     

Thermophilic dark fermentation of untreated rice straw using mixed cultures for hydrogen production

Biohydrogen production from untreated rice straw using different heat-treated sludge, initial cultivation pH, substrate concentration and particle size was evaluated at 55 °C. The peak hydrogen production yield of 24.8 mL/g TS was obtained with rice straw concentration 90 g TS/L, particle size <0.297 mm and heat-treated sludge S1 at pH 6.5 and 55 °C in batch test. Hydrogen production using sludge S1 resulted from acetate-type fermentation and was pH dependent. The maximum hydrogen production (P), production rate (R_m) and lag (λ) were 733 mL, 18 mL/h and 45 h respectively. Repeated-batch operation showed decreasing trend in hydrogen production probably due to overloading of substrate and its non-utilization. PCR-DGGE showed both hydrolytic and fermentative bacteria (Clostridium pasteurianum, Clostridium stercorarium and Thermoanaerobacterium saccharolyticum) in the repeated-batch reactor, which perhaps in association led to the microbial hydrolysis and fermentation of raw rice straw avoiding the pretreatment step.     

Contents of various sources of glucose and fructose in rice straw, a potential feedstock for ethanol production in Japan

The major carbohydrates of rice straw samples were determined in order to evaluate the potential of using rice straw as a feedstock for ethanol production in Japan. Straw samples were harvested by cutting the plants at ground level when the grain was mature and immediately heating or chilling the samples. In all cases, significant amounts (62-303 g kg⁻¹) of soft carbohydrates defined as consisting of glucose, fructose, sucrose, starch and ??-1,3-1,4-glucan were detected, in addition to structural carbohydrates (cellulose and xylan). These results indicate that rice straw is a rich source of fermentable sugars from both soft carbohydrates and lignocellulosic portions of the cell wall.     

Alkali-stable cellulase from a halophilic isolate,Gracilibacillus sp. SK1 and its application in lignocellulosic saccharification for ethanol production

A halophilic strain SK1 showing cellulolytic activity was isolated from Yuncheng Salt Lake, and was identified as the genus of Gracilibacillus by 16S rRNA gene sequence analysis. Cellulase production was strongly influenced by the salinity of culture medium with maximal level in the presence of 10% NaCl. Substrate specificity test indicated the crude cellulase was a multi-component enzyme system, showing a combined activity of endoglucanase, exoglucanase and β-glucosidase. Zymogram analysis indicated six different endoglucanases were secreted by this strain. The crude enzyme was highly active and stable over broad ranges of temperature (40–70 °C), pH (6.0–10.0) and NaCl concentration (7.5–17.5%), with an optimum at 60 °C, pH 8.0 and 12.5% NaCl, which showed excellent thermostable, alkali-stable and halostable properties. Moreover, it displayed high stability in the presence of hydrophobic organic solvents. Saccharification of corn stover and rice straw by the cellulase resulted in respective yields of 0.678 and 0.502 g g⁻¹ dry substrate of reducing sugars. The enzymatic hydrolysates of corn stover were then used as the substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.186 g g⁻¹ dry substrate, and the efficiency of reducing sugars conversion to ethanol was about 52.8%, which suggested the prospects of the crude enzyme from Gracilibacillus sp. SK1 in application for bio-ethanol production.     

Conversion of rice straw to sugars by dilute-acid hydrolysis

Hydrolysis of rice straw by dilute sulfuric acid at high temperature and pressure was investigated in one and two stages. The hydrolyses were carried out in a 10-l reactor, where the hydrolysis retention time (3–10 min), pressure (10–35 bar) and acid concentration (0–1%) were examined. Optimization of first stage hydrolysis is desirable to achieve the highest yield of the sugars from hemicellulose and also as a pretreatment for enzymatic hydrolysis. The results show the ability of first stage hydrolysis to depolymerize xylan to xylose with a maximum yield of 80.8% at hydrolysis pressure of 15 bar, 10 min retention time and 0.5% acid concentration. However, the yield of glucose from glucan was relatively low in first stage hydrolysis at a maximum of 25.8%. The solid residuals were subjected to further dilute-acid hydrolysis in this study. This second-stage hydrolysis without addition of the acid could not increase the yield of glucose from glucan beyond 26.6%. On the other hand, the best results of the hydrolysis were achieved, when 0.5% sulfuric acid was added prior to each stage in two-stage hydrolysis. The best results of the second stage of the hydrolysis were achieved at the hydrolysis pressure and the retention time of 30 bar and 3 min in the second stage hydrolysis, where a total of 78.9% of xylan and 46.6% of glucan were converted to xylose and glucose, respectively in the two stages. Formation of furfural and HMF were functions of the hydrolysis pressure, acid concentration, and retention time, whereas the concentration of acetic acid was almost constant at pressure of higher than 10 bar and a total retention time of 10 min.     

Characterization of a Trichoderma atroviride strain isolated from switchgrass bales and its use to saccharify ammonia-pretreated switchgrass for biobutanol production

The feedstock-specific enzyme systems for saccharification of biofuel feedstocks like switchgrass may potentially provide better enzymatic systems for production of second-generation biofuels. One strategy to develop these enzyme systems could be to harness the microorganisms growing naturally on specific feedstocks. This study presents the isolation and screening of fungal cultures from switchgrass bales for saccharification of ammonia-pretreated switchgrass for subsequent biobutanol production. The best performing fungal isolate during screening was identified through Sanger sequencing of its ITS region to be a unique strain of Trichoderma atroviride and further characterized for production of an enzyme system for saccharification of ammonia pretreated switchgrass. The maximum FPase, CMCase and xylanase activity produced by T. atroviride CUA1 were 0.25 fpu/mL, 0.18 IU/mL and 5.8 IU/mL, respectively. T. atroviride CUA1 also produced considerable amount of β-glucosidase activity. This isolate was used to produce an enzyme system to convert switchgrass to soluble sugars that were then fermented to butanol, ethanol, acetate and butyrate. Glucose was the major product of hydrolysis of ammonia-pretreated switchgrass performed using the enzyme system produced by the isolate. This fungus may be useful for the hydrolysis for the bioenergy crop of switchgrass to overcome this rate-limiting step in the overall conversion of biomass to biofuels.     

Biohydrogen production by Thermoanaerobacterium thermosaccharolyticum KKU-ED1: Culture conditions optimization using xylan as the substrate

Thermophilic hydrogen production from xylan by Thermoanaerobacterium thermosaccharolyticum KKU-ED1 isolated from elephant dung was investigated using batch fermentation. The optimum conditions for hydrogen production from xylan by the strain KKU-ED1 were an initial pH of 7.0, temperature of 55 °C and xylan concentration of 15 g/L. Under the optimum conditions, the hydrogen yield (HY), hydrogen production rate (HPR) and xylanase activity were 120.05 ± 15.07 mL H₂/g xylan, 11.53 ± 0.19 mL H₂/L h and 0.41 units/mL, respectively. The optimum conditions were then used to produce hydrogen from 62.5 g/L sugarcane bagasse (SCB) (equivalent to 15 g/L xylan) in which the HY and HPR of 1.39 ± 0.10 mL H₂/g SCB (5.77 ± 0.41 mL H₂/g xylan) and 0.66 ± 0.04 mL H₂/L h, respectively, were achieved. In comparison to the other strains, the HY of the strain KKU-ED1 (120.05 ± 15.07 mL H₂/g xylan) was close to that of Clostridium sp. strain X53 (125.40 mL H₂/g xylan) and Clostridium butyricum CGS5 (90.70 mL H₂/g xylan hydrolysate).     

Efficient hydrogen production from lignocellulosic feedstocks by a newly isolated thermophlic Thermoanaerobacterium sp. strain F6

Consolidated bioprocessing (CBP) is a promising approach for hydrogen production from lignocellulose owing to its lower cost and higher efficiency. In this study, the newly isolated theromphilic Thermoanaerobacterium sp. strain F6 exhibited the capability of direct utilization of various hemicellulosic and cellulosic materials for hydrogen production, including xylan, Avicel and filter paper etc. Especially, the maximum cumulative hydrogen production reached 370.7 mmoL/L from 60 g/L of xylan. In addition, natural lignocellulosic materials, such as corn cob and sugarcane bagasse without any hydrolytic pretreatment could also be directly utilized as the sole carbon source for hydrogen production. 1822.6 and 826.3 mL H₂/L of hydrogen were produced from corn cob and sugarcane bagasse, respectively. The high hydrogen production from cellulosic and hemicellulosic materials were both benefit from its efficient secretion of hydrolytic enzymes. Thus, Thermoanaerobacterium sp. strain F6 is a potential candidate for effective conversion of lignocellulose to hydrogen through CBP.     

Fed batch enzymatic saccharification of newspaper cellulosics improves the sugar content in the hydrolysates and eventually the ethanol fermentation by Saccharomyces cerevisiae

The newspaper is comprised of (w w^?1) holocellulose (70.0%) with substantial amount of lignin (16.0%). Bioconversion of the carbohydrate component of newspaper to sugars by enzymatic saccharification, and its fermentation to ethanol was investigated. Of various enzymatic treatments using cellulase, xylanase and laccase, cellulase enzyme system was found to deink the newspaper most efficiently. The saccharification of deinked paper pulp using enzyme cocktail containing exoglucanase (20 U g^?1), β-glucosidase (60 U g^?1) and xylanase (80 U g^?1) resulted in 59.8% saccharification. Among additives, 1% (v v^?1) Tween 80 and 10 mol m^?3 CoCl₂ improved the enzymatic hydrolysis of newspaper maximally, releasing 14.64 g L^?1 sugars. The fed batch enzymatic saccharification of the newspaper increased the sugar concentration in hydrolysate from 14.64 g L^?1 to 38.21 g L^?1. Moreover, the batch and fed batch enzymatic hydrolysates when fermented with Saccharomyces cerevisiae produced 5.64 g L^?1 and 14.77 g L^?1 ethanol, respectively.     

Optimization of the medium composition for the improvement of hydrogen and butanol production using Clostridium saccharoperbutylacetonicum DSM 14923

The dark fermentation process promises a sustainable route for biohydrogen production. But the low substrate conversion efficiency hinders its commercial feasibility. The present study aims towards simultaneous hydrogen and butanol production to enhance the net energy recovery. Process parameters optimization revealed that pH of 6.5, temperature of 37 °C and inoculum size of 7% (v/v) were suitable for obtaining maximum hydrogen and butanol yields by C. saccharoperbutylacetonicum. Starch and xylan were observed to be preferred carbon sources suggesting effective utilization of C₅ and C₆ sugars. The maximum hydrogen yield of 264.3 mL g⁻¹ starch and 216 mL g⁻¹ xylan and butanol yield of 0.27 g g⁻¹ starch and 0.24 g g⁻¹ xylan were obtained with overall energy recovery of 85.61% (starch) and 75.22% (xylan), respectively. The present research work indicates the potency of bi-phasic fermentation to boost energy recovery from starch/xylan based feedstock.     

Optimization of concomitant production of cellulase and xylanase from Rhizopus oryzae SN5 through EVOP-factorial design technique and application in Sorghum Stover based bioethanol production

Current study deals with the production of cellulases and xylanases from the Rhizopus oryzae SN5 isolated from composed soil of Himalayan pine forest, in order to meet the challenges of lignocellulosic biomass based biorefineries. Culture parameters for concomitant production of cellulase and xylanase were optimized through EVOP-factorial design technique under solid state fermentation. And maximum yield of cellulase and xylanase were obtained 437.54 U/gds and 273.83 U/gds, respectively at 30 °C and pH 6.0 after 5 days of incubation. On applying these enzymes for the saccharification of the dilute acid pretreated Sorghum Stover (SS), 0.407 g/g sugar was yielded. This hydrolysate on fermentation, yielded 0.411 g/g ehanol with Saccharomyces cerevisiae (NCIM 3288), which could be considered a good conversion. Therefore, Rhizopus oryzae SN5 was found as potent strain for the production of the cocktail of lignocellulosic biomasss hydrolytic enzymes and would be promising tool in the area of lignocellulose based bio-refineries.     

Biohydrogen production from diluted-acid hydrolysate of rice straw in a continuous anaerobic packed bed biofilm reactor

Bio-hydrogen production in a continuously operated anaerobic packed bed biofilm reactor (APBR) using acid-hydrolysate of rice straw as feedstock and inoculated with an anaerobic mesophilic sludge from a municipal wastewater treatment plant was investigated at three different HRTs (17, 8.2 and 2 h). Fermentable sugars solution achieved from a two-stage diluted acid hydrolysis of rice straw was used as the feedstock. First, rice straw was treated with 1% w v^?1 sulfuric acid at 120 °C for 30 min with a yield of 58.5% xylose. Higher temperature of 180 °C for 10 min at 0.5% w v^?1 sulfuric acid was applied in the second stage in which cellulosic crystalline structure was partially depolymerized to glucose with a yield of 19.3% glucose. Hydrogen production rate and yield were enhanced as the hydraulic retention time was decreased with a maximum production rate of 252 mL L^?1 h^?1 and yield of 1 mol H₂ mol^?1 sugar consumed at 2 h HRT. Experimental results illustrated the increase of COD conversion from 44% to 47% by shortening the HRT from 17 to 2 h. Furthermore, acetic acid and butyric acid production were reduced slower than other soluble metabolites like ethanol.     

Parthenium sp. as a plant biomass for the production of alkalitolerant xylanase from mutant Penicillium oxalicum SAUE-3.510 in submerged fermentation

The use of congress grass (Parthenium sp.) and water hyacinth (Eichhornia crassipes) as low cost raw materials for xylanase production from mutant Penicillium oxalicum SAU_E-3.510 in submerged fermentation was investigated. For development of mutant from wild type P. oxalicum SA-8 ITCC 6024, a strategy of mixed mutagenesis was followed using UV-irradiation and ethidium bromide, which had resulted into 1.87 fold increases in the activity of the enzyme. For enzyme production, the fungus was cultivated in mineral medium containing congress grass as carbon source. Considerably higher levels of production (475.2 ± 6.0 IU ml^?1) were achieved in media containing congress grass, although it was slightly less than that was obtained (488.5 ± 6.5 IU ml^?1) in presence of commercial oat spelt xylan. This fact confirms the feasibility of using this low cost non-food resource as an alternative carbon source to save costs of the enzyme production process. Maximum xylanase activity was reported at 55 °C with its stability at 80 °C for 2 h. The highest activity of xylanase at pH 9.0 and its stability at similar pH for 24 h denote the alkalitolerant nature of enzyme.     

Enzymatic saccharification of pretreated rice straw by cellulase produced from Bacillus carboniphilus CAS 3 utilizing lignocellulosic wastes through statistical optimization

A marine bacterium, Bacillus carboniphilus CAS 3 was subjected to optimization for cellulase production utilizing cellulosic waste through response surface methodology. Plackett – Burman and Central composite design was employed and the optimal medium constituents for maximum cellulase production (4040.45 U/mL) were determined as rice bran, yeast extract, MgSO₄·7H₂O and KH₂PO₄ at 6.27, 2.52, 0.57 and 0.39 g/L, respectively. The cellulase produced was purified to the specific activity of 434.94 U/mg and 11.46% of recovery with the molecular weight of 56 kDa. The optimum temperature, pH and NaCl for enzyme activity was determined as 50 °C, 9 and 30% and more than 70% of its original activity was retained even at 80 °C, 12 and 35% respectively. Further, enzymatic saccharification of pretreated rice straw yielded about 15.56 g/L of reducing sugar at 96 h, suggesting that the purified cellulase could be useful for production of reducing sugars from cellulosic biomass into ethanol.     

Biohydrogen production from pure and natural lignocellulosic feedstock with chemical pretreatment and bacterial hydrolysis

Pretreatment and saccharification of lignocellulosic materials is the key technology affecting the efficiency of cellulosic biohydrogen production. In this work, two pure cellulosic materials (i.e., carboxymethyl-cellulose (CMC) and xylan) were directly hydrolyzed (without pretreatment) by a cellulolytic isolate Cellulomonas uda E3-01 able to release extracellular cellulolytic enzymes. Natural cellulosic feedstock (i.e., sugarcane bagasse) was chemically pretreated prior to the bacterial hydrolysis.A temperature-shift strategy (35 °C for cellulolytic enzymes production and 45 °C for hydrolysis reaction) was used to increase the production of reducing sugars during the bacterial hydrolysis. The hydrolysates of CMC, xylan, and bagasse were efficiently converted to H₂ via dark fermentation with Clostridium butyricum CGS5. The maximum hydrogen yield was 8.80 mmol H₂/g reducing sugar (i.e., 1.58 mol H₂/mol hexose) for CMC, 6.03 mmol H₂/g reducing sugar (i.e., 0.91 mol H₂/mol pentose) for xylan, and 6.01 mmol H₂/g reducing sugar for bagasse.