Carboxymethyl Cellulase Research Articles

Bioethanol Production from Lignocellulosic Biomass Using Aspergillus niger and Aspergillus flavus Hydrolysis Enzymes through Immobilized S. cerevisiae

Lignocellulose, the main component of a plant cell wall, is a potential renewable bioenergy source. It is composed of cellulose, hemicellulose, and lignin structures. Cellulose is a linear polysaccharide that is hydrolyzed chemically or enzymatically by cellulase. The addition of lignocellulosic biomass, such as wheat bran and coffee pulp, into the fermentation culture, induces the production of cellulases. Cellulose accounts for 20% of the enzyme market worldwide, demonstrating benefits in diverse applications, especially bioethanol and biogas generation. The aim is to evaluate the optimal condition for bioethanol production by previously isolated fungal species from different soil types in the eastern region of the Kingdom of Saudi Arabia. This study attempts to evaluate and optimize the culture conditions of lignocellulosic biomass under SSF using the highest cellulases-producer strains in the region: Aspergillus niger and Aspergillus flavus (GenBank Accession No. MT328516 and MT328429, respectively) to produce raw sugar that consequently is used in the next step of bioethanol production. This process has two parts: (1) hydrolyze lignocellulosic biomass to obtain raw sugar using A. niger and A. flavus that produce cellulase, and (2) produce bioethanol through the conversion of the raw sugar produced from the cellulolysis into ethanol using Saccharomyces cerevisiae. The optimal conditions under SSF were seven days of incubation, 5% glucose as a carbon source, 1% ammonium sulfate as a nitrogen source, and 80% moisture for both isolates. Biochemical characterization showed stability for the immobilized enzyme in all temperature ranges (from 20 °C to 70 °C), while the free enzyme exhibited its maximum at 20 °C of 1.14 IU/mL. CMCase production was the highest at pH 4.0 (1.26 IU/mL) for free enzyme and at pH 5.0 (2.09 IU/mL) for the immobilized form. The CMCase activity increased steadily with an increase in water level and attained a maximum of 80% moisture content. The maximum enzyme activity was with coffee pulp as a substrate of 7.37 IU/mL and 6.38 IU/mL for A. niger and A. flavus after seven days of incubation, respectively. The Carboxymethyl Cellulase (CMCase) activity in immobilized enzymes showed good storage stability under SSF for six weeks, maintaining 90% of its initial activity, while the free enzyme retained only 59% of its original activity. As a carbon source, glucose was the best inducer of CMCase activity with coffee pulp substrate (7.41 IU/mL and 6.33 IU/mL for A. niger and A. flavus, respectively). In both fungal strains, ammonium sulfate caused maximum CMCase activities with coffee pulp as substrate (7.62 IU/mL and 6.47 IU/mL for A. niger and A. flavus, respectively). Immobilized S. cerevisiae showed an increase in ethanol production compared to free cells. In the case of immobilized S. cerevisiae cells, the concentration of ethanol was increased steadily with increasing fermentation time and attained a maximum of 71.39 mg/mL (A. niger) and 11.73 mg/mL (A. flavus) after 72 h of fermentation.

Integration of agricultural residues as biomass source to saccharification bioprocess and for the production of cellulases from filamentous fungi.

The production of second-generation bioethanol has several challenges, among them finding cheap and efficient enzymes for a sustainable process. In this work, we analyzed two native fungi, Cladosporium cladosporioides and Penicillium funiculosum, as a source of cellulolytic enzyme production, and corn stover, wheat bran, chickpeas, and bean straw as a carbon source in two fermentation systems: submerged and solid fermentation. Corn stover was selected for cellulase production in both fermentation systems, because we found the highest enzymatic activities when carboxymethyl cellulase activity (CMCase) was assessed using CMC as substrate. C. cladosporioides showed the highest CMCase activity (1.6U/mL), while P. funiculosum had the highest filter paper activity (Fpase) (0.39U/mL). The ß-glucosidase activities produced by both fungi were similar in submerged fermentation using corn stover as substrate. Through in-gel zymography, three polypeptides with cellulolytic activities were identified in each fungus: with molecular weights of ~ 38, 45 and 70kDa in C. cladosporioides and ~ 21, 63 and 100kDa in P. funiculosum. The best results for saccharification (10.11g/L of reducing sugars) of diluted acid pretreated corn stover were obtained after 36h of the hydrolytic process at pH 5 and 50°C using the enzyme extract of P. funiculosum. This is the first report of cellulase identification in C. cladosporioides and the saccharification of corn stover using enzymes of this fungus. Enzymatic extracts of C. cladosporioides and P. funiculosum obtained from low-cost lignocellulosic biomass have great potential for use in the production of second-generation bioethanol.

Isolation and Characterization of Bacteria from Natural Hot Spring and Insights into the Thermophilic Cellulase Production.

A thermophilic cellulase-producing bacterium,Bacillus velezensisstrain MRC 5958, from Bakra natural hot Springs, India was characterized through genome sequencing. It has a genome size of 4,467,129bp and a GC content of 45.7%. A cellulase purified from its fermentation broth has a molecular weight of about 18kDa. The optimum temperature and pH for carboxymethyl cellulase activity were at 55°C and pH ~ 7.0. The enzyme is stable over a wide range of temperatures from 30°C to 70°C with maximum activity observed at 48h of incubation. The strain produces cellulase on alkali-treated sugarcane bagasse, rice straw, rice husk, rice bran, and sawdust. The sugarcane bagasse exhibited the most effective carbon source for cellulase production at (85 U/ml) followed by rice bran (68 U/ml), rice husk (60 U/ml), rice straw (48 U/ml), and sawdust (39 U/ml). Therefore, this strain can be a potential thermostable cellulase-producing candidate for converting the waste biomass into biofuel and other industrial enzymes.

Active polysaccharides from Lentinula edodes and Pleurotus ostreatus by addition of corn straw and xylosma sawdust through solid-state fermentation

In this study, Lentinula edodes and Pleurotus ostreatus were selected as representatives to explore the effects of corn straw and xylosma sawdust on the production and activity of polysaccharides by edible fungi during solid-state fermentation. The results clearly indicated that the addition of lignocellulose could promote the polysaccharide content compared to the control group. Meanwhile, the hydroxyl radical scavenging activity of polysaccharides reached the maximum when the glucose concentration was 1.5 %, and among them, the xylosma sawdust groups for two fungi (L. edodes-32.37 %, P. ostreatus-25.86 %) both performed better than corn straw groups (L. edodes-24.96 %, P. ostreatus-20.80 %). In addition, structural characterization and degradation mode analysis were carried out. The results showed that the structure of the xylosma had a stronger destruction than corn straw. The activities of lignocellulolytic enzymes such as carboxymethyl cellulase, filter paper enzyme, β-glucosidase and xylanase increased rapidly in the early stage of fermentation, which could degrade the cellulose and hemicellulose as raw materials for the synthesis of active polysaccharides.

Optimization of Production Conditions of Cellulase Enzyme from Micro-Fungi Aspergillus Fumigatus for Agriculture Application

Cellulase enzymes are belonging to the hydrolytic group of enzymes facilitates the sugar release and its bioconversion into different valuable industrial products. Isolated micro fungi from rice straw by dilution plating pouring method studied for playing a various role in industries as well as in agriculture application. Various micro-fungi show enzymatic degradation of lignocellulosic material. The present study optimized the growing conditions for cellulase enzymes production from A.fumigatus. Optimization of various growth conditions such as temperature, different pH level and nitrogen source were studied for the production of enzyme carboxymethyl cellulase during this study. The result showed that A. fumigatus produced highest cellulase activity (3.546 IU/ml) at pH 7.0 and temperature 300C with yeast extract and Fpase activity (0.653 IU/ml) through solid state fermentation. In future agriculture applications and in industries the cellulase enzyme production attains a crucial role to acquire biodegradable yield.

Two novel phosphorus/potassium-degradation bacteria: Bacillus aerophilus SD-1/Bacillus altitudinis SD-3 and their application in two-stage composting of corncob residue

For effective utilization of corncob residue to realize green circular production, using composting to obtain a high-quality and low-cost biomass fertilizer has become a very important transformation avenue. In this paper, two novel phosphorus/potassium-degradation bacterial strains were isolated from tobacco straw and identified as Bacillus aerophilus SD-1/Bacillus altitudinis SD-3 (abbreviated as SD-1/SD-3). These identified two novel bacteria SD-1/SD-3 show that the soluble phosphorus content of SD-1/SD-3 reached 360.89mgL-1/403.56mgL-1 in the shake flask test, and the mass concentration of soluble potassium is 136.56mgL-1/139.89mgL-1. In addition, the Laccase (Lac), Lignin peroxidase (LiP), and Manganese peroxidase (MnP) activities of SD-1 and SD-3 are 54.45UL-1/394.84UL-1/222.79UL-1 and 46.27UL-1/395.26UL-1/203.98UL-1 respectively, with the carboxy-methyl cellulase (CMCase) of 72.07UmL-1 and 52.69UmL-1. Meanwhile, the effects of three different combinations of cultures, i.e., no inoculation (K1), inoculation of SD-1/SD-3 on day 21 (K2) and on day 0 (G) are investigated to understand the influence on the degradation degree of corncob residue compost. The results of K2 compost treatment showed that the effective P/K content increased nearly 3.1/2.4 times, the degradation of cellulose/lignin was 49.1/68.0%, and the germination rate was 110.23%, which were higher than other experiment groups K1/G. In conclusion, knowledge of this paper will be very useful for the industrial sector for the treatment of complex corncob residue.

Isolation and Screening of potent cellulolytic soil fungi from Raipur city of Chhattisgarh State, India

The current research focuses on the diversity of fungal isolates obtained from the soil and their ability to produce cellulolytic enzymes. Out of 76 colonies obtained, 11 fungal colonies were isolated viz. Aspergillus , Humicola and Rhizopus being the major genera of the isolates. From the 11 isolates, the identification of potent cellulase producing cultures was done using qualitative carboxy methyl cellulase screening test. Following the preliminary screening, five cultures viz., Aspergillus sydowii , Humicola sp - 1, Aspergillus niger , Aspergillus ustus , Aspergillus flavus were identified to be potent with enzymatic indices ranging from 1.25 to 2.29. These were further selected for quantitative enzyme analysis. Aspergillus sydowii was found to have highest enzymatic activity and Aspergillus flavus was found to have the least enzymatic activity. Our findings revealed that Aspergillus sydowii appeared to be a promising candidate for cellulase production and can be utilized for waste recycling and other biotechnological applications.

Isolation, purification and characterization of carboxymethyl cellulase (CMCase) from psychrotolerant yeast Rhodotorula mucilaginosa BPT1

Psychrotrophic yeast Rhodotorula mucilaginosa BPT1 originally isolated from Baramullah (J&K) was used to obtain carboxymethyl cellulase (CMCase). Partially purified CMCase exhibited highest activity at 20˚C, and pH 5.0. The enzyme showed residual activity above 60 % in the temperature range of 4–60 °C. It was sensitive to pH showing residual activity below 40 % at other pH. The enzyme was highly sensitive to metal ions (10 mM), Ca2+ causing more than 70 % inhibition. The cold active CMCase is an excellent enzyme for bioprocessing at low temperature. Its acidophilic nature makes it especially suitable for applications in food processing, and biofuel industries.

Establishment of a Highly Efficient Corn Stock-Degrading Microbial Consortium and Its Degradation Effect

Corn stalks are not easily degradable and thus have a low utilization rate. In this paper, a highly efficient corn stalk-degrading microbial consortium, designated as SDP, was established using the strains Z22 (Bacillus subtilis), Z15 (Bacillus megaterium), and Z08 (Aspergillus tubingensis). Moreover, the fermentation conditions for enzyme production by SDP were optimized through orthogonal experiments. The Van Soest method was used to determine the cellulose, hemicellulose, and lignin contents in the degraded corn stalks using the SDP consortium to evaluate its degrading effect. Scanning electron microscopy was used to analyze the micromorphological structure changes of the stalks to evaluate the degradation effects of SDP on corn stalks. The carboxymethyl cellulase activity of SDP reached 6.37 U/mL. Moreover, the enzyme production effect was optimal when the culture conditions were as follows: temperature, 30°C; time, 5 d; nitrogen source, NH4NO3; and initial pH, 7.0. The SDP consortium showed an improved ability to degrade corn stalks. The cellulose, hemicellulose, and lignin contents were reduced by 32.15%, 23.87%, and 7.98%, respectively, after culturing for 20 d. This study provides insights to guide further research and development of consortia for efficient corn stalk degradation.

Screening of cellulose-degrading yeast and evaluation of its potential for degradation of coconut oil cake.

Coconut oil cake (COC), a byproduct of oil extraction, contains high levels of cellulose. The aim of this study was to isolate a cellulose-degrading yeast from rotten dahlia that can effectively use COC as the only carbon source for cellulase secretion. Based on screening, Meyerozyma guillermondii CBS 2030 (M. guillermondii) was identified as a potential candidate, with the highest cellulolytic activity among the yeast strains isolated, with the carboxymethyl cellulase (CMCase) activity reaching 102.96 U/mL on day 5. The cellulose in COC samples was evaluated before and after degradation by M. guillermondii. Analysis based on field emission scanning electron microscopy (FESEM) revealed that the COC structure was changed significantly during the treatment, indicating effective hydrolysis. Fourier transform infrared spectroscopy (FTIR) of the modified functional groups indicated successful depolymerization of coconut cake. X-ray diffraction (XRD) and analysis of color differences established effective degradation of COC by M. guillermondii. The results demonstrate that M. guillermondii effectively secretes CMCase and degrades cellulose, which has important practical significance in COC degradation.

Evaluation of Enzymatic Activity during Growth of Pleurotus HK 37 on Saba comorensis Exocarp

Mushrooms degrade lignocellulosic biomass by releasing lignolytic and hydrolytic enzymes which convert lignocellulosic material into soluble and low molecular weight compounds which are then absorbed as nutrients. In the present study, enzymatic activities of Pleurotus HK 37 during growth on Saba comorensis exocarps were evaluated. It was observed that, Pleurotus HK 37 has ability to produce lignolytic enzyme (Laccase) and hydrolytic enzymes (Carboxymethyl cellulase, xylanase and filterpaperase). Maximum laccase activity of 3.33 ± 0 UmL-1 was observed during colonization period and the activity dropped during fruitification phase. Similar to hydrolytic enzymes, the activity was observed during colonization period and decreased during fruitification. However, higher filterpaperase activity of 0.93 ± 0.13 UmL–1 was observed compared to other hydrolytic enzymes (CMCase 0.78 ± 0.13 UmL–1, and Xylanase 0.56 ± 0.07 UmL–1). Pleurotus HK 37 showed ability to degrade Saba comorensis exocarps and to release enzymes which can be used in biotechnological industries.
 Keywords: Mushroom, Lignolytic, Hydrolytic, Enzyme, Saba comorensis

Effects of dietary concentrate level and feeding length on nutrient digestibility, rumen hydrolytic enzymes activity, intermediary metabolites, and feeding behavior in growing fat-tailed lambs: Iranian feedlot system

High concentrate diets (feedlot) are commonly used in the intensive lamb finishing systems. However, the use of high concentrate diets increases the cost of production especially when feeding length (FL) is prolonged. Both levels of concentrate and FL need to be adjusted in order to get the best economic profit in feedlot systems. This experiment aimed to evaluate the effects of dietary concentrate level (CL) and FL on nutrients digestibility, activity of rumen hydrolytic enzymes, circulating intermediary metabolites, and feeding behavior of growing fat-tailed lambs. Twenty-seven male Lori-Bakhtiari lambs (26.3 ± 3.3 kg body weight, age 90 ± 15 days) were maintained in individual pens and fed diets with 55% (CL55), 70% (CL70), and 85% (CL85) concentrate level and the remaining with alfalfa hay. The experiment lasted 105d including a 15d of adaptation period. Daily feed intakes, feed conversion ratio (FCR), average daily gain (ADG), dry matter digestibility (DMD), feeding behavior were measured three times during the 90d feeding period. Intermediary metabolites in blood and rumen fluid volatile fatty acid, ammonia, protozoa number, α-amylase, carboxymethyl cellulase (CMCase), microcrystalline cellulase (MCCase), and filter paper-degrading (FPD) activity were measured at 30, 60 and 90 d of feeding. At the end of feeding, final body weight (FBW), carcass weight and relative weight of offal were recorded. Lambs fed CL85 had higher (P < 0.05) ADG, DMD, activities of α-amylase, CMC, MCC, FDP and greater FBW, carcass weight and subcutaneous fat and lower acetate/propionate (AP) ratio, and less blood protein as compared to lambs fed CL55 or CL70. Lambs fed CL55 spent more (P < 0.05) time on eating and chewing than those fed CL70 and CL85. Overall, toward the end of feeding period, DMI, DMD, CPD and activity of α-amylase, CMC, MCC, FDP significantly increased (P < 0.05), while circulating glucose, cholesterol and total protein were decreased(P < 0.05) when compared with early feeding period. Extended feeding worsened FCR values and decreased the utilization efficiency of energy and protein for gain by 31% and 37% respectively. In conclusion, increased feeding length more than 60 days significantly reduces feed efficiency per unit of gain presumably through a shift towards elevated dietary energy usage for fat deposition in the body, evidenced by greater subcutaneous fat content and fat tail weight. The study indicates that the use of concentrate level more than 70% in the ration along with extended feeding length may not be profitable in finishing of fat-tailed lambs.

Improving saccharification of ramie stalks by synergistic effect of in-house cellulolytic enzymes consortium

The high cost of cellulase is one of the main obstacles hindering the large-scale biorefining of lignocellulosic biomass. Therefore, developing efficient method for preparation of cellulase is promising. In the present study, the production of cellulase by Trichoderma reesei, Trichoderma harzianum, and Aspergillus niger was optimized, and the synergistic effect of these cellulase on enzymatic hydrolysis of pretreated ramie stalks was also evaluated. The maximum CMCase (Carboxymethyl Cellulase) and filter paper activity (FPA) produced by T. reesei reached to 3.12 IU/mL and 0.13 IU/mL, respectively. The maximum activities of CMCase (3.68 IU/mL), FPA (0.04 IU/mL) and β-glucosidase (8.44 IU/mL) were obtained from A. niger. The results also showed that under the premise of the same FPA activity, the contribution of β-glucosidase activity to yield of reducing sugar was greater than that of CMCase. Besides, cellulase produced by T. reesei and A. niger had the best synergistic effect on enzymatic hydrolysis of pretreated ramie stalks. The highest reducing sugars yield (417 mg/g dry substrate) was achieved when enzyme cocktail was prepared at the ratio of 1:1, which was 1.36–3.35 folds higher than that of different single enzymes. The present research has provided a novel method for efficient preparation of enzymes consortium for enzymatic hydrolysis of ramie stalks.Graphical

Variation in community structure and network characteristics of spent mushroom substrate (SMS) compost microbiota driven by time and environmental conditions

Global mushroom production is growing rapidly, raising concerns about polluting effects of spent mushroom substrate (SMS) and interest in uses in composts. In this study, SMS composting trials and high-throughput sequencing were carried out to investigate to better understand how the structure, co-occurrence patterns, and functioning of bacterial and fungal communities vary through compost time and across environmental conditions. The results suggested that both bacterial and fungal microbiota displayed significant variation in community composition across different composting stages. Enzyme activity levels showed both directional and fluctuating changes during composting, and the activity dynamics of carboxymethyl cellulase, polyphenol oxidase, laccase, and catalase correlated significantly with the succession of microbial community composition. The co-occurrence networks are “small-world” and modularized and the topological properties of each subnetwork were significantly influenced by the environmental factors. Finally, seed germination and seedling experiments were performed to verify the biosafety and effectiveness of the final composting products.

Halophilic and Halotolerant Actinomycetes of Sambhar Salt Lake, India: Screening and Optimization of Cellulolytic Activity

Actinomycetes are Gram-positive filamentous bacteria well known for the production of bioactive compounds. Recently, many halophilic habitats have been explored for isolation of actinomycetes that exhibit biotechnological potentials. In this investigation, a saline habitat of Rajasthan, Sambhar Salt Lake (SSL) was selected to study the actinomycetes population and Carboxy Methyl Cellulase (CMCase) production by native isolates. A total of sixteen actinomycete isolates, halotolerant and moderately halophilic, were obtained using culture-dependent methods and characterized morphologically and biochemically. They were identified as members of Streptomyces, Nocardiopsis, Pseudonocardia, Saccharospolyspora, and Microbispora. Streptomyces was the most dominating genus, followed by Nocardiopsis. Agar plate assay was used for screening the isolates for CMCase production. Thirteen were found to produce the enzyme, apparent by hydrolysis observed on media plates. The highest relative activity of 22.04 was shown by isolate SSL 14 identified as Nocardiopsis sp. by 16S rDNA sequencing studies and thus selected for further optimization studies. Maximum enzyme (1.08 ± 0.09 U/ml) was produced using medium containing Carboxy Methyl Cellulose (Carbon source) and yeast extract (nitrogen source) at 12% NaCl and pH 9.0, incubated at 30 °C for 96 h. Maximum CMCase production at high salt concentration and pH suggests that Nocardiopsis SSL 14 can be used for industrial processes that operate under excessive saline and alkaline conditions.

Biodegradation of Gramineous Lignocellulose by Locusta migratoria manilensis (Orthoptera: Acridoidea)

Exploring an efficient and green pretreatment method is an important prerequisite for the development of biorefinery. It is well known that locusts can degrade gramineous lignocellulose efficiently. Locusts can be used as a potential resource for studying plant cell wall degradation, but there are few relative studies about locusts so far. Herein, some new discoveries were revealed about elucidating the process of biodegradation of gramineous lignocellulose in Locusta migratoria manilensis. The enzyme activity related to lignocellulose degradation and the content of cellulose, hemicellulose, and lignin in the different gut segments of locusts fed corn leaves were measured in this study. A series of characterization analyses were conducted on corn leaves and locust feces, which included field emission scanning electron microscopy (FE-SEM), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction pattern (XRD), and thermogravimetric (TG) analysis. These results showed that the highest activities of carboxymethyl cellulase (CMCase), filter paper cellulase (FPA), and xylanase were obtained in the foregut of locusts, which strongly indicated that the foregut was the main lignocellulose degradation segment in locusts; furthermore, the majority of nutritional components were absorbed in the midgut of locusts. The activity of CMCase was significantly higher than that of xylanase, and manganese peroxidase (MnPase) activity was lowest, which might be due to the basic nutrition of locusts being cellulose and hemicellulose and not lignin based on the results of FE-SEM, FTIR, XRD, and TG analysis. Overall, these results provided a valuable insight into lignocellulosic degradation mechanisms for understanding gramineous plant cell wall deconstruction and recalcitrance in locusts, which could be useful in the development of new enzymatic pretreatment processes mimicking the locust digestive system for the biochemical conversion of lignocellulosic biomass to fuels and chemicals.

Enhancing the Activity of Carboxymethyl Cellulase Enzyme Using Highly Stable Selenium Nanoparticles Biosynthesized by Bacillus paralicheniformis Y4.

The inorganic selenium is absorbed and utilized inefficiently, and the range between toxicity and demand is narrow, so the application is strictly limited. Selenium nanoparticles have higher bioactivity and biosafety properties, including increased antioxidant and anticancer properties. Thus, producing and applying eco-friendly, non-toxic selenium nanoparticles in feed additives is crucial. Bacillus paralicheniformis Y4 was investigated for its potential ability to produce selenium nanoparticles and the activity of carboxymethyl cellulases. The selenium nanoparticles were characterized using zeta potential analyses, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). Additionally, evaluations of the anti-α-glucosidase activity and the antioxidant activity of the selenium nanoparticles and the ethyl acetate extracts of Y4 were conducted. B. paralicheniformis Y4 exhibited high selenite tolerance of 400 mM and the selenium nanoparticles had an average particle size of 80 nm with a zeta potential value of −35.8 mV at a pH of 7.0, suggesting that the particles are relatively stable against aggregation. After 72 h of incubation with 5 mM selenite, B. paralicheniformis Y4 was able to reduce it by 76.4%, yielding red spherical bio-derived selenium nanoparticles and increasing the carboxymethyl cellulase activity by 1.49 times to 8.96 U/mL. For the first time, this study reports that the carboxymethyl cellulase activity of Bacillus paralicheniforis was greatly enhanced by selenite. The results also indicated that B. paralicheniformis Y4 could be capable of ecologically removing selenite from contaminated sites and has great potential for producing selenium nanoparticles as feed additives to enhance the added value of agricultural products.

Design and application of an efficient cellulose-degrading microbial consortium and carboxymethyl cellulase production optimization

Microbial consortia with high cellulase activities can speed up the composting of agricultural wastes with high cellulose contents and promote the beneficial utilization of agricultural wastes. In this paper, rabbit feces and sesame oil cake were used as feedstocks for compost production. Cellulose-degrading microbial strains were isolated from compost samples taken at the different composting stages and screened via Congo red staining and filter paper degradation test. Seven strains, Trichoderma reesei, Escherichia fergusonii, Proteus vulgaris, Aspergillus glaucus, Bacillus mycoides, Corynebacterium glutamicum, and Serratia marcescens, with high activities of carboxymethyl cellulase (CMCase), filter paper cellulase (FPase), and β-glucosidase (β-Gase) were identified and selected for consortium design. Six microbial consortia were designed with these strains. Compared with the other five consortia, consortium VI composed of all seven strains displayed the highest cellulase activities, 141.89, 104.56, and 131.18 U/ml of CMCase, FPase, and β-Gase, respectively. The single factor approach and response surface method were employed to optimize CMCase production of consortium VI. The optimized conditions were: culture time 4.25 days, culture temperature 35.5°C, pH 6.6, and inoculum volume 5% (v/v). Under these optimized conditions, the CMCase activity of consortium VI was up to 170.83 U/ml. Fermentation experiment of rabbit feces was carried out by using the consortium VI cultured under the optimal conditions. It was found that the application effect was better than other treatments, and the fermentation efficiency and nutrient content of the pile were significantly improved. This study provides a basis for the design of microbial consortia for the composting of agricultural wastes with high cellulose contents and provides a support for beneficial utilization of agricultural wastes.

Saccharification of pre-treated wheat straw via optimized enzymatic production using Aspergillus niger: Chemical analysis of lignocellulosic matrix

Bioconversion of lignocellulosic wastes to valuable end-products via multi-enzymatic hydrolysis is a potential low costing process for large-scale application in many industrial sectors. Thus, in this study, among thirty local fungal isolates, Aspergillus niger which gave the highest cellulase production, was identified under the accession number MZ062603 in GeneBank. Three types of pre-treatments (0.0–0.7%) acid, (0.0–2%) alkali, and (70–90 °C) hot water were applied to increase wheat straw (WS) digestibility by A. niger, and 1% NaOH treated WS was superior to the other pre-treatments (acid and hot water). During solid-state fermentation, the total cellulolytic activity of [filter-paper cellulase (FPase), carboxy-methyl cellulase (CMCase), and β- glucosidase (βGase)] increased about 2.8-fold. While reducing sugar was increased by 3.1 times. The optimum values of total cellulases activities and reducing sugar (8907.2 and 92.4 mg/gds) were obtained after 3 days of incubation at 30 °C and pH 5.2 at 75% v/w moisture using 3 days old inoculum (106 spores/mL/gds). The WS substrate which was subjected to alkali pre-treatment subsequent to fungal bioconversion was varied in its chemical composition and detailed structure compared to the raw and alkali pre-treatment ones as indicated by its chemical analysis, Scanning electron microscopy (SEM) observation, Fourier Transform Infra-red Spectroscopy (FTIR), and X-Ray Diffraction (XRD). All these analyses revealed that the lignocellulosic matrix was completely destroyed after the fungal treatment.

Cellulolytic Properties of a Potentially Lignocellulose-Degrading Bacillus sp. 8E1A Strain Isolated from Bulk Soil

Cellulolytic enzymes produced by spore-forming bacteria seem to be a potential solution to the degradation of lignocellulosic waste. In this study, several dozen bacterial spore-forming strains were isolated from soil and one of them was selected for further studies. The studied bacterial strain was identified to genus Bacillus (strain 8E1A) by 16S rRNA gene sequencing. Bacillus sp. 8E1A showed an activity of carboxymethyl cellulase (CMCase) with visualization with Congo Red-25 mm (size of clear zone). To study CMCase, filter paper hydrolase (FPase), and microcrystalline cellulose Avicel hydrolase (Avicelase) production, three different cellulose sources were used for bacterial strain cultivation: carboxymethyl cellulose (CMC), filter paper (FP), and microcrystalline cellulose Avicel. The highest CMCase (0.617 U mL−1), FPase (0.903 U mL−1), and Avicelase (0.645 U mL−1) production of Bacillus sp. 8E1A was noted for using CMC (after 216 h of incubation), Avicel cellulose (after 144 h of incubation), and CMC (after 144 h of incubation), respectively. Subsequently, the cellulases’ activity was measured at various temperatures and pH values. The optimal temperature for CMCase (0.535 U mL−1) and Avicelase (0.666 U mL−1) activity was 70 °C. However, the highest FPase (0.868 U mL−1) activity was recorded at 60 °C. The highest CMCase and Avicelase activity was recorded at pH 7.0 (0.520 and 0.507 U mL−1, respectively), and the optimum activity of FPase was noted at pH 6.0 (0.895 U mL−1). These results indicate that the cellulases produced by the Bacillus sp. 8E1A may conceivably be used for lignocellulosic waste degradation in industrial conditions.