Cellulase Fermentation Research Articles

Effect of exogenous cellulase pretreatment of straw-type fermented total mixed ration on digestibility and ruminal fermentation in growing beef cattle.

This study aimed to assess the impact of corn straw-based unfermented and fermented total mixed rations (TMR) supplemented with exogenous cellulase on the in vitro fermentation characteristics, growth performance, feeding behavior, apparent digestibility, rumen fermentation and digestive enzyme activities of Chinese Simmental bulls. Unfermented (direct spraying of exogenous cellulase onto TMR, TMR) and fermented (exogenous cellulase fermentation for more than 7 d, fermented total mixed rations [FTMR]) TMR were collected, dried, powdered and used as fermentation substrates. The fermentation liquid was ruminal fluid collected from Chinese Simmental bulls. The artificial rumen culture fluid were continuously cultured in vitro for 48 h. Based on the diets they were fed, 24 healthy Chinese Simmental bulls (average weight of 495.93 ±10.89 kg) were randomly divided into two groups, with 12 bulls in each group, which were fed TMR or FTMR. The study lasted 56 d. In in vitro experiments, the neutral detergent fiber degradability and total volatile fatty acid, propionate, iso-butyrate, iso-valerate and valerate concentrations were greater in the FTMR group (p<0.05) than in the TMR group. However, the methane production, pH and A/P of the FTMR group tended to be lower (p<0.05) than those of the TMR group. In the in vivo experiments, the average daily gain, eating rate, and feed efficiency of the FTMR groups were greater (p<0.05) than those of the TMR group. Similarly, the NDF degradability of the FTMR group was greater (p<0.05) than that of the TMR group. Compared to those in the TMR group, the concentrations of total volatile fatty acids, iso-butyrate, propionate and butyrate were greater in the FTMR group (p<0.05), and the A/P ratio was lower (p<0.05). Similarly, cellulase, xylanase, and β-glucosidase activities were greater (p<0.05) in the FTMR group than in the TMR group. Corn straw-based fermented total mixed rations supplemented with exogenous cellulase play a vital role in decreasing the structural carbohydrate content of TMR and ruminal methane production in vitro, improving nutrient digestion and absorption, optimizing rumen fermentation, and improving the growth performance of beef cattle.

English

Springtails are considered as an important candidate bioindicator to assess soil quality spiked with trace metals, but little is known of their endophytic bacteria. In this study, a kind of Tomoceridae springtail was used, and a total of 45 effective sequences were obtained through the process of endophytic bacteria isolation, culture, polymerase chain reaction (PCR) amplification and sequencing. After NCBI-BLAST, the results showed that there were 20 bacterial colonies belonging to the genus Staphylococcus, 12 belonging to the genus Bacillus, 7 belonging to the genus Paenibacillus, 1 belonging to the genus Exiguobacterium and 5 belonging to Acinetobacter lwoffii. Furthermore, five bacterial strains from these five genera (named TomoRZH14, TomoRZH26, TomoRZH30, TomoRZH37, TomoRZH40) were selected for cellulose degradation analysis. The results showed that TomoRZH26 (Bacillus sp.) seemed to have a stronger cellulose degradation ability than those of the other four strains, while the three main components cellulase endo-&beta;-glucanase, exo-&beta;-glucanase and &beta;-glucosidase in TomoRZH26 showed significantly higher enzymatic activity than in the other strains. Viscosity analysis also showed that the TomoRZH26 bacterium degraded relatively quickly in cellulase fermentation medium. In general, in this study, we preliminarily revealed several endophytic bacteria of Tomoceridae springtails and found that they had potentially strong cellulose degradation activity, which may be one of the important reasons behind springtail adaptation to this kind of soil ecological environment. &nbsp; Key words: springtail, endophytic bacteria, cellulose.

Use of fusion transcription factors to reprogram cellulase transcription and enable efficient cellulase production in Trichoderma reesei

BackgroundTrichoderma reesei is widely used for cellulase production and accepted as an example for cellulase research. Cre1-mediated carbon catabolite repression (CCR) can significantly inhibit the transcription of cellulase genes during cellulase fermentation in T. reesei. Early efforts have been undertaken to modify Cre1 for the release of CCR; however, this approach leads to arrested hyphal growth and decreased biomass accumulation, which negatively affects cellulase production.ResultsIn this study, novel fusion transcription factors (fTFs) were designed to release or attenuate CCR inhibition in cellulase transcription, while Cre1 was left intact to maintain normal hyphal growth. Four designed fTFs were introduced into the T. reesei genome, which generated several transformants, named Kuace3, Kuclr2, Kuace2, and Kuxyr1. No obvious differences in growth were observed between the parent and transformant strains. However, the transcription levels of cel7a, a major cellulase gene, were significantly elevated in all the transformants, particularly in Kuace2 and Kuxyr1, when grown on lactose as a carbon source. This suggested that CCR inhibition was released or attenuated in the transformant strains. The growth of Kuace2 and Kuxyr1 was approximately equivalent to that of the parent strain in fed-batch fermentation process. However, we observed a 3.2- and 2.1-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of the parent strain. Moreover, we observed a 6.1- and 3.9-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of Δcre1 strain.ConclusionsA new strategy based on fTFs was successfully established in T. reesei to improve cellulase titers without impairing fungal growth. This study will be valuable for lignocellulosic biorefining and for guiding the development of engineering strategies for producing other important biochemical compounds in fungal species.

Enzymatic activity and protein expression of cellulase from rice straw produced by Trichoderma reesei in the presence of oxygen vectors

Oxygen mass transfer is one of the most essential factors affecting microbial cell growth and cellulase fermentation. In this study, an effective approach capable of improving oxygen mass transfer was developed by adding oxygen vectors into the fermentation broth by Trichoderma reesei. With the addition of 1% (v/v) n-dodecane, the volumetric mass transfer coefficient (kLa) was increased from 142h−1 to 185h−1 at the beginning of the fermentation. In response to the improved oxygen transfer, 44.6% higher FPase activity, 29.2% higher xylanase activity and 42.5% higher saccharification yield of rice straw were obtained. The scanning electron microscopy showed that the addition of n-dodecane did not cause obvious variations on the cell morphology, whereas the comparative proteomics analysis identified 30 protein spots with different expression levels. Among them, exoglucanases (CBH I, CBH II), endoglucanase (EG VI), xylanases (Xyn I, Xyn II, Xyn III), acetyl xylan esterase, swollenin and cellulose induced protein (Cip1) were highly expressed after the addition of n-dodecane, whereas more EG III and β-xylosidase were produced without the addition of n-dodecane. The present study indicated that the addition of n-dodecane was advantageous for cellulase fermentation by T. reesei.

Purification of cellulase fermentation broth via low cost ceramic microfiltration membranes with nanofibers-like attapulgite separation layers

Abstract This paper reports the study of low cost ceramic microfiltration membranes with a separation layer of attapulgite nanofibers (attapulgite microfiltration membrane), used to clarify the cellulase fermentation fluid in order to enhance the quality of cellulase products. Based on resistance-in series model, the influence of membrane microstructure and operating conditions on the decline in membrane flux was investigated. The appropriate method for cleaning membranes that had undergone fouling was also found. The results show that an attapulgite microfiltration membrane with mean pore size of 0.12 μm is suitable for treating a cellulase fermentation fluid. The appropriate operating conditions were found to be the following: T = 30 ± 3 °C, ΔP = 0.10 MPa, and v = 4 m/s. The dominant component of the total resistance ( R t ) was the reversible fouling resistance ( R rev ), whereas the irreversible fouling resistance ( R irr ) was less important. In conditions of low trans-membrane pressure and high velocity, the fouled attapulgite microfiltration membrane could be satisfactorily regenerated using water rinsing and chemical cleaning. The results obtained in this work indicated that attapulgite microfiltration membrane separation may represent a feasible pretreatment for cellulase fermentation broth. The resistance-in-series analysis may provide additional insight into the relative importance of different fouling mechanisms.

Effects of dietary addition of cellulase and a Saccharomyces cerevisiae fermentation product on nutrient digestibility, rumen fermentation and enteric methane emissions in growing goats

ABSTRACTThis study was designed to assess the effectiveness of dietary cellulase (243 U/g, derived from Neocallimastix patriciarum) and a Saccharomyces cerevisiae fermentation product (yeast product) on ruminal fermentation characteristics, enteric methane (CH4) emissions and methanogenic community in growing goats. The experiment was conducted in a 5 × 5 Latin square design using five Xiangdong black wether goats. The treatments included a Control and two levels of cellulase (0.8 g and 1.6 g/kg dry matter intake (DMI), i.e. 194 U/kg and 389 U/kg DMI, respectively) crossed over with two levels (6 g or 12 g/kg DMI) of the yeast product. There were no significant differences regarding feed intake, apparent digestibility of organic matter, neutral detergent fibre and acid detergent fibre among all the treatments. In comparison with the Control, the ruminal ammonia N concentration was decreased (p = 0.001) by cellulase and yeast product addition. The activities of carboxymethylcellulase and xylanase were decreased after cellulase addition. Moreover, dietary cellulase and yeast product addition led to a significant reduction (p < 0.05) of enteric CH4 emissions although the diversity and copy numbers of methanogens among treatments were not dissimilar. The present results indicate that the combination of cellulase and yeast fermentation product can reduce the production of CH4 energy and mitigate the enteric CH4 emissions to a certain degree.

Characterization of cellulase enzyme produced by Chaetomium sp. isolated from books and archives

Background: Cellulase is an important industrial enzyme used to degrade cellulosic biomass. The demand for cellulase enzyme is continuously increasing because of its applications in various industries. Hence, screening of cellulase producing microorganisms from different sources has gained significant importance. Material and Methods: In this study, fungi isolated from books and archives were screened for their cellulase producing abilities. Four different fungi were isolated from books and archives using potato dextrose agar. Screening of these isolates for cellulase production was carried out using carboxymethyl cellulose broth. The most efficient fungus was subjected to cellulase fermentation and enzymes produced were purified and partially characterized. Results: Four different fungi, Chaetomium sp., Aspergillus niger, Aspergillus nidulans and Penicillium sp., were isolated from books and archives. All the isolates were tested for their ability to produce cellulase enzyme. During the primary screening Chaetomium sp. showed good growth and higher cellulase activity (155.3±25.6 U/mL) in carboxymethyl cellulose medium than the other fungi. The cellulase fermentation study was conducted with Chaetomium sp. using carboxymethyl cellulose as a substrate. During the stationary phase (144 h) of the growth, the cellulase activity of Chaetomium sp. was significantly high. The maximum mycelial weight of this fungi was obtained at 168 h. Viscosity of the Chaetomium sp. inoculated fermentation medium continuously decreased until 144 h because of the degradation of carboxymethyl cellulose. During cellulase fermentation, pH increased from the initial neutral pH to 8.5. Purified cellulase showed a specific activity of 7.3 U/mg. It exhibited maximum activity at 20°C and was stable between pH 5 and 9. Conclusions: Books and archives could be a good source for the isolation of cellulase producing fungi.

Kinetic studies on batch cultivation of Trichoderma reesei and application to enhance cellulase production by fed-batch fermentation

Reducing the production cost of cellulase as the key enzyme for cellulose hydrolysis to fermentable sugars remains a major challenge for biofuel production. Because of the complexity of cellulase production, kinetic modeling and mass balance calculation can be used as effective tools for process design and optimization. In this study, kinetic models for cell growth, substrate consumption and cellulase production in batch fermentation were developed, and then applied in fed-batch fermentation to enhance cellulase production. Inhibition effect of substrate was considered and a modified Luedeking–Piret model was developed for cellulase production and substrate consumption according to the growth characteristics of Trichoderma reesei. The model predictions fit well with the experimental data. Simulation results showed that higher initial substrate concentration led to decrease of cellulase production rate. Mass balance and kinetic simulation results were applied to determine the feeding strategy. Cellulase production and its corresponding productivity increased by 82.13% after employing the proper feeding strategy in fed-batch fermentation. This method combining mathematics and chemometrics by kinetic modeling and mass balance can not only improve cellulase fermentation process, but also help to better understand the cellulase fermentation process. The model development can also provide insight to other similar fermentation processes.

Impact of cellulase production on environmental and financial metrics for lignocellulosic ethanol

AbstractThe cost of cellulases remains a key issue in the production of cellulosic ethanol, and the impact of enzymes on greenhouse gas (GHG) emissions of cellulosic ethanol has received little attention. This study evaluates life cycle emissions and cellulase production costs for bioethanol production, considering on‐site and off‐site production options. A complete enzyme production process was simulated using AspenPlus, generating mass and energy balance information required to calculate GHG emissions and financial metrics.GHG emissions for cellulase production range from 10.2 to 16.0 g CO2 eq g–1 enzyme protein, depending on on‐site or off‐site production and the method of transportation. Enzyme GHG emissions are predicted to be 258 g CO2 eq. L–1 of ethanol for on‐site production, versus 403 g CO2 eq. L–1 for off‐site production, based on a 150 MMLY ethanol plant using 11.5 mg enzyme g–1 substrate and a cellulase fermentation yield of 90%.Cellulase production costs were estimated for a range of conditions, including ethanol plant size, enzyme dose and protein yield for on‐site production, and enzyme plant size, protein yield and return on investment for off‐site production. On‐site production costs range between $3.80 and $6.75 kg–1 protein, versus $4.00 to $8.80 kg–1 for off‐site production. In both scenarios, the lowest cost corresponds to a 90% protein yield, and a high enzyme demand and production capacity. An enzyme production cost of $4.70 USD kg–1 corresponds to an enzyme cost of 0.46 USD gal–1 ($0.12 L–1) of ethanol in a 150 MMLY plant using 11.5 mg enzyme g–1 substrate. © 2013 Society of Chemical Industry and John Wiley &amp; Sons, Ltd

Cellulase production from palm oil mill effluent in Malaysia: Economical and technical perspectives

AbstractThe demand for cellulases has increased tremendously over the last few decades. This is due to its numerous applications in industry and also because it can be used to hydrolyze cellulosic materials into sugars that can be fermented into bioethanol and bio‐based products. This does not only open up a big and significant market for cellulases, but also provides another source of biofuel and bioenergy in the future. Nevertheless, the cost of the existing substrates for cellulase fermentation is very high if required for large‐scale production. Sustainable supplies and an economically feasible biomass are needed to reduce the cost of cellulase production. Palm oil mill effluent (POME) is rich in carbohydrates, proteins, nitrogenous compounds, lipids, minerals, cellulose, hemicelluloses and lignin. It can be used naturally as a fermentation medium, either for cellulase or other value‐added product fermentation. In Malaysia, a large and continually increasing amount of POME is produced every year because of the high global demand for palm oil. Hence, the development of cellulase production from POME is reviewed, covering the POME production, cellulase production and the major challenges together with the future prospects of these processes.

Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30

Coupling fermentation with in situ foam fractionation may be beneficial to cellulase production in optimizing oligomer inducer generation, minimizing catabolite repression and reducing cellulase degradation by proteases. In this study, the potential factors that may affect the foaming behavior of broth from Trichoderma reesei Rut C-30 fermentation were examined. These factors included solid (both cell and cellulose) concentrations, cellulase activity and extracellular protein concentration. The loss of cellulase activity caused by the foaming process was minimal. The foamate generation was lower in the presence of higher solids (cell and/or cellulose) concentrations. Cellulase appeared to promote the broth foaming ability but its enrichment ratio was not high (lower than 1.2). The enrichment ratios for the individual component enzymes (β-glucosidase, endo- and exo-glucanases) were found to be similarly low. None of the cellulase components were likely the primary foaming factors. The foam also carried out cells and cellulose solids. The hydrophobicity of cell surface, studied at various fermentation stages and in both media with and without cellulose, increased as the fermentation approached the stationary phase and then decreased gradually after entering the stationary phase.

Cellulase fermentation on a novel substrate (waste cardboard) and subsequent utilization of home-produced cellulase and commercial amylase in a rabbit feeding trial

Cellulase and amylase enzymes are increasingly being used as feed additives in animal nutrition. Those are mainly applied in order to improve the nutritional value of feed by partially degrading its fiber content, which has major importance during the early post-weaning period of young animals possessing inadequate digestive enzyme activities. Due to their commercial availability at low cost, the utilization of amylases as animal feed additive can be highly recommended, while the high price of cellulases actually makes their use as a feed additive unreasonable. A promising way to gain cheap cellulases is to conduct the enzyme fermentation process on novel substrates, which are readily available at no cost. In this study, the utilization of old corrugated cardboard (OCC) as a novel fermentation substrate has been investigated for cellulase enzyme fermentation by Trichoderma reesei Rut C-30. Batch fermentation experiments were carried out in shake-flasks and in a 30 l laboratory fermenter. The OCC feedstock proved to be a suitable fermentation substrate, a filter paper activity (FPA) of as high as 2.27 FPU ml −1 was achieved in the fermenter, which corresponds to a yield of 227 FPU g −1 cellulose. The maximum productivity achieved was 35.7 FPU l −1 h −1. The subsequent utilization of this cellulase enzyme preparation was investigated as additive of commercial rabbit compound feed in order to improve its digestibility during the early post-weaning period. In parallel, commercial amylase preparations were also tested for the same purposes. The results of animal feeding experiments showed, that enzyme supplementation of the complete feed improved production traits, as it increased the digestibility of nutrients and thus reduced the mortality caused by digestive disorders.

Electrostimulation of cellulase fermentation by pulsatile electromagnetically induced currents

Electrostimulation has been an established method in medicine for many years. However, the application of electrostimulation to fermentation processes in biotechnology is a recent development. Although there are many publications 111 and patents in this field, only a few applications on the industrial scale have been reported. Electrostimulation in biology has been reviewed by Berg [1,21. Examples of electrostimulation in biotechnology are the increase in CO, formation at Succharomyces cerevisiae [31 and the influence of pulsatile electromagnetically induced currents (PEMIC) on the production of the antibiotic Nourseothricin from Streptomyces noursei [4]. One reason why electrostimulation is seldom applied in fermentation processes may be the complicated mechanisms and the unknown limiting step on the molecular level of most microbial fermentation processes, particularly in the case of syntheses of extracellular products. We are interested in the production of fungal cellulase which is a very complex system. The limiting step in the formation and secretion of the cellulase enzyme complex is governed by different biochemical and genetic control mechanisms, mainly the induction of enzyme formation and carbon catabolite repression. Carbon catabolite repression suppresses cellulase formation even if the concentration of easily utilizable sugars in the fermentation medium is very low, e.g. less than 20 mg 1-r of glucose. The mechanism of these regulatory processes on the molecular level is not yet completely understood. Many fungal mutant strains with improved cellulase formation have altered regulation of cellulase formation [5,6]. By using mutants with “switched off’ carbon catabolite repression it is possible to enhance cellulase formation significantly if the induction is optimal. We have previously developed such a Tricho-