Effects of agitation level on the adsorption, desorption, and activities on cotton fabrics of full length and core domains of EGV (Humicola insolens) and CenA (Cellulomonas fimi)

The activities (at pH 7 and 50 degrees C) of purified EGV (Humicola insolens) and CenA (Cellulomonas fimi) were determined on cotton fabrics at high and low levels of mechanical agitation. Similar activity measurements were also made by using the core domains of these cellulases. Activity experiments suggested that the presence of cellulose binding domains (CBDs) is not essential for cellulase performance in the textile processes, where high levels of mechanical agitation are applied. The binding reversibilities of these cellulases and their cores were studied by dilution of the treatment liquor after equilibrium adsorption. EGV showed low percentage of adsorption under both levels of agitation. It was observed that the adsorption/desorption processes of cellulases are enhanced by higher mechanical agitation levels and that the binding of cellulase with CBD of family I (EGV) is more reversible than that of CBD of the cellulase of family II (CenA).     

Hydrodynamic and kinetic study of cellulase production by Trichoderma reesei with pellet morphology

Numerical simulations and experimental validation were performed to understand the effects of hydrodynamics on pellet formation and cellulase production by filamentous T. reesei. The constructed model combined a steady-state multiple reference frame (MRF) approach describing mechanical mixing, oxygen mass transfer, and non-Newtonian flow field with a transient sliding mesh approach and kinetics of oxygen consumption, pellet formation, and enzyme production. The model was experimentally validated at various agitation speeds in a two-impeller Rushton turbine fermentor. Results from simulation and experimentation showed that higher agitation speeds led to increases in the pellet diameter and the proportion of pelletized (vs. filamentous) forms of the biomass. It also led to increase in dissolved oxygen mass transfer rate in shear-thinning fluid and cellulase productivity. The extent of these increases varied considerably among agitation speeds. Pellet formation and morphology were presumably affected within a viscosity-dependent shear-rate range. Cellulase activity and cell viability were shown to be sensitive to impeller shear. A maximum cellulase activity of 3.5 IU/mL was obtained at 400 rpm, representing a twofold increase over that at 100 rpm.     

一株产纤维素酶菌株的分离、鉴定及产酶特性

【目的】筛选并鉴定一株产纤维素酶的菌株,初步探究该菌的产酶特性,为综合利用纤维素筛选菌源。【方法】在常温条件下,采用滤纸培养基对菌种富集,采用CMC-Na初筛纤维素降解菌,采用LB培养基分离纯化菌株,经形态学、生理生化特征试验、16S r RNA基因序列测定等分析筛选菌株的系统分类地位。单因素试验确定培养时间、培养温度、初始p H及Na Cl浓度对筛选菌株产酶活力的影响。【结果】从腐烂的玉米秸秆中分离出一株在常温下产纤维素酶细菌KZ-2,根据菌落形态特征、生理生化特征鉴定以及16S r RNA基因序列分析,初步鉴定KZ-2为肠杆菌(Enterobacter sp.),为潜在新种。产酶条件实验显示:该菌使用产酶发酵培养基120 h产酶量达到最大值,在25–35°C、初始p H 4.5–5.5、Na Cl浓度1.0%–2.0%范围内为最佳产酶条件,在最适条件下酶活可达80.93 U/m L。该菌株所产纤维素酶最适反应p H为7.0,最适反应温度为50°C。【结论】KZ-2是一株具有降解纤维素能力的细菌,在常温下即可分泌纤维素酶,并且该菌株为潜在新种,具有潜在的开发价值。     

高产纤维素酶枯草芽胞杆菌S-16的筛选及其发酵工艺优化

利用刚果红鉴别培养基及基础液体筛选培养基进行菌种筛选,从新疆盐碱地分离得到的16株菌株中筛选获得一株产纤维素酶活力较高的菌株S-16,对该菌株进行16SrDNA鉴定,确定该菌为枯草芽胞杆菌(Bacillus subtilis)。对S-16发酵产纤维素酶的主要影响因素进行研究,分别考察了碳源、氮源、培养基初始pH和接种量等因素对发酵产纤维素酶的影响。结合单因素影响实验得到优化后的培养基配方为:羧甲基纤维素钠1.5%,酵母粉1%,NaCl 1%,MgSO_4·7H_2O 2‰,KH_2PO_4·3H_2_O 1‰。优化后的发酵条件为:初始pH为8,接种量1%,种龄8h,培养时间48h。经过发酵工艺优化,S-16产生的羧甲基纤维素酶活(CMCase)和滤纸酶活(FPase)分别达到4.64IU/mL和0.46IU/mL,与初始培养条件下的酶活相比分别提高了3.14倍和1.30倍。本研究得到的枯草芽胞杆菌S-16及其优化发酵工艺为秸秆的快速腐熟和高产纤维素酶的应用奠定了基础。     

Cellulase finishing of woven, cotton fabrics in jet and winch machines

Some authors have reported that as the applied agitation rate increases, the apparent activity of the endoglucanases from Trichoderma reesei towards cotton cellulose increases more markedly than does the apparent activity of the cellobiohydrolases. This suggests that the quality of cellulase finishing effects on cellulosic textiles may be machine-type dependent. The present work using total crude, endoglucanase-rich and cellobiohydrolase-rich cellulases from T. reesei confirmed that the final properties of woven, cotton fabrics treated under realistic processing conditions in a jet machine, were measurably and perceivably different from those of the same fabrics, treated using the same processing conditions of temperature, time, pH, enzyme concentration and fabric to liquor ratio, but in a winch machine. The results are interpreted in terms of the effects of agitation rate on the adsorption–desorption behaviour of the T. reesei endoglucanases and cellobiohydrolases.     

Augmented cellulase production by Bacillus subtilis strain MU S1 using different statistical experimental designs

The production of cellulase by Bacillus subtilis MU S1, a strain isolated from Eravikulam National Park, was optimized using one-factor-at-a-time (OFAT) and statistical methods. Physical parameters like incubation temperature and agitation speed were optimized using OFAT and found to be 40?°C and 150?rpm, respectively, whereas, medium was optimized by statistical tools. Plackett-Burman design (PBD) was employed to screen the significant variables that highly influence cellulase production. The design showed carboxymethyl cellulose (CMC), yeast extract, NaCl, pH, MgSO₄ and NaNO₃ as the most significant components that affect cellulase production. Among these CMC, yeast extract, NaCl and pH showed positive effect whereas MgSO₄ and NaNO₃ were found to be significant at their lower levels. The optimum levels of the components that positively affect enzyme production were determined using response surface methodology (RSM) based on central composite design (CCD). Three factors namely CMC, yeast extract and NaCl were studied at five levels whilst pH of the medium was kept constant at 7. The optimal levels of the components were CMC (13.46?g/l), yeast extract (8.38?g/l) and NaCl (6.31?g/l) at pH 7. The maximum cellulase activity in optimized medium was 566.66?U/ml which was close to the predicted activity of 541.05?U/ml. Optimization of physical parameters and medium components showed an overall 3.2-fold increase in activity compared to unoptimized condition (179.06?U/ml).     

Purification and characterization of a carboxymethyl cellulase from Artemia salina

Brine shrimp (Artemia salina) belong to a group of crustaceans that feed on microalgae and require a cellulase enzyme that can be used in ethanol production from marine algae. Protein with potential cellulase activity was purified and the activity analyzed under different conditions. After initial identification of cellulase activity by CMC cellulase, surface sterilization and PCR using 16s rRNA primers was conducted to confirm that the cellulase activity was not produced from contaminating bacteria. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. After the final purification, a 70-fold increase in specific enzyme activity was observed. SDS–PAGE results revealed that the cellulase enzyme had a molecular mass of 96 kDa. Temperature, pH, and salinity values were found to be optimal at 55 °C, pH 8.0, and 600 mM NaCl, respectively. Specifically, the enzyme showed a fivefold increase in enzyme activity in seawater compared to 600 mM NaCl in phosphate buffer. Further analysis of the purified enzyme by molecular spectrometry showed no match to known cellulases, indicating this enzyme could be a novel halophilic cellulase that can be used for the production of bioethanol from marine macroalgae.     

Removal of lignin and reuse of cellulases for continuous saccharification of lignocelluloses

Method of the removal of lignin and reuse of cellulases for a continuous saccharification of lignocelluloses were investigated. Only lignin could be separated from hydrolysates by differences in the settling velocity; it was removed from the saccharification process by flocculation with chitosan without loss of cellulases. The ultra-filtration membrane PM10 (Amicon) could be used for recovery of cellulases, but the membrane UH-1 (Toyo Roshi) was better for this purpose, because no cellulases leaked from the membrane, and the amount of cellulase adsorbed to the membrane was less. The cellulases were inactivated by vigorous agitation of the solution in an ultra-filtration device. The loss of cellulase activity by such agitation increased with agitation time, but could be controlled by recovery at a low speed of agitation, so the cellulases could be reused.     

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.     

Effect of cellobiose, glucose, ethanol, and metal ions on the cellulase enzyme complex of Thermomonospora fusca

The saccharification of cellulosic substrates by cellulase from Thermomonospora fusca, strain YX, was influenced by the presence of various substances. Cellobiose was strongly inhibitory, reducing the activity against swollen cellulose to 25% at 5% concentration. Glucose had much less effect, reducing activity to 40% at 20% concentration. Ethanol was found to be only slightly inhibitory to the cellulase, reducing activity by about 15% at 6% concentration, but much more inhibitory to the cell-bound beta-glucosidase activity. Of the numerous metal ions examined, Ca(2+) and Co(2+) at 0.1mM-1.0mM concentration were found to be slightly activating under assay conditions, while 1.0mM Pb(2+) and Hg(2+) were the most inhibitory ions. The implications of these results for the design of commercial processes for ethanol production from cellulose are discussed.     

Effect of aeration and agitation on the production of mycelial biomass and exopolysaccharides in an enthomopathogenic fungus Paecilomyces sinclairii

AIMS: The objective of the present study was to investigate the influence of aeration rate and agitation intensity on the production of mycelial biomass and exopolysaccharide (EPS) in Paecilomyces sinclairii. METHODS AND RESULTS: The P. sinclairii was cultivated under various aeration and agitation conditions in a 5 l stirred-tank bioreactor. The highest mycelial biomass (30.5 g l-1) and EPS production (11.5 g l-1) were obtained at a high aeration rate (3.5 v.v.m.) and at a high agitation speed (250 rev min-1). The apparent viscosities (6000-8000 cP) of fermentation broth increased rapidly towards the end of fermentations at high aeration and agitation conditions. CONCLUSIONS: The high level of dissolved oxygen achieved at a high aeration rate (3.5 v.v.m.) associated with higher hyphal density eventually resulted in enhanced EPS production. Agitation intensity was also proved to be a critical factor influencing on both the mycelial biomass and EPS production: high agitation speeds up to 250 rev min-1 were preferred to the yields of biomass and EPS production. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effects of aeration and agitation in the culture process of P. sinclairii were found, which is widely applicable to other kinds of basidiomycetes or ascomycetes in their submerged culture processes.     

Cellulase from Bacillus licheniformis and Brucella sp. isolated from mangrove soils of Mahanadi river delta,Odisha, India

In the present study, two cellulose-degrading bacteria (CDB-5 and CDB-12) were isolated from mangrove soils of Mahanadi river delta, based on halo zone formation in Congo red agar medium and evaluation for cellulase production in CMC broth medium. Based on morphological, biochemical and 16S rRNA gene sequencing, the two strains, CDB-5 and CDB-12, were identified as Brucella sp. and Bacillus licheniformis, respectively. The gene bank accession number of the strains CDB-5 and CDB-12 are KR632646 and KR632645, respectively. The strain Brucella sp. and B. licheniformis showed an enzyme activity of 96.37?U/ml and 98.25?U/ml, respectively, after 72?h of incubation period. Enzyme production was optimized under different growth conditions such as pH, temperature, agitation rate, carbon source, sodium chloride (NaCl), and nitrogen sources. Maximum cellulase production by both the strains was obtained in the same parameter condition such as pH (7.0), rpm (150), and NaCl (2%, w/v) which varies for other parameters. The strain, CDB-5, produced maximum cellulase at 35?°C temperature, maltose as a carbon source, and yeast extract as a nitrogen source where as the strain CDB-12 produces maximum cellulase at 45?°C temperature, carboxyl methyl cellulose (CMC) as carbon source and trypton as a nitrogen source. The bacterial crude enzyme was purified by ammonium sulfate precipitation followed by overnight dialysis. SDS-PAGE analysis of the partially purified cellulase enzyme exhibited band sizes of approximately 55 and 72?kDa.     

Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene

The potential of Pseudomonas aeruginosa expressing the Vitreoscilla hemoglobin gene (vgb) for rhamnolipid production was studied. P. aeruginosa (NRRL B-771) and its transposon mediated vgb transferred recombinant strain, PaJC, were used in the research. The optimization of rhamnolipid production was carried out in the different conditions of cultivation (agitation rate, the composition of culture medium and temperature) in a time-course manner. The nutrient source, especially the carbon type, had a dramatic effect on rhamnolipid production. The PaJC strain and the wild type cells of P. aeruginosa started producing biosurfactant at the stationary phase and its concentration reached maximum at 24 h (838 mg/l(-1)) and at 72 h (751 mg l(-1)) of the incubation respectively. Rhamnolipid production was optimal in batch cultures when the temperature and agitation rate were controlled at 30 degrees C and 100 rpm. It reached 8373 mg l(-1) when the PaJC cells were grown in 1.0% glucose supplemented minimal media. Genetic engineering of biosurfactant producing strains with vgb may be an effective method to increase its production.     

低温产纤维素酶菌株的筛选、鉴定及纤维素酶学性质

[目的]筛选一株低温产纤维素酶菌株并进行鉴定,初步探索其酶学性质,为微生物肥料生产筛选菌种资源.[方法]常温条件下,采用CMC-刚果红染色法初筛纤维素降解菌株.采用低温条件诱导的方法,筛选耐低温且产纤维素酶能力最强的菌株,经形态学、生理生化特征试验、ITS序列等方面分析系统分类地位.单因素试验确定温度、pH及金属离子对纤维素酶活力的影响.[结果]从秸秆还田土壤中分离出一株在13℃低温环境下高效分解纤维素的真菌M11,鉴定M11为草酸青霉(Penicillium oxalicum).发酵试验表明:以玉米秸秆粉为唯一碳氮源,13℃、200 r/min摇床发酵培养9d时,纤维素酶活力最高为33.08 U/mL.对其酶学性质初步研究表明:该酶最适pH为5.0,最适反应温度为20℃,在5℃-20℃间酶活力仍能保持在90％以上.[结论]Penicillium oxalicum M11是一株高效的纤维素降解菌株,在低温条件下可分泌纤维素酶且活性显著,具有潜在的开发价值.     

Effect of various process parameters on morphology, rheology, and polygalacturonase production by Aspergillus sojae in a batch bioreactor

The effects of pH, agitation speed, and dissolved oxygen tension (DOT), significant in common fungal fermentations, on the production of polygalacturonase (PG) enzyme and their relation to morphology and broth rheology were investigated using Aspergillus sojae in a batch bioreactor. All three factors were effective on the response parameters under study. An uncontrolled pH increased biomass and PG activity by 27% and 38%, respectively, compared to controlled pH (pH 6) with an average pellet size of 1.69 +/- 0.48 mm. pH did not significantly affect the broth rheology but created an impact on the pellet morphology. Similarly, at constant agitation speed the maximum biomass obtained at 500 rpm and at 30 h was 3.27 and 3.67 times more than at 200 and 350 rpm, respectively, with an average pellet size of 1.08 +/- 0.42 mm. The maximum enzyme productivity of 0.149 U mL-1 h-1 was obtained at 200 rpm with an average pellet size of 0.71 +/- 0.35 mm. Non-Newtonian and pseudoplastic broth rheology was observed at 500 rpm agitation speed, broth rheology exhibited dilatant behavior at the lower agitation rate (200 rpm), and at the medium agitation speed (350 rpm) the broth was close to Newtonian. Furthermore, a DOT range of 30-50% was essential for maximum biomass formation, whereas only 10% DOT was required for maximum PG synthesis. Non-Newtonian shear thickening behavior (n > 1.0) was depicted at DOT levels of 10% and 30%, whereas non-Newtonian shear thinning behavior (n < 1.0) was dominant at 50% DOT. The overall fermentation duration (50-70 h) was considerably shorter compared to common fungal fermentations, revealing the economic feasibility of this particular process. As a result this study not only introduced a new strain with a potential of producing a highly commercially significant enzyme but also provided certain parameters significant in the design and mathematical modeling of fungal bioprocesses.     

Effect of agitation rate on biomass and protease production by a marine bacterium Vibrio harveyi cultured in a fermentor

A marine bacterium Vibrio harveyi was adapted to grow and produce extracellular proteases in a seawater/Zobell-based medium, supplemented with skim milk under different hydrodynamic conditions, namely agitation and aeration rates. The addition of skim milk to Zobell medium enhanced fivefold the extracellular enzyme production. Protease production seemed to take place after maximum luminescence had been produced. Specific growth rate increased as a consequence of increasing agitation rates. The maximum activity of 4.28 units mg^–1 protein were formed with 700 rev min^–1 and 0.5 v/v/m. Protease activity detected has a molecular weight of 34 kDa. Another minor band of protease activity was found at 40 kDa.     

液态发酵条件下拟康宁木霉8985产纤维素酶能力初探

液态发酵条件下,以微晶纤维素为唯一碳源,比较了拟康宁木霉Trichoderma koningiopsis 8985和里氏木霉T.reesei QM9414产纤维素酶的能力。8985发酵12 h开始产生纤维素酶,36 h时酶活达到产酶峰值的50%,此时QM9414尚未诱导产酶。测定8985发酵84 h时上清液中滤纸纤维素酶、羧甲基纤维素酶、β-葡萄糖苷酶和木聚糖酶的酶活分别为1.06、3.62、1.80和6.67 IU/mL,分别是QM9414上述酶活的1.72、1.70、6.35和1.12倍。8985滤纸纤维素酶酶活的最适反应条件为pH 4.5,反应温度50℃,在Fe³⁺(≤4 mmol/L)和Cu²⁺(0–10 mmol/L)存在条件下酶活稳定。     

Statistical optimization of process conditions for cellulase production by liquid state bioconversion of domestic wastewater sludge

A two-level fractional factorial design (FFD) was used to determine the effects of six factors, i.e. substrate (domestic wastewater sludge - DWS) and co-substrate concentration (wheat flour - WF), temperature, initial pH, inoculum size and agitation rate on the production of cellulase enzyme by Trichoderma harzianum in liquid state bioconversion. On statistical analysis of the results from the experimental studies, optimum process conditions were found to be temperature 32.5 degrees C, substrate concentration (DWS) 0.75% (w/w), co-substrate (WF) concentration 2% (w/w), initial pH 5, inoculum size 2% (v/w) and agitation 175 rpm. Analysis of variance (ANOVA) showed a high coefficient of determination (R2) of 0.975. Cellulase activity reached 10.2 FPU/ml at day 3 during the fermentation process which indicated about 1.5-fold increase in production compared to the cellulase activity obtained from the results of design of experiment (6.9 FPU/ml). Biodegradation of DWS was also evaluated to verify the efficiency of the bioconversion process as a waste management method.     

Fungal morphology and fragmentation behavior in a fed-batch Aspergillus oryzae fermentation at the production scale

It is well known that high-viscosity fermentation broth can lead to mixing and oxygen mass transfer limitations. The seemingly obvious solution for this problem is to increase agitation intensity. In some processes, this has been shown to damage mycelia, affect morphology, and decrease product expression. However, in other processes increased agitation shows no effect on productivity. While a number of studies discuss morphology and fragmentation at the laboratory and pilot scale, there are relatively few publications available for production-scale fungal fermentations. The goal of this study was to assess morphology and fragmentation behavior in large-scale, fed-batch, fungal fermentations used for the production of protein. To accomplish this, a recombinant strain of Aspergillus oryzae was grown in 80 m(3) fermentors at two different gassed, impeller power-levels (one 50% greater than the other). Impeller power is reported as energy dissipation/circulation function (EDCF) and was found to have average values of 29.3 +/- 1.0 and 22.0 +/- 0.3 kW m(-3) s(-1) at high and low power levels, respectively. In all batches, biomass concentration profiles were similar and specific growth rate was < 0.03 h(-1). Morphological data show hyphal fragmentation occurred by both shaving-off of external clump hyphae and breakage of free hyphae. The fragmentation rate constant (k(frag)), determined using a first-order model, was 5.90 and 5.80 h(-1) for high and low power batches, respectively. At the end of each batch, clumps accounted for only 25% of fungal biomass, most of which existed as small, sparsely branched, free hyphal elements. In all batches, fragmentation was found to dominate fungal growth and branching. We speculate that this behavior was due to slow growth of the culture during this fed-batch process.