Optimization of Xylanase Production by Thermomyces Lanuginosus in Tomato Seed Meal Using Response Surface Methodology

Summary A 3² central composite experimental design was performed with the aim of optimizing xylanase production by Thermomyces lanuginosus grown on corn cobs in submerged cultures. Xylanase production was first tested on different nitrogen sources (tomato skin, tomato seed meal, corn steep liquor, meat peptone, bacto-tryptone and yeast extract). Tomato seed meal was the selected substrate to test the effect of two variables on xylanase production (corn cobs and tomato seed meal concentrations). A second-order quadratic model and a response surface method showed that the optimum condition for xylanase production was corn cobs 4.6% (w/v) and tomato seed meal 2.1% (w/v). The optimum conditions found were transferred to 7-l bioreactors, where activities as high as 1630 U/ml were obtained.     

New and different lignocellulosic materials from Turkey for laccase and manganese peroxidase production by Trametes versicolor

In the present study, the production of laccase (Lac) and manganese‐dependent peroxidase (MnP) by the white‐rot fungus Trametes versicolor grown in submerged cultures with different agricultural residues was investigated. The lignocellulosic materials studied were almond shells, hazelnut husks, sunflower stems, clover straw and hazelnut cobs, because they are common agricultural wastes in Turkey. Among the different lignocellulosic materials studied, hazelnut cobs provided the highest Lac and MnP activities (47.09 and 109.21 U/L, respectively). The optimum conditions were determined for Lac and MnP production in submerged cultures of T. versicolor by using hazelnut cobs as substrate. For Lac production, the optimum incubation time, hazelnut cob concentration, pH, and shaking rate were found as 4 days, 2% w/v, 6.0 and 130 rpm, respectively. For MnP production, the optimum incubation time, hazelnut cob concentration, pH and shaking rate were found as 5 days, 2% w/v, 6.0 and 90 rpm, respectively.     

Production and characterization of xylanases of a Bacillus strain isolated from soil

Xylanase production was performed by growing a Bacillus isolate on agricultural by-products, wheat straw, wheat bran, corn cobs and cotton bagasse. A maximum xylanase activity of 180 U/ml was obtained together with a cellulase activity of 0.03 U/ml on 4 (w/v) corn cobs. Electrophoretic analysis showed the presence of three endo--1, 4-xylanases having molecular weights of about 22, 23 and 40 kDa. Xylanolytic activity was stable up to 50 °C in the pH range of 4.5–10 and the highest activity was observed at 70 °C and pH 6.5.     

Statistical optimization of alkaline xylanase production from <Emphasis Type="Italic">Streptomyces violaceoruber</Emphasis> under submerged fermentation using response surface methodology

Response surface methodology employing central composite design (CCD) was used to optimize fermentation medium for the production of cellulase-free, alkaline xylanase from Streptomyces violaceoruber under submerged fermentation. The design was employed by selecting wheat bran, peptone, beef extract, incubation time and agitation as model factors. A second-order quadratic model and response surface method showed that the optimum conditions for xylanase production (wheat bran 3.5 % (w/v), peptone 0.8 % (w/v), beef extract 0.8 % (w/v), incubation time 36 h and agitation 250 rpm) results in 3.0-fold improvement in alkaline xylanase production (1500.0 IUml⁻¹) as compared to initial level (500.0 IUml⁻¹) after 36 h of fermentation, whereas its value predicted by the quadratic model was 1347 IUml⁻¹. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.9718, ensuring a satisfactory adjustment of the quadratic model with the experimental data. The economical and cellulase-free nature of xylanase would enhance its applicability in pulp and paper industry.     

Improved xylanase production using apple pomace waste by Aspergillus niger in koji fermentation

Xylanase production by Aspergillus niger NRRL‐567 in solid‐state fermentation (koji fermentation) was optimized using 2⁴ factorial design and response surface methodology. The evaluated variables were the initial moisture level and concentration of inducers [veratryl alcohol (VA), copper sulphate (CS), and lactose (LAC)], leading to the response of xylanase production. Initial moisture level and LAC were found to be the most significant variable for xylanase production (p<0.05). The highest xylanase production was observed with 3578.8 ± 65.3 IU/gds (gram dry substrate) under optimal conditions using initial moisture of 85% (v/w), pH 5.0 and inducers VA (2 mM/kg), LAC 2% (w/w), and CS (1.5 mM/kg) after 48 h of incubation time. Higher xylanase activity of 3952 ± 78.3 IU/gds was attained during scale‐up of the process in solid‐state tray fermentation under optimum conditions after 72 h of incubation time. The present study demonstrates that A. niger NRRL‐567 can efficiently be used to achieve xylanase production with an economical and environmental benefit in solid‐state tray fermentation. The developed process can be used to develop an effective process for commercially feasible bioproduction of xylanases for speciality applications, such as conversion of lignocellulosic biomass to biofuels and other value‐added products.     

Production of xylanase by Emericella nidulans NK-62 on low-value lignocellulosic substrates

Thermotolerant Emericella nidulans NK-62 was isolated from bird nesting material and was tested for its ability to produce xylanase. The fungus when grown on a medium containing wheat bran (2% w/v) supplemented with Czapek's mineral salt solution at 45 °C for 7 days produced 362 IU/ml of xylanase (EC 3.2.1.8). The specific activity of E. nidulans NK-62 xylanase was found to be 275 IU/mg of total protein. The enzyme was found to be active over a broad temperature and pH range with 60 °C as optimum temperature for enzyme activity. The enzyme was stable at 50 °C and its half-life at 55 °C was 45 min. -xylosidase (EC 3.2.1.37) and carboxymethylcellulase (EC 3.2.1.4) activities, 0.018 and 0.21 IU/ml respectively, were also noticed. The fungus was screened for its ability to produce xylanase on four different lignocellulosic substrates. It produced 318.9 IU/ml of cellulase-free xylanase on corn cobs. The fungus could also utilize lentil bran (seed husk of Lens esculentus) and meal of groundnut shells to produce 84.8 and 17.3 IU/ml xylanase respectively.     

Optimizing conditions for poly(β-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> LC1 in batch culture with sucrose as carbon source

Halomonas boliviensis LC1 is able to accumulate poly(β-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l⁻¹ CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l⁻¹ and 14 g l⁻¹, respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l⁻¹ in 17 h but reduced the PHB content in the cells to 52 wt.%.     

Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads

In the present study, the optimum conditions for the production of xylanase by immobilized spores of Trichoderma reesei SAF3 in calcium alginate beads were determined. The operational stability of the beads during xylanase production under semi-continuous fermentation was also studied. The influence of alginate concentration (1, 2, 3, and 4%) and initial cell loading (100, 200, 300, 400, and 500 beads per flask) on xylanase production was considered. The production of xylanase was found to increase significantly with increasing concentration of alginate and reached a maximum yield of 3.12 ± 0.18 U ml⁻¹ at 2% (w/v). The immobilized cells produced xylanase consistently up to 10 cycles and reached a maximum level at the forth cycle (3.36 ± 0.2 U ml⁻¹).     

樟绒枝霉(Malbranchea cinnamomea)固体发酵产木聚糖酶的发酵条件优化

[目的] 研究樟绒枝霉(Malbranchea cinnamomea) CAU521利用农业废弃物固体发酵产木聚糖酶的发酵条件.[方法]采用单因素试验法优化影响菌株产酶的各个条件,包括碳源种类、氮源种类、初始pH、初始水分含量、培养温度及发酵时间共6个因素.[结果]获得的最佳产酶条件为:稻草为发酵碳源、2％(W/W)的酵母提取物为氮源、初始pH 7.0、初始水分含量80％和发酵温度45℃.在此条件下发酵6d后木聚糖酶的酶活力达到13 120 U/g干基碳源.[结论]樟绒枝霉固体发酵产木聚糖酶的产酶水平高,生产成本低,具有潜在的工业化应用前景.     

Biodegradation of 2,4,5-trichlorophenoxyacetic acid in liquid culture and in soil by the white rot fungus Phanerochaete chrysosporium

Summary Extensive biodegradation of [¹⁴C]-2,4,5-trichlorophenoxyacetic acid ([¹⁴C]-2,4,5-T) by the white rot fungus Phanerochaete chrysosporium was demonstrated in nutrient nitrogen-limited aqueous cultures and in [¹⁴C]-2,4,5-T-contaminated soil inoculated with this fungus and supplemented with ground corn cobs. After incubation of [¹⁴C]-2,4,5-T with aqueous cultures of the fungus for 30 days, 62.0%±2.0% of the [¹⁴C]-2,4,5-T initially present was degraded to ¹⁴CO₂. Mass balance analysis demonstrated that water soluble metabolites were formed during degradation, and HPLC and thin layer chromatography (TLC) of methylene chloride-extractable material revealed the presence of polar and non-polar [¹⁴C]-2,4,5-T metabolites. It was also shown that only 5% of the [¹⁴C]-2,4,5-T initially present in cultures remained as undegraded [¹⁴C]-2,4,5-T. In incubations composed of [¹⁴C]-2,4,5-T-contaminated soil, ground corn cobs, and 40% (w/w) water, 32.5%±3.6% of the [¹⁴C]-2,4,5-T initially present was converted to ¹⁴CO₂ after 30 days of incubation. These results suggest that it may be possible to develop practical systems based on the use of this fungus to detoxify 2,4,5-T-contaminated water and soil.     

Production of a high level of cellulase-free xylanase by the thermophilic fungus Thermomyces lanuginosus in laboratory and pilot scales using lignocellulosic materials

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was able to produce a very high level of cellulase-free xylanase in shake cultures using inexpensive lignocellulosic biomass. Of the nine lignocellulosic substrates tested, corn cobs were found to be the best inducer of xylanase activity. The laboratory results of xylanase production have been successfully scaled up to VABIO (Voest-Alpine Biomass Technology Center) scale using a 15-m³ fermentor for industrial production and application of xylanase. In addition, some properties of the enzyme in crude culture filtrate produced on corn cobs are presented. The enzyme exhibited very satisfactory storage stability at 4–30°C either as crude culture filtrate or as spray- or freeze-dried powder. The crude enzyme was active over a broad range of pH and had activity optima at pH 6.5 and 70–75°C. The enzyme was almost thermostable (91–92%) at pH 6.5 and 9.0 after 41 h preincubation at 55°C and lost only 20–33% activity after 188 h. In contrast, it was much less thermostable at pH 5.0 and 11.0. Xylanases produced on different lignocellulosic substrates exhibited differences in thermostability at 55°C and pH 6.5. Correspondence to: J. Gomes     

Optimization of conditions for the production of lignocellulolytic enzymes by Sphingobacterium sp. ksn-11 utilizing agro-wastes under submerged condition

Abstract

The present work was aimed at studying the production of lignocellulolytic enzymes, namely cellulase, xylanase, pectinase, mannanase, and laccase by a newly isolated bacterium Sphingobacterium sp. ksn-11, utilizing various agro-residues as a substrate under submerged conditions. The production of lignocellulolytic enzymes was found to be maximum at the loading of 10%(w/v) agro-residues. The enzyme secretion was enhanced by two-fold at 2?mM CaCO_3, optimum pH 7, and temperature 40°. The Field Emission Gun-Scanning Electron Microscope (FEG-SEM) results have shown the degradative effect of lignocellulases; cellulase, xylanase, mannanase, pectinase, and laccase on corn husk with 3.55?U/ml, 79.22?U/ml, 12.43?U/ml, 64.66?U/ml, and 21.12?U/ml of activity, respectively. The hydrolyzed corn husk found to be good adsorbent for polyphenols released during hydrolysis of corn husk providing suitable conditions for stability of lignocellulases. Sphingobacterium sp. ksn is proved to be a promising candidate for lignocellulolytic enzymes in view of demand for enzymes in the biofuel industry.     

Effect of shear rate and culture pH on the production of xylanase by Thermomyces lanuginosus

Summary The pH-value and the stirrer speed during cultivation of the thermophilic fungus Thermomyces lanuginosus were found to have a pronounced influence on xylanase production using corn cobs as carbon source. The highest xylanase activity of 32500 nkat/ml was produced in labscale fermentation within 118 hours at a stirrer speed of 50 rpm and a controlled pH-value of 7.5.     

Improvement in the bleaching of kraft pulp with xylanase from Penicillium crustosum FP 11 isolated from the Atlantic forest

Xylanase produced from the newly isolated Penicillium crustosum FP 11 and its potential in the prebleaching of kraft pulp were evaluated using a statistical approach. A Plackett–Burman design (PBD) was carried out to select the significant variables of the medium, these being NaNO₃, KH₂PO₄, MgSO₄, KCl, Fe₂(SO₄)₃, yeast extract, corn stover, and initial pH, in a liquid culture under static conditions for 6 d at 28?°C. Statistical analysis with a central composite design and response surface methodology showed that 0.15% (w/v) KH₂PO₄, 2% (w/v) corn stover, and an initial pH of 6.0 provided the best conditions for xylanase production. Furthermore, xylanase from P. crustosum FP 11 was effective in the bleaching of Eucalyptus kraft pulp, with a significant kappa efficiency of 35.04%. Therefore, the newly isolated P. crustosum FP 11 from the Atlantic Forest biome in Brazil showed two advantages: xylanase production with agricultural residue (corn stover) as a carbon source and an improvement in the bleaching of kraft pulp. Environmental pollution could thus be minimized because of a reduction in the use of chlorine as a bleaching agent.     

Culture conditions for the production of amylolytic enzymes by a thermophilic Clostridium sp.

The growth of a thermophilic Clostridium sp. and the production of α-glucosidase, α-amylase and pullulanase were studied under anaerobic conditions using different carbon and nitrogen sources and varying pH values and temperatures. Growth and enzyme activities were highest with soybean meal as the nitrogen source. The optimum concentration was 2.5% [w/v] for the production of α-amylase as well as pullulanase and 2% [w/v] for α-glucosidase. The best carbon source proved to be soluble starch for α-amylase, and pullulanase and maltose for α-glucosidase. Growth and enzyme production reached their optimum at pH 6.5 to 7.0 and 70°C. Under these conditions, the enzyme activities followed exponential growth with maximum yields of α-glucosidase, α-amylase and pullulanase at 28, 36, and 44 h.     

The production of hemicellulases by Thermomyces lanuginosus strain SSBP: influence of agitation and dissolved oxygen tension

Shake-flask cultivation of T. lanuginosus strain SSBP on coarse corn cobs yielded β-xylanase levels of 56,500 nkat/ml at 50 °C, whereas other hemicellulases (β-xylosidase, β-glucosidase, and α-l-arabinofuranosidase) were produced at levels less than 7 nkat/ml. Cultivation on d-xylose yielded much lower levels of xylanase (350 nkat/ml), although other hemicellulase levels were similar to those produced on corn cobs. The influence of agitation rate and dissolved oxygen tension (DOT) on hemicellulase production was studied further in a bioreactor. On xylose, xylanase activities of 4,330 nkat/ml and 4,900 nkat/ml were obtained at stirrer speeds up to 1,400 rpm to control DOT. At a constant stirrer speed of 400 rpm, xylanase activities of 10,930 nkat/ml and 15,630 nkat/ml were obtained when cultivated on xylose and beechwood xylan respectively, despite DOT levels below 5% for the duration of fermentation. The results indicate that there is an interaction between agitation rate and DOT, impacting on xylanase and accessory enzyme production. Higher agitation rates favoured the production of xylosidase, arabinofuranosidase and glucosidase by T. lanuginosus strain SSBP, whereas the lower agitation rates favoured xylanase production. Rheological difficulties precluded cultivation on corn cobs in the bioreactor. Volumetric xylanase productivities of 1,060,000 nkat/l · h and 589,000 nkat/l · h obtained on beechwood xylan and xylose indicate that T. lanuginosus strain SSBP is a hyper-xylanase producer with considerable industrial potential. Received: 5 May 1999 / Received revision: 31 May 2000 / Accepted: 3 June 2000     

Optimization of nutrient medium containing agricultural wastes for xylanase production by Aspergillus niger B03 using optimal composite experimental design

The xylanase biosynthesis is induced by its substrate - xylan. The high xylan content in some of the wastes like corn cobs and wheat bran makes them an accessible and cheap source of inducers. Nutrient medium for xylanase biosynthesis in submerged cultivation of Aspergillus niger B03 has been optimized. The optimization process was analyzed using optimal composite experimental design and response surface methodology. The predicted by the regression model optimum components of nutrient medium are as follows (g/l): (NH(4))(2)HPO(4) 2.6, urea 0.9, corn cobs 24.0, wheat bran 14.6 and malt sprout 6.0. Five parallel experiments have been carried out, at definite, optimum components concentrations of the nutrient medium, and a mean value of the activity Y=996.30 U/ml has been obtained. The xylanase activity, obtained with the optimized nutrient medium is 33% higher than the activity, achieved with the basic medium.     

Production of Sophorolipids from Candida bombicola ATCC 22214 Using Turkish Corn Oil and Honey

The ability of Candida bombicola ATCC 22214 to produce sophorolipids using Turkish corn oil and honey was investigated. Shake flask experiments were carried out both with and without the addition of glucose as the second carbon source. The organism could produce sophorolipids under both conditions but higher production was obtained when corn oil was combined with glucose. The 3 L bioreactor was first operated in batch mode, using both corn oil and glucose. When all the glucose was consumed, ¹/₄th of the broth was pumped out and was replaced by freshly prepared medium containing 10 % [w/v] of cheap market honey as the sole carbon source. Feed was comprised of corn oil. High concentrations of sophorolipids (&#62; 400 g/L) were produced. The crude products obtained from the batch cultivation could be solidified as very light brown solids when unused oil was removed by hexane, while the products of the two‐stage cultivation remained as viscous, honey‐like liquids after identical treatments.     

Fermentation of molasses using a thermotolerant yeast,Kluyveromyces marxianus IMB3: simplex optimisation of media supplements

 The use of molasses as a substrate for ethanol production by the thermotolerant yeast Kluyveromyces marxianus var. marxianus was investigated at 45°C. A maximum ethanol concentration of 7.4% (v/v) was produced from unsupplemented molasses at a concentration of 23% (v/v). The effect on ethanol production of increasing the sucrose concentration in 23% (v/v) molasses was determined. Increased sucrose concentration had a similar detrimental effect on the final ethanol produced as the increase in molasses concentration. This indicated that the effect may be due to increased osmotic activity as opposed to other components in the molasses. The optimum concentration of the supplements nitrogen, magnesium, potassium and fatty acid for maximum ethanol production rate was determined using the Nelder and Mead (Computer J 7:308–313, 1965) simplex optimisation method. The optimum concentrations of the supplements were 0.576 g l^-1 magnesium sulphate, 0.288 g l^-1 potassium dihydrogen phosphate and 0.36% (v/v) linseed oil. Added nitrogen in the form of ammonium sulphate did not affect the ethanol production rate. Received: 29 January 1996/Received revision: 23 April 1996/Accepted: 29 April 1996     

Xylanases of marine fungi of potential use for biobleaching of paper pulp

Microbial xylanases that are thermostable, active at alkaline pH and cellulase-free are generally preferred for biobleaching of paper pulp. We screened obligate and facultative marine fungi for xylanase activity with these desirable traits. Several fungal isolates obtained from marine habitats showed alkaline xylanase activity. The crude enzyme from NIOCC isolate 3 (Aspergillus niger), with high xylanase activity, cellulase-free and unique properties containing 580 U l^–1 xylanase, could bring about bleaching of sugarcane bagasse pulp by a 60 min treatment at 55°C, resulting in a decrease of ten kappa numbers and a 30% reduction in consumption of chlorine during bleaching. The culture filtrate showed peaks of xylanase activity at pH 3.5 and pH 8.5. When assayed at pH 3.5, optimum activity was detected at 50°C, with a second peak of activity at 90°C. When assayed at pH 8.5, optimum activity was seen at 80°C. The crude enzyme was thermostable at 55°C for at least 4 h and retained about 60% activity. Gel filtration of the 50–80% ammonium sulphate-precipitated fraction of the crude culture filtrate separated into two peaks of xylanase with specific activities of 393 and 2,457 U (mg protein)^–1. The two peaks showing xylanase activity had molecular masses of 13 and 18 kDa. Zymogram analysis of xylanase of crude culture filtrate as well as the 50–80% ammonium sulphate-precipitated fraction showed two distinct xylanase activity bands on native PAGE. The crude culture filtrate also showed moderate activities of -xylosidase and -l-arabinofuranosidase, which could act synergistically with xylanase in attacking xylan. This is the first report showing the potential application of crude culture filtrate of a marine fungal isolate possessing thermostable, cellulase-free alkaline xylanase activity in biobleaching of paper pulp.