Laccase production in solid‐state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×10⁵ U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.     

Effect of growth substrate,method of fermentation,and nitrogen source on lignocellulose-degrading enzymes production by white-rot basidiomycetes

The exploration of seven physiologically different white rot fungi potential to produce cellulase, xylanase, laccase, and manganese peroxidase (MnP) showed that the enzyme yield and their ratio in enzyme preparations significantly depends on the fungus species, lignocellulosic growth substrate, and cultivation method. The fruit residues were appropriate growth substrates for the production of hydrolytic enzymes and laccase. The highest endoglucanase (111 U ml⁻¹) and xylanase (135 U ml⁻¹) activities were revealed in submerged fermentation (SF) of banana peels by Pycnoporus coccineus. In the same cultivation conditions Cerrena maxima accumulated the highest level of laccase activity (7,620 U l⁻¹). The lignified materials (wheat straw and tree leaves) appeared to be appropriate for the MnP secretion by majority basidiomycetes. With few exceptions, SF favored to hydrolases and laccase production by fungi tested whereas SSF was appropriate for the MnP accumulation. Thus, the Coriolopsis polyzona hydrolases activity increased more than threefold, while laccase yield increased 15-fold when tree leaves were undergone to SF instead SSF. The supplementation of nitrogen to the control medium seemed to have a negative effect on all enzyme production in SSF of wheat straw and tree leaves by Pleurotus ostreatus. In SF peptone and ammonium containing salts significantly increased C. polyzona and Trametes versicolor hydrolases and laccase yields. However, in most cases the supplementation of media with additional nitrogen lowered the fungi specific enzyme activities. Especially strong repression of T. versicolor MnP production was revealed.     

Effects of exogenous fibrolytic enzymes on in vitro ruminal fermentation of substrates with different forage:concentrate ratios

Batch cultures of mixed rumen micro-organisms were used to study the effects of three fibrolytic enzymes (xylanase from Trichoderma viride (XYL) and fibrolytic enzymes from Aspergillus niger (ASP) and Trichoderma longibrachiatum (TR)) on the fermentation of three substrates composed of grass hay:concentrate in the proportions (dry matter (DM) basis) of 0.7:0.3 (HF), 0.5:0.5 (MF) and 0.3:0.7 (LF). Enzymes were characterized for xylanase, endoglucanase, exoglucanase and amylase activities, and were supplied at rates of 40 and 80 enzymatic units/g substrate DM. In 8 h incubations, all enzymes increased (P=0.048 to P<0.001) the true degradability of substrate DM and the production of acetate, propionate, total volatile fatty acids (VFA) and gas. After 24 h incubation, some of the observed effects disappeared, but all enzymes still increased (P=0.028 to P<0.001) the degradability of substrate acid detergent fibre and the production of acetate, propionate and total VFA. For all enzymes, the effects on ruminal variables were less marked at 24 than at 8 h of incubation. Only few significant (P=0.044 to P=0.001) enzyme × substrate interactions were detected, although the magnitude of the response for each substrate varied with the enzyme. When considering the amount of organic matter apparently fermented (OMAF) and the methane:OMAF ratio as main variables, TR80 produced the greatest increase in OMAF (17.0%) for HF substrate, with ASP80 and TR40 having similar values (11.1 and 12.6%), and XYL and ASP40 showing no effects (P>0.05). A decrease (P<0.05) of methane:OMAF ratio was only found for TR80 at 8 h (17.4%). All enzymes, with the exception of ASP40, increased (P<0.05) OMAF at 8 h for MF substrate (11.3–25.4%), TR80 showing the greatest response. After 24 h of incubation, both doses of XYL and TR increased (P<0.05) OMAF (mean value 8.2%) and decreased methane:OMAF ratio (mean value 9.5%). All enzymes increased significantly OMAF with LF substrate at 8 h (7.5–19.9%), but after 24 h no effect (P>0.05) was detected on OMAF and methane:OMAF ratio. In general, few differences were detected between both doses of enzymes, which indicate than the used enzymes would be effective in enhancing ruminal degradation of substrates at a dose lower than 80 enzymatic units/g substrate DM.     

Lignocellulose-degrading enzyme production by white-rot <Emphasis Type="Italic">Basidiomycetes</Emphasis> isolated from the forests of Georgia

The production of lignocellulolytic enzymes by eleven basidiomycetes species isolated from two ecosystems of Georgia was investigated for the first time under submerged (SF) and solid-state fermentation (SSF) of lignocellulosic by-products. Notable intergeneric and intrageneric differences were revealed with regard to the extent of hydrolase and oxidase activity. Several fungi produced laccase along with hydrolases in parallel with growth during the trophophase, showing that the synthesis of this enzyme is not connected with secondary metabolism. The lignocellulosic substrate type had the greatest impact on enzyme secretion. Some of the substrates significantly stimulated lignocellulolytic enzyme synthesis without supplementation of the culture medium with specific inducers. Exceptionally high carboxymethyl cellulase (CMCase, 122 U ml⁻¹) and xylanase (195 U ml⁻¹) activities were revealed in SF of mandarin peelings by Pseudotremella gibbosa IBB 22 and of residue after ethanol production (REP) by Fomes fomentarius IBB 38, respectively. The SSF of REP by T. pubescens IBB 11 ensured the highest level of laccase activity (24,690 U l⁻¹), whereas the SSF of wheat bran and SF of mandarin peels provided the highest manganese peroxidase activity (570–620 U l⁻¹) of Trichaptum biforme IBB 117. Moreover, the variation of lignocellulosic growth substrate provides an opportunity to obtain enzyme preparations containing different ratios of individual enzymes.     

Isolation of cellulose-hydrolytic bacteria and applications of the cellulolytic enzymes for cellulosic biohydrogen production

Nine cellulolytic bacterial strains were isolated from soil sample taken in southern Taiwan. Through 16S rRNA sequence matching; eight of those isolates belong to Cellulomonas sp., while the other one belongs to Cellulosimicrobium cellulans. The activity of cellulolytic enzymes (cellulases and xylanase) produced from those strains was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (xylan, rice husk and rice straw) used for growth. HPLC analysis confirmed the bacterial hydrolysis of these cellulosic substrates for soluble sugars production. The efficiency of fermentative H₂ production from the enzymatically hydrolyzed rice husk was examined with seven H₂-producing pure bacterial isolates. With an initial reducing sugar concentration of 0.36 g l⁻¹, only Clostridium butyricum CGS5 exhibited efficient H₂ production from the rice husk hydrolysates with a cumulative H₂ production and H₂ yield of 88.1 ml l⁻¹ and 19.15 mmol H₂ (g reducing sugar)⁻¹ (or 17.24 mmol H₂ (g cellulose)⁻¹), respectively.     

Use of sugarcane bagasse and grass hydrolysates as carbon sources for xylanase production by Bacillus circulans D1 in submerged fermentation

The use of sugarcane bagasse and grass as low cost raw material for xylanase production by Bacillus circulans D1 in submerged fermentation was investigated. The microorganism was cultivated in a mineral medium containing hydrolysate of bagasse or grass as carbon source. High production of enzyme was obtained during growth in media with bagasse hydrolysates (8.4 U/mL) and in media with grass hydrolysates (7.5 U/mL). Xylanase production in media with hydrolysates was very close to that obtained in xylan containing media (7.0 U/mL) and this fact confirm the feasibility of using this agro-industrial byproducts by B. circulans D1 as an alternative to save costs on the enzyme production process.     

Production of acidic lipase by a mutant of Aspergillus niger NCIM 1207 in submerged fermentation

Mutants of Aspergillus niger NCIM 1207, isolated by subjecting conidia to UV-irradiation, were tested for the production of lipase (glycerol ester hydrolase EC 3.1.1.3). Mutants UV-10 and ANCR-1 showed seven fold and five fold enhanced productivity of enzyme, respectively, over the wild strain in shake flask culture when grown in SOB medium containing 1% olive oil. Maximum lipase activity (41 IU/ml) was obtained in the culture broth when UV-10 was grown in medium supplemented with 0.5% Triton X-100. A higher concentration of oil in the medium did not help lipase production in the case of mutant UV-10. Similarly no increase in enzyme levels was observed when mutant UV-10 was grown in medium supplemented with glucose. However, the addition of glucose in the medium resulted in increased levels of lipase production by wild strain, Aspergillus niger NCIM 1207.     

Immobilization of fungus Aspergillus sp. by a novel cryogel technique for production of extracellular hydrolytic enzymes

Aerobic cells of a fungus isolate Aspergillus sp. CX-1 have been immobilized in macroporous cryoPAG and in different composite cryoPAGs — fibrous adjunct carriers. The productivity of the extracellular enzymes (exo-1.4-β-glucanase, endo-1.4-β-glucanase, β-glucosidase and xylanase), and the viability, growth and ultrastructure of the immobilized fungus have been studied. The enzyme activities and stability during long-term repeated batch cultivation in the immobilized fungus were higher than in free mycelia when batch cultivated. The fungus immobilized in the composite cryoPAG, containing polypropylene non-woven fabric, possessed the highest exo-1.4-β-glucanase activity, the longest durability of enzyme production (85 days) and the most reliable mechanical strength. The fungus immobilized in porous composite cryogel possessed a variety of advantages including easy control of cryogel porosity, improved mechanical strength and durability, simplicity of construction, high enzyme productivity and high stability.     

Exopolysaccharides, proteins and lipids in Pleurotus pulmonarius submerged culture using different carbon sources

For many years mushrooms have been consumed and appreciated by their nutritional value, and medicinal properties. The traditional mushroom cultivation takes too long and the macrofungi biotechnology has not been explored in its full potential yet. The goal of this work was to observe if different carbon sources could improve the yield and diversify fungi nutrient composition in submerged culture.Pleurotus pulmonarius mycelia and exopolysacharide productions were evaluated using glucose, galactose, xylose and arabinose. The mycelia yield varied depending on the culture medium, and galactose showed to be the best carbon source to produce EPS. Samples that showed the highest protein contents were grown with xylose (19.44%) and arabinose (26.05%). Furthermore, the biomass cultivated with these carbohydrates and with galactose showed five essential amino acids. All cultured biomass showed low lipid contents (∼1%), being composed mainly of unsaturated fatty acids. All EPS fractions showed as main structures glucans and mannogalactans.     

Optimization of submerged culture conditions for mycelial polysaccharide production in Cordyceps pruinosa

Cordyceps pruinosa is an entomogenous fungus noteworthy for its various bioactivities. The influence of synthetic medium and cultural conditions on polysaccharides production was investigated in shake flask culture. In the present study, optimal medium and submerged culture conditions were investigated using an orthogonal layout. Media and cultural conditions including potato starch 2% (w/v), sucrose 2.5%, soybean 0.5%, beef extract 0.5%, yeast extract 0.1%, KCl 0.02%, K₂HPO₄ 0.1%, MgSO₄·7H₂O 0.05%, pH 7.0, inoculum size 5%, medium capacity 50 ml/250 ml flask, dispersant 15 beads, culture time 7 days were employed. In fermentation medium, sucrose, beef extract and yeast extract were replaced with molasses of sucrose, groundnut and Vitamin B complex, respectively. Under optimal culture conditions, the yield of polysaccharides production was 9.51 g l⁻¹ after 54 h of fermentation in a 25 l fermenter, which was approximately twice as high as that in shake flask cultures. In addition the entire period of fermentation was shorted to around 1/4 of flask culture time (9 days). Thus, it will meet closely the requirements of industrial fermentation scale of polysaccharides production in C. pruinosa.     

Effect of nitrogen source on lignocellulolytic enzyme production by white-rot basidiomycetes under solid-state cultivation

Summary The effect of additional nitrogen sources on lignocellulolytic enzyme production by four species of white-rot fungi (Funalia trogii IBB 146, Lentinus edodes IBB 363, Pleurotus dryinus IBB 903, and P. tuberregium IBB 624) in solid-state fermentation (SSF) of wheat straw and beech tree leaves was strain- and substrate-dependent. In general, the yields of hydrolytic enzymes and laccase increased by supplementation of medium with an additional nitrogen source. This stimulating effect of additional nitrogen on enzyme accumulation was due to higher biomass production. Only xylanase specific activity of P. dryinus IBB 903 and laccase specific activity of L. edodes IBB 363 increased significantly (by 66% and 73%, respectively) in SSF of wheat straw by addition of nitrogen source to the control medium. Additional nitrogen (20 mM) repressed manganese peroxidase (MnP) production by all fungi tested. The study of the nitrogen concentration effect revealed that 10 mM peptone concentration was optimal for cellulase and xylanase accumulation by P. dryinus IBB 903. While variation of the peptone concentration did not cause the change in MnP yield, elevated concentrations of this nutrient (20–40 mM) led to a 2–3-fold increase of P. dryinus IBB 903 laccase activity. About 10–20 mM concentration of NH₄NO₃ was optimal for cellulase and xylanase production by F. trogii IBB 146. However, neither the laccase nor the MnP yield was significantly changed by the additional nitrogen source.     

Using spectrophotometry and spectral mapping technique for the study of the production of manganese-dependent and manganese-independent peroxidases by Pleurotus ostreatus

The activity of manganese-dependent and manganese-independent peroxidases produced by Pleurotus ostreatus in culture media composed of agro-residues was measured by visible spectrophotometry. The overall enzyme activity and its selectivity were separated by using spectral mapping technique followed by nonlinear mapping. The relationships between the parameters of enzyme production and the composition of culture media and fermentation time was assessed by stepwise regression analysis. Calculations proved that the addition of extract of straw to the culture media significantly decreased the overall production of both enzymes, whereas the selectivity of enzyme production was influenced by amount of potato extract and the concentration of total sugar in the culture media. Enzyme activity depended quadratically on the fermentation time.     

Laccase and Manganese Peroxidase Activities of Phellinus Robustus and Ganoderma Adspersum Grown on Food Industry Wastes in Submerged Fermentation

Phellinus robustus produced both laccase (700–4,000 U l⁻¹) and manganese peroxidase (MnP) (1,000–11,300 U l⁻¹) in fermentation of nine food wastes, whereas Ganoderma adspersum produced only laccase (600–34,000 U l⁻¹). Glucose provided high laccase and MnP activity of P. robustus but repressed enzyme production by G. adspersum. Ammonium sulphate and ammonium tartrate increased the P. robustus laccase yield (3-fold), whereas the accumulation of MnP was not enhanced by additional nitrogen.     

蛹虫草多糖和D-甘露醇深层发酵的非结构动力学模型

多糖和D-甘露醇是蛹虫草的重要药理活性成分。本文开展了蛹虫草在分批发酵过程中同时生产多糖和D-甘露醇的发酵动力学研究。利用Sigmoid函数构建了蛹虫草菌丝生长、糖基质消耗、多糖和D-甘露醇的非结构动力学模型,并根据Boltzmann方程拟合求解出各模型参数。结果显示,各模型的实测值和预测值拟合度较好。蛹虫草比生长速率在第1.0天达到最大值（μ_max）1.244d^-1;底物葡萄糖的比消耗速率在第0.6天达到最大值（q_{S, max}）2.163d^-1;多糖比合成速率在第2.0天达到最大值（q_{P, max}）51.852mg/(g·d);D-甘露醇比合成速率在第0.99天左右达到最大值（q_{D, max}）37.963mg/(g·d)。蛹虫草多糖的形成与菌丝细胞的生长呈现部分生长关联型,而D-甘露醇的形成与细胞生长呈现生长关联型关系。研究结果为利用分批发酵规模化同时发酵生产蛹虫草多糖和D-甘露醇提供了理论依据。     

Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation

The kinetics of β-fructofuranosidase (Ffase) production by Aspergillus niger in submerged (SmF) and solid-state fermentation (SSF) systems was investigated. The maximum productivity of Ffase (81.8 U/l per h) was obtained in SSF for 72 h while it was 18.3 U/l per h in SmF for 120 h. The productivity of extra cellular Ffase produced in SSF was 5-fold higher than in SmF. Optimization of fermentation medium for Ffase production was carried out using De Meo's fractional factorial design with seven components such as (NH₄)₂SO₄, KH₂PO₄, FeSO₄, MgSO₄ · 7H₂O, sucrose, urea and yeast extract. The media designed for SmF after two steps of optimization supported the growth of A. niger and higher productivity of Ffase (58.3 U/l per h) than with the medium before optimization. The optimized medium of SmF when used in SSF, did not improve the Ffase productivity and therefore medium for SSF was optimized independent of SmF. After two optimization steps, the media was defined for SSF which supported the growth and high level of Ffase productivity (149.1 U/l per h) in SSF compared to the medium before optimization (81.8 U/l per h) and optimized medium for SmF (58.3 U/l per h). Our results suggested that the optimized media for SmF and SSF for the production of Ffase have to be different.     

Algal biomass: A sustainable,economical and renewable approach for microbial production of pectinolytic enzymes using submerged and solid state fermentation techniques

In the past decade, algal waste has been used as useful natural resource for production of enormous range of products that have wide economical and commercial importance. Pectinases are group of enzymes that have wide commercial applications. Hence, current study was designed to utilize algal biomass for the production of pectinases using submerged (SmF) and solid state fermentation (SSF) techniques. Different algal sources including brown (Dictyopteris polypodioides, Sargassum wightii and Dictyopteris divaricata) and green algae (Ulva lactuca and Codium tomentosum) were used and U. lactuca was found to be the most suitable substrate. Several bacterial and fungal strains were screened and among them Bacillus licheniformis KIBGE-IB4 was selected based on maximum pectinase production. SmF and SSF were studied utilizing U. lactuca as a substrate and results revealed that enzyme production was favoured by SmF (2457?±?3.31?U?mg^?1) as compared to SSF (1432?±?1.46?U?mg^?1). Parametric optimization of pectinase production indicated that B. licheniformis KIBGE-IB4 requires 10.0?g L^–1 U. lactuca as a biomass in the medium with a pH 7.0 when incubated at 37?°C for 24 hours. Likewise, production of pectinase using algal resource was also compared with that of the conventional agricultural biomass and it was observed that when U. lactuca was used, the selected bacterial isolate produced a higher yield of enzyme than sugarcane bagasse and rice husk. Hence, it is anticipated that algal biomass can be efficiently utilized as an environmental friendly bioresource for the production of industrially important hydrolytic enzymes.     

普鲁兰酶在需钠弧菌中的分泌表达与发酵优化

普鲁兰酶是一种淀粉脱支酶,因其分子量较大,胞外分泌表达难度较高。需钠弧菌(Vibrionatriegens)是一种新型的蛋白表达宿主,拥有高效的蛋白合成效率。本研究使用基因组整合T7 RNA聚合酶表达框的V.natriegens VnDX为宿主,构建了产全长普鲁兰酶PulA及其截短突变体PulN2的重组需钠弧菌,分析了信号肽、发酵温度、诱导剂浓度、甘氨酸浓度及发酵时间等条件对产酶的影响,并且对比了2种普鲁兰酶在V.natriegens VnDX与大肠杆菌(Escherichia coli)BL21(DE3)中的胞外产酶能力。研究结果显示,普鲁兰酶PulA和PulN2在V.natriegens VnDX中的胞外酶活为61.6 U/mL和64.3 U/mL,分别为E.coli BL21(DE3)最大酶活力的110%和62%。上述结果表明V.natriegens VnDX可以分泌表达大分子量的全长普鲁兰酶PulA,本研究可为其他大分子量蛋白在V.natriegens VnDX中的分泌表达提供参考和借鉴。     

Experimental design to enhance the production of l-(+)-lactic acid from steam-exploded wood hydrolysate using Rhizopus oryzae in a mixed-acid fermentation

The fermentation of hemicellulosic hydrolysate from Pinus taeda chips, using the fungal culture Rhizopus oryzae, was carried out to produce l-(+)-lactic acid and to optimize and enhance the biological conversion of reducing sugar into l-(+)-lactic acid using the experimental design to evaluate the culture conditions. The first factorial design based on surface response with five factors (agitation level, substrate concentration, CaCO₃ concentration, C/N and C/P ratios) at low levels and one medium point was performed to optimize culture conditions. The second study tested two factors (substrate concentration and C/N ratio) at three levels. The statistical analysis of the data obtained from the factorial study showed that a C/N ratio of 35 and substrate concentration of 90 g/litre were the best conditions to produce l-(+)-lactic acid with R. oryzae on P. taeda hydrolysate, but in this case the statistical projection was not correct and the real optimized conditions were C/N ratio of 55 and substrate concentration of 75 g/litre of reducing sugar.