Use of diluted medium in repeated-batch fermentation for production of lignin peroxidase by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Lignin peroxidase has been extensively studied due to the potential use of this enzyme in environmental pollution control. Important aspects of the production of the enzyme by the white rot fungus, Phanerochaete chrysosporium, include the improvement of yield results and cell maintenance. In the present work, Phanerochaete chrysosporium was immobilized in polyurethane foam and used for repeated-batch fermentations with various dilution of the initial medium (D), and lignin peroxidase production was investigated. The peak of 283 ± 17.5 U lignin peroxidase/l production rate was obtained at a D of 1/5, with significantly lower production rates seen at higher and lower dilution ratios. When six cycles of repeated-batch fermentation were conducted using a D of 1/5, the results revealed that at least four cycles of repeated-batch fermentation were possible with a high lignin peroxidase production rate under a cut-off value of 178 ± 3.87 U/l. Furthermore, the cell-free culture broth could be successfully concentrated to 2,800 U/l by ultrafiltration. Thus, the present study shows that optimizing the dilution of the utilized nutritional medium can improve repeated batch production of lignin peroxidase from immobilized P. chrysosporium, in terms of both cycle number and output.     

Fungal pretreatment of lignocellulose by Phanerochaete chrysosporium to produce ethanol from rice straw

Phanerochaete chrysosporium is a wood‐rot fungus that is capable of degrading lignin via its lignolytic system. In this study, an environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods. To maximize the production of manganese peroxidase, which is the primary lignin‐degrading enzyme, culture medium was optimized using response surface methodologies including the Plackett–Burman design and the Box–Behnken design. Fermentation of 100 g of rice straw feedstock containing 35.7 g of glucan (mainly in the form of cellulose) by cultivation with P. chrysosporium for 15 days in the media optimized by response surface methodology was resulted in a yield of 29.0 g of glucan that had an enzymatic digestibility of 64.9% of the theoretical maximum glucose yield. In addition, scanning electronic microscopy, confocal laser scanning microscopy, and X‐ray diffractometry revealed significant microstructural changes, fungal growth, and a reduction of the crystallinity index in the pretreated rice straw, respectively. When the fungal‐pretreated rice straw was used as a substrate for ethanol production in simultaneous saccharification and fermentation (SSF) for 24 h, the ethanol concentration, production yield and the productivity were 9.49 g/L, 58.2% of the theoretical maximum, and 0.40 g/L/h, respectively. Based on these experimental data, if 100 g of rice straw are subjected to fungal pretreatment and SSF, 9.9 g of ethanol can be produced after 96 h, which is 62.7% of the theoretical maximum ethanol yield. Biotechnol. Bioeng. 2009; 104: 471–482 © 2009 Wiley Periodicals, Inc.     

Hyperactivation and thermostabilization of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> lignin peroxidase by immobilization in xerogels

This is a continuation of our previous paper on production of lignin peroxidase (LiP) by Phanerochaete chrysosporium in solid substrate fermentation (SSF) medium of corncobs. The enzyme was purified by ammonium sulphate precipitation and ion-exchange fast protein liquid chromatography. Maximum yield of LiP was 13.7 U/gds (units per gram dry substrate) after 5 days of SSF with 70% moisture and 20% (v/w) inoculum. The approximate molecular mass of purified LiP, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 38 kDa. The pH and temperature optima for the LiP were 4 and 40°C, respectively. Immobilization of LiP in hydrophobic xerogels caused hyperactivation of LiP and enhanced its thermostability properties. The K _M and V _max values for immobilized LiP were 10.56 mg/ml and 16.67 μmol/min (120.49 U/mg of protein) as compared to 13 mg/ml and 11.76 μmol/min (85 U/mg of protein), respectively, for free LiP using veratryl alcohol as substrate.     

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.     

Screening of a fungus capable of powerful and selective delignification on wheat straw

Aims: To screen and characterize a novel fungus with powerful and selective delignification capability on wheat straw. Methods and Results: A fungus capable of efficient delignification under solid‐state fermentation (SSF) conditions on wheat straw was screened. After 5 days of incubation, 13·07% of the lignin was removed by fungal degradation, and 7·62% of the holocellulose was lost. Furthermore, 46·53% of the alkali lignin was removed after 2 days of liquid fermentation. The fungus was identified as Fusarium concolor based on its morphology and an analysis of its 18S rDNA gene sequence. The molecular weight distribution of lignin was evaluated by gel permeation chromatography. Enzyme assay indicated that the fungus produced laccase, cellobiose dehydrogenase, xylanase and cellulase during the incubation period. Intracellular lignin peroxidase, manganese peroxidase and laccase were produced during liquid fermentation. Conclusions: We have successfully screened a fungus, F. concolor, which can efficiently degrade the lignin of wheat straw, with slight damage to the cellulose, after 5 days of SSF. Significance and Impact of the Study: The newly isolated strain could be used in pretreatment of lignocellulose materials prior to biopulping, bioconversion into fuel and substrates for the chemical industry.     

Bioethanol production from tension and opposite wood of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> using organosolv pretreatment and simultaneous saccharification and fermentation

During tree growth, hardwoods can initiate the formation of tension wood, which is a strongly stressed wood on the upper side of the stem and branches. In Eucalyptus globulus, tension wood presents wider and thicker cell walls with low lignin, similar glucan and high xylan content, as compared to opposite wood. In this work, tension and opposite wood of E. globulus trees were separated and evaluated for the production of bioethanol using ethanol/water delignification as pretreatment followed by simultaneous saccharification and fermentation (SSF). Low residual lignin and high glucan retention was obtained in organosolv pulps of tension wood as compared to pulps from opposite wood at the same H-factor of reaction. The faster delignification was associated with the low lignin content in tension wood, which was 15% lower than in opposite wood. Organosolv pulps obtained at low and high H-factor (3,900 and 12,500, respectively) were saccharified by cellulases resulting in glucan-to-glucose yields up to 69 and 77%, respectively. SSF of the pulps resulted in bioethanol yields up to 35 g/l that corresponded to 85–95% of the maximum theoretical yield on wood basis, considering 51% the yield of glucose to ethanol conversion in fermentation, which could be considered a very satisfactory result compared to previous studies on the conversion of organosolv pulps from hardwoods to bioethanol. Both tension and opposite wood of E. globulus were suitable raw materials for organosolv pretreatment and bioethanol production with high conversion yields.     

Comparative studies on lignin and polycyclic aromatic hydrocarbons degradation by basidiomycetes fungi

A total of 130 wild basidiomycetes fungi were collected and identified. The polycyclic aromatic hydrocarbons (PAHs) degradation by the potential Phellinus sp., Polyporus sulphureus (in liquid state fermentation (LSF), solid state fermentation (SSF), in soil) and lignin biodegradation were compared with those of a bacterial isolate and their corresponding cocultures. The PAHs degradation was higher in LSF and the efficiency of the organisms declined in SSF and in soil treatment. Phellinus sp. showed better degradation in SSF and in soil. Bacillus pumilus showed higher degradation in LSF. B. pumilus was seen to have lower lignin degradation than the fungal cultures and the cocultures could not enhance the degradation. Phellinus sp. which had higher PAHs and lignin degradation showed higher biosurfactant production than other organism. Manganese peroxidase (MnP) was the predominant enzyme in Phellinus sp. while lignin peroxidase (Lip) was predominant in P. sulphureus.     

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

     

Optimization of fermentation conditions for the production of ethanol from sago starch using response surface methodology

The quantitative effects of temperature, pH and time of fermentation were investigated on simultaneous saccharification and fermentation (SSF) of ethanol from sago starch with glucoamylase (AMG) and Zymomonas mobilis ZM4 using a Box–Wilson central composite design protocol. The SSF process was studied using free enzyme and free cells and it was found that with sago starch, maximum ethanol concentration of 70.68 g/l was obtained using a starch concentration of 140 g/l, which represents an ethanol yield of 97.08%. The optimum conditions for the above yield were found to be a temperature of 36.74 °C, pH of 5.02 and time of fermentation of 17 h. Thus by using the central composite design, it is possible to determine the accurate values of the fermentation parameters where maximum production of ethanol occurs.     

Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation

Alkalophilic Bacillus subtilis ASH produced high levels of xylanase using easily available inexpensive agricultural waste residues such as wheat bran, wheat straw, rice husk, sawdust, gram bran, groundnut and maize bran in solid-state fermentation (SSF). Among these, wheat bran was found to be best substrate. Xylanase production was highest after 72 h of incubation at 37 °C and at a substrate to moisture ratio of 1:2 (w/v). The inoculum level of 15% resulted in maximum production of xylanase. The enzyme production was stimulated by the addition of nutrients such as yeast extract, peptone and beef extract. In contrast, addition of glucose and xylose repressed the production of xylanase. The extent of repression by glucose (10%, w/v) was 81% and it was concentration-dependent. Supplementation of the medium with 4% xylose caused 59% repression. Under optimized conditions, xylanase production in SSF (8,964 U of xylanase/g dry wheat bran) was about twofold greater than in submerged fermentation. Thus, B. subtilis produced a very high level of xylanase in SSF using inexpensive agro-residues, a level which is much higher than that reported by any other bacterial isolate. Furthermore, the enzyme was produced at room temperature and with tap water without the addition of any mineral salt in SSF, leading to a marked decrease in the cost of xylanase production, which enhances its industrial potential.     

Solid state fermentation for the production of α-amylase from Penicillium chrysogenum using mixed agricultural by-products as substrate

Production of α-amylase from local isolate, Penicillium chrysogenum, under solid-state fermentation (SSF) was carried out in this study. Different agricultural by-products, such as wheat bran (WB), sunflower oil meal (SOM), and sugar beet oil cake (SBOC), were used as individual substrate for the enzyme production. WB showed the highest enzyme activity (750 U/gds). Combination of WB, SOM, and SBOC (1:3:1 w/w/w) resulted in a higher enzyme yield (845 U/gds) in comparison with the use of the individual substrate. This combination was used as mixed solid substrate for the production of α-amylase from P. chrysogenum by SSF. Fermentation conditions were optimized. Maximum enzyme yield (891 U/gds) was obtained when SSF was carried out using WB + SOM + SBOC (1:3:1 w/w/w), having initial moisture of 75%, inoculum level of 20%, incubation period of 7 days at 30°C. Galactose (1% w/w), urea and peptone (1% w/w), as additives, caused increase in the enzyme activity.     

Solid state fermentation of Achras zapota lignocellulose by Phanerochaete chrysosporium

The biological transformation of lignocellulose of Achras zapota by white rot fungi, Phanerochaete chrysosporium, in solid state fermentation (SSF) was studied for 28 days. The kinetic transformation of lignocellulose was monitored through the determination of acid soluble and acid insoluble lignin content, total organic carbon (TOC) and chemical oxygen demand (COD). The lignolytic enzymes, lignin peroxidase (LiP) and manganese peroxidase (MnP) were quantified on weekly intervals. The degradation of lignin and other structural moieties of A. zapota lignocellulose were confirmed by high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The degradation of lignin was increased after 7 days of fermentation with the release of water soluble and fermentable products. The LiP and MnP activities were increased in the first week of SSF and lignin degradation was also set to increase. This was accompanied with increase in COD by 94.6% and TOC by 80% and lignin content was decreased by 76%. The maximum activities of the enzymes LiP and MnP in extracellular fluid of SSF under nitrogen limitation, at pH 5.0, at temperature 37 degrees C and at 60% humidity were 2100 U/L and 1200 U/L.     

Solid-state production of lignin peroxidase (LiP) and manganese peroxidase (MnP) by Phanerochaete chrysosporium using steam-exploded straw as substrate

In the used media mainly consisting of steam-exploded wheat straw, the straw, which could replace expensive veratryl alcohol, might act not only as nutrient, but also as inducer of lignin enzymes. The activities of the enzymes lignin peroxidase (LiP) and manganese peroxidase (MnP) in solid-state fermentation (SSF) were far higher than in submerged fermentation (SmF). Under optimal conditions of SSF, the maximum activities of the enzymes Lip and MnP were 2600 and 1375 U/L, respectively. Thus, this would pave the way for production and application of lignin enzymes on a large scale.     

Kinetics of wheat straw solid-state fermentation with Trametes versicolor and Pleurotus ostreatus — lignin and polysaccharide alteration and production of related enzymatic activities

Summary The kinetics of straw solid-state fermentation (SSF) with Trametes versicolor and Pleurotus ostreatus was investigated to characterize the delignification processes by these white-rot fungi. Two successive phases could be defined during straw transformation, characterized by changes in respiratory activity, changes in lignin and polysaccharide content and composition, increase in in-vitro digestibility, and enzymatic activities produced by the fungi. Lignin composition was analysed after CuO alkaline degradation, and decreases in syringyl/guaiacyl and syringyl/p-hydroxyphenyl ratios and cinnamic acid content were observed during the fungal treatment. An increase in the phenolic acid yield, revealing fungal degradation of side-chains in lignin, was produced by P. ostreatus. The highest xylanase level was produced by P. ostreatus, and exocellulase activity was nearly absent from straw treated with this fungus. Lactase activity was found in straw treated with both fungi, but lignin peroxidase was only detected during the initial phase of straw transformation with T. versicolor. High levels of H₂O₂-producing aryl-alcohol oxidase occurred throughout the straw SSF with P. ostreatus. Offprint requests to: A. T. Martínez     

Degradation of the herbicide bentazon as related to enzyme production by Phanerochaete chrysosporium in two solid substrate fermentation systems

Enzyme production and degradation of the herbicide bentazon by Phanerochaete chrysosporium growing on straw (solid substrate fermentation, SSF) and the effect of nitrogen and the hydraulic retention time (HRT) were studied using a small bioreactor and batch cultures. The best degradation of bentazon was obtained in the low nitrogen treatments, indicating participation of the ligninolytic system of the fungus. The treatments that degraded bentazon also had manganese peroxidase (MnP) activity, which seemed to be necessary for degradation. Pure MnP (with Mn(II) and H₂O₂) did not oxidize bentazon. However, in the presence of MnP, Mn(II) and Tween 80, bentazon was slowly oxidized in a H₂O₂-independent reaction. Bentazon was a substrate of pure lignin peroxidase (LiP) and was oxidized significantly faster (22,000–29,000 times) as compared to the MnP-Tween 80 system. Although LiP was a better enzyme for bentazon oxidation in vitro, its role in the SSF systems remains unclear since it was detected only in treatments with high nitrogen and high HRT where no degradation of bentazon occurred. Inhibition of LiP activity may be due to phenols and extractives present in the straw.     

利用农业废弃物固态发酵裂褶菌F17产锰过氧化物酶的基质优化(英文)

锰过氧化物酶(MnP)在环保领域有着广阔的应用前景。目前,利用廉价基质生产MnP,尤其是利用工农业废弃物生产MnP的研究受到了国内外学者的广泛关注。本实验利用响应面方法从几种不同的农业废弃物中筛选裂褶菌F17(Schizophyllum sp.F17)产MnP的固态发酵基质。结果表明,以0.52∶0.15∶0.33的比例组成的松木屑、稻草和黄豆粉的混合基质为发酵产MnP的最佳基质,发酵第6天MnP的活力最高,达到11.18 U/g。因此,利用农业废弃物固态发酵产锰过氧化物酶在减低酶的成本和环境污染物治理方面具有重要的意义。     

Solid-state fermentation for production of griseofulvin on rice bran using Penicillium griseofulvum

Griseofulvin is a secondary metabolite produced from fungal species that have morphology suitable for solid-state fermentation (SSF). Reports on production of griseofulvin by SSF are scarce. The present work investigates SSF for griseofulvin production, optimization of its process parameters vis-à-vis the conventional submerged fermentation and its downstream processing from the same. Rice bran adjusted to an initial moisture content (IMC) of 50% (v/w) inoculated with 1 mL of a suspension of 10(6) spores/mL under agitation at 250 rpm containing the modified Czapek-Dox medium and additional 0.1% choline chloride as a precursor gave a yield of griseofulvin in 9 days that was comparable to submerged fermentation after 28 days. The yield of griseofulvin (microg/g dry biomass) was comparable in SSF and submerged fermentation. The biomass was estimated by estimation of chitin. Discussions on the effect of each parameter in SSF have also been included.     

Tannase production by Aspergillus fumigatus MA under solid-state fermentation

A tannase yielding fungal culture identified as Aspergillus fumigatus MA was isolated from the effluent collected from a local small scale tannery. The fungal culture produced high yields of extracellular tannase under solid-state fermentation (SSF) using different agro forest residues such as Amla leaves (Phyllanthus emblica), Ber leaves (Zyzyphus mauritiana), Jamun leaves (Syzygium cumini), Jamoa leaves (Syzygium sp.) and Keekar leaves (Acacia nilotica). Among different substrates used, Jamun leaves yielded maximal extra-cellular production of tannase. Various parameters were studied to optimize the extracellular yield of tannase under SSF. The maximum yield of 174.32 U g⁻¹ was obtained at 25°C after 96 h of incubation at pH 5.0. The tap water was used as a moistening agent. A substrate to tap water ratio of 1:1 was found to best for tannase production. Supplementation of the medium with ammonium sulfate as nitrogen source had enhanced tannase production whereas glucose had decreased the enzyme production. This is the first report on production of tannase by Aspergillus fumigatus MA, giving a much higher yield of enzyme under SSF with Jamun leaves as the substrate.     

Bioconversion of grape must into modulated gluconic acid production by Aspergillus niger ORS-4.410

AIMS: Analysis of regulators for modulated gluconic acid production under surface fermentation (SF) condition using grape must as the cheap carbohydrate source, by mutant Aspergillus niger ORS-4.410. Replacement of conventional fermentation condition by solid-state surface fermentation (SSF) for semi-continuous production of gluconic acid by pseudo-immobilization of A. niger ORS-4.410. METHODS AND RESULTS: Grape must after rectification was utilized for gluconic acid production in batch fermentation in SF and SSF processes using mutant strain of A. niger ORS-4.410. Use of rectified grape must led to the improved levels of gluconic acid production (80-85 g l(-1)) in the fermentation medium containing 0.075% (NH4)2HPO4; 0.1% KH2PO4 and 0.015% MgSO4.7H2O at an initial pH 6.6 (+/-0.1) under surface fermentation. Gluconic acid production was modulated by incorporating the 2% soybean oil, 2% starch and 1% H2O2 in fermentation medium at continuously high aeration rate (2.0 l min(-1)). Interestingly, 95.8% yield of gluconic acid was obtained when A. niger ORS-4.410 was pseudo-immobilized on cellulose fibres (bagasse) under SSF. Four consecutive fermentation cycles were achieved with a conversion rate of 0.752-0.804 g g(-1) of substrate into gluconic acid under SSF. CONCLUSIONS: Use of additives modulated the gluconic acid production under SF condition. Semi-continuous production of gluconic acid was achieved with pseudo-immobilized mycelia of A. niger ORS-4.410 having a promising yield (95.8%) under SSF condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioconversion of grape must into modulated gluconic acid production under SSF conditions can further be employed in fermentation industries by replacing the conventional carbohydrate sources and expensive, energy consuming fermentation processes.     

Laccase, cellulase and xylanase activities during growth ofPleurotus sajor-caju on sagohampas

Sagohampas, the fibrous pith residue left after starch extraction from sago palm, is abundant at sago-processing factories and can be used as a substrate for the production of laccase by solid substrate fermentation (SSF) withPleurotus sajorcaju, an edible mushroom. The fungus grown onhampas with an adjusted carbon : nitrogen ratio of 35:1, exhibited high laccase activity together with variable cellulase (0.3-2.8 U/g) and xylanase (0.9-10.1 U/g) activity. The maximum amount of laccase produced was approximately 17.7 U/g after 6 days of SSF using 4-week-old inoculum at a density of 10%. With the mature four-week inoculum, laccase activity increased 12-fold compared to that achieved with two-week-old inoculum. The optimum pH and temperature of the crude laccase were 6.0 and 50‡C, respectively. The apparent K_m and V_max values obtained were 0.073 mM and 0.962 U/min, respectively. The maximum laccase activity could be almost doubled after 6 days of fermentation by addition of 0.2 mM vanillin or ferulic acid; the cellulose to lignin ratio increased significantly during the 12 days of SSF, from 2.74 in the control to 3.3, when 0.2 mM of either vanillin or ferulic acid was added to the substrate.