Effect of manganese on preferential degradation of lignin by Pleurotus ostreatus during solid-state fermentation.

Practical utilization of the polysaccharides in the lignocellulosic complex is limited because of the high lignin content of the complex. In this study we focused on the effect of Mn on lignin and cellulose biodegradation during solid-state fermentation by the edible mushroom Pleurotus ostreatus. Preferential degradation of lignin was enhanced by the addition of Mn(II) to cotton stalks at concentrations ranging from 30 to 620 micrograms of Mn per g. This effect was most apparent when we compared mineralization rates of [14C] lignin with mineralization rates of [14C] cellulose. Enhanced selectivity was also observed when we analyzed residual organic matter at the end of the fermentation period by using crude fiber analysis. The cellulose fraction in the original material was 1.8 times larger than the cellulose fraction of lignin. The cellulose/lignin ratio increased during 32 days of solid-state fermentation from 2.5 in the control to 3.3 following the addition of Mn to the medium. The in vitro digestibility value for fermented cotton stalks was 53% of the dry matter. Addition of 600 micrograms of Mn per g to the cotton stalks resulted in a digestibility value of 65.4%. Enhancement of preferential lignin degradation could be result of either increased activity of the ligninolytic enzymes or production of Mn (III), which might preferentially degrade aromatic structures in the lignocellulosic complex.     

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.     

黄孢原毛平革菌对松木、稻草和芦苇去木质化作用的研究

为确定黄孢原毛平革菌对不同植物材料的去木质化作用,以pH、干物质重、半纤维素、纤维素和木质素为主要技术指标,比较黄孢原毛平革菌对松木、稻草和芦苇降解能力的差异。松木、芦苇在发酵过程中pH呈下降趋势,稻草呈上升趋势。在干物质重、半纤维素、纤维素降解率三个指标上皆为松木〈芦苇〈稻草,在木质素降解率上则为松木〈稻草〈芦苇,且差异显著。表明黄孢原毛平革菌对不同植物材料去木质化能力有较大差异,其中芦苇的木质素降解率为13％,是三种材料中最易于被去木质化的。     

Solid-state fermentations for biological delignification

Biological delignification, using white-rot fungi to liberate cellulose and hemicellulose from their complex with lignin, can aid the use of lignocellulose as ruminant animal feed, a source of sugars, and pulp. Solid-state fermentation is the method of choice for biological delignification. Lignin-degrading fungi, their ecophysiological requirements, and the design of solid-state fermenters for delignification are discussed. Capital and operating costs for solid-state fermentation can be kept low, and the lignocellulosic substrate is likely to be the major component of the cost of the delignified product. Experience in the operation of biological delignification processes at pilot plant or larger scale is needed to establish realistic process costs.     

Growth of Azotobacter vinelandii in a solid-state fermentation of technical lignin

Azotobacter vinelandii was cultured on technical lignin, derived from Kraft pulping processes, for biofertilizer production in solid-state fermentation. The effects of the ratio of technical lignin to corn straw, initial water content, and material bed depth on the microorganisms were studied in detail. At 30 degrees C, technical lignin to corn straw at the ratio of 1:0.75, the bed depth of 5 cm, and 67% moisture content, A. vinelandii was grown and reached 4.2 x 10(10) cfu g(-1) dry rot after 36 h.     

白腐菌混合菌降解木质素最佳条件的优化

通过正交试验对3种白腐菌混合菌降解竹材木质素的条件进行优化,结果表明,在温度为32℃、pH3.0、固体发酵时间20 d、培养液与竹材基质质量百分比110%时降解木质素的效率最高.在此基础上,研究了两种诱导剂对白腐菌混合菌降解木质素的影响.结果表明,两种诱导剂均能促进木质素的降解,其中H_2O_2在浓度1%时,木质素降解率高达62.9%,苯甲酸在浓度0.1%时,木质素降解率最高,为67.8%.     

木质素降解菌的筛选及混合菌发酵降解秸秆的研究

农作物秸秆是农业生产的副产品,也是一项重要的生物资源。由于其成分结构的特殊性所导致的难降解问题,一直成为了转化利用秸秆的难题。目前,利用混合菌将秸秆纤维素转化为蛋白质、乙醇、乙酸、乳酸等研究已逐渐为人们所重视。本文通过马铃薯琼脂平板培养、马铃薯液体摇瓶培养和稻草秸秆固态发酵,从6株常见的食用白腐菌中筛选出了生长优势较强、产漆酶酶活高的平菇HF。为了让秸秆得到更好的降解和利用,采用平菇和康氏木霉二步混合发酵法;通过不同的组合方式,发现H6-T10组合得出的降解效果最好,其木质素降解率达到44.77%,纤维素降解率达到41.48%。     

Preferential degradation of phenolic lignin units by two white rot fungi.

The differential biodegradation of phenolic and nonphenolic (C-4-etherified) lignin units in wheat straw treated with the white rot fungi Pleurotus eryngii and Phanerochaete chrysosporium was investigated under solid-state fermentation conditions. Two analytical techniques applied to permethylated straw were used for this purpose, i.e., alkaline CuO degradation and analytical pyrolysis (both followed by gas chromatography-mass spectrometry for product identification). Despite differences in the enzymatic machinery produced, both ligninolytic fungi caused a significant decrease in the relative amount of phenolic lignin units during the degradation process. Nevertheless, no differences in the biodegradation rates of phenolic and etherified cinnamic acids were observed. Changes in lignin composition and cinnamic acid content were also analyzed in the phenolic and nonphenolic lignin moieties. The results obtained are discussed in the context of the enzymatic mechanisms of lignin biodegradation.     

Lignocellulose Degradation during Solid-State Fermentation: Pleurotus ostreatus versus Phanerochaete chrysosporium

Lignocellulose degradation and activities related to lignin degradation were studied in the solid-state fermentation of cotton stalks by comparing two white rot fungi, Pleurotus ostreatus and Phanerochaete chrysosporium. P. chrysosporium grew vigorously, resulting in rapid, nonselective degradation of 55% of the organic components of the cotton stalks within 15 days. In contrast, P. ostreatus grew more slowly with obvious selectivity for lignin degradation and resulting in the degradation of only 20% of the organic matter after 30 days of incubation. The kinetics of ¹⁴C-lignin mineralization exhibited similar differences. In cultures of P. chrysosporium, mineralization ceased after 18 days, resulting in the release of 12% of the total radioactivity as ¹⁴CO₂. In P. ostreatus, on the other hand, 17% of the total radioactivity was released in a steady rate throughout a period of 60 days of incubation. Laccase activity was only detected in water extracts of the P. ostreatus fermentation. No lignin peroxidase activity was detected in either the water extract or liquid cultures of this fungus. 2-Keto-4-thiomethyl butyric acid cleavage to ethylene correlated to lignin degradation in both fungi. A study of fungal activity under solid-state conditions, in contrast to those done under defined liquid culture, may help to better understand the mechanisms involved in lignocellulose degradation.     

Solubilisation and mineralisation of [14C]lignocellulose from wheat straw by Streptomyces cyaneus CECT 3335 during growth in solid-state fermentation

Nine Streptomyces strains were screened for their ability to solubilise and mineralise ¹⁴C-labelled lignin during growth in solid-state fermentation. Streptomyces viridosporus was confirmed as an active lignin-degrading organism along with a new isolate, Streptomyces sp. UAH 15, further classified as Streptomyces cyaneus CECT 3335. This organism was able to solubilise and mineralise the [¹⁴C]lignin fraction of lignocellulose (44.96 ± 1.77% and 3.41 ± 0.48% respectively) after 21 days of incubation. Cell-free filtrates from Streptomyces sp. grown in solid-state fermentation were capable of solubilising up to 20% of the [¹⁴C]lignin after 2 days incubation, with most of the product detected in the acid-soluble rather than in the water-soluble fraction. Identification of the extracellular enzymes produced during growth of S. cyaneus CECT 3335 revealed that extracellular peroxidase and phenol oxidase activities were present, with the activity of phenol oxidase being 100 times greater than peroxidase activity. The activity of these two enzymes was found to correlate with both solubilisation and mineralisation rates. This is the first report of phenol oxidase activity produced by a Streptomyces strain during growth in solid-state fermentation. A role for the enzyme in the solubilisation and mineralisation of lignocellulose by S. cyaneus is suggested. Received: 12 May 1997 / Accepted: 19 May 1997     

Analysis of a Modern Hybrid and an Ancient Sugarcane Implicates a Complex Interplay of Factors in Affecting Recalcitrance to Cellulosic Ethanol Production

Abundant evidence exists to support a role for lignin as an important element in biomass recalcitrance. However, several independent studies have also shown that factors apart from lignin are also relevant and overall, the relative importance of different recalcitrance traits remains in dispute. In this study we used two genetically distant sugarcane genotypes, and performed a correlational study with the variation in anatomical parameters, cell wall composition, and recalcitrance factors between these genotypes. In addition we also tracked alterations in these characteristics in internodes at different stages of development. Significant differences in the development of the culm between the genotypes were associated with clear differential distributions of lignin content and composition that were not correlated with saccharification and fermentation yield. Given the strong influence of the environment on lignin content and composition, we hypothesized that sampling within a single plant could allow us to more easily interpret recalcitrance and changes in lignin biosynthesis than analysing variations between different genotypes with extensive changes in plant morphology and culm anatomy. The syringyl/guaiacyl (S/G) ratio was higher in the oldest internode of the modern genotype, but S/G ratio was not correlated with enzymatic hydrolysis yield nor fermentation efficiency. Curiously we observed a strong positive correlation between ferulate ester level and cellulose conversion efficiency. Together, these data support the hypothesis that biomass enzymatic hydrolysis recalcitrance is governed by a quantitative heritage rather than a single trait.     

裂褶菌F17固态发酵产锰过氧化物酶及其发酵动力学模型的建立

白腐真菌分泌的锰过氧化物酶是木质素降解酶系统的主要组分,对木质素解聚,纸浆和染料的脱色均有重要作用.利用裂褶菌F17在自行设计的通气托盘式反应器中,以松木屑、稻草及黄豆粉为混合营养基质进行固态发酵生产锰过氧化物酶.在自制通气托盘式反应器中,裂褶菌F17能够产生锰过氧化物酶,发酵96 h时,最高酶活力达到13.51 U/...     

Lignin Peroxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes Produced by Phlebia radiata during Solid-State Fermentation of Wheat Straw

The white rot fungus Phlebia radiata 79 (ATCC 64658) produces lignin peroxidase (LiP), manganese peroxidase (MnP), glyoxal oxidase (GLOX), and laccase in the commonly used glucose low-nitrogen liquid medium. However, the enzymes which this fungus utilizes for selective removal of lignin during degradation of different lignocellulosic substrates have not been studied before. Multiple forms of LiP, MnP, GLOX, and laccase were purified from P. radiata culture extracts obtained after solid-state fermentation of wheat straw. However, the patterns of extracellular lignin-modifying enzymes studied were different from those of the enzymes usually found in liquid cultures of P. radiata. Three LiP isoforms were purified. The major LiP isoform from solid-state cultivation was LiP2. LiP3, which has usually been described as the major isoenzyme in liquid cultures, was not expressed during straw fermentation. New MnP isoforms have been detected in addition to the previously reported MnPs. GLOX was secreted in rather high amounts simultaneously with LiP during the first 2 weeks of growth. GLOX purified from P. radiata showed multiple forms, with pIs ranging from 4.0 to 4.6 and with a molecular mass of ca. 68 kDa.     

Decolorization of synthetic textile dyes by lignin peroxidase of Phanerochaete chrysosporium

Neem hull waste (containing a high amount of lignin and other phenolic compounds) was used for lignin peroxidase production byPhanerochaete chrysosporum under solid-state fermentation conditions. Maximum decolorization achieved by partially purified lignin peroxidase was 80% for Porocion Brilliant Blue HGR, 83 for Ranocid Fast Blue, 70 for Acid Red 119 and 61 for Navidol Fast Black MSRL. The effects of different concentrations of veratryl alcohol, hydrogen peroxide, enzyme and dye on the efficiency of decolorization have been investigated. Maximum decolorization efficiency was observed at 0.2 and 0.4 mmol/L hydrogen peroxide, 2.5 mmol/L veratryl alcohol and pH 5.0 after a 1-h reaction, using 50 ppm of dyes and 9.96 mkat/L of enzyme.     

木质纤维素酶高产菌株的筛选及其对玉米秸秆降解效果的影响

为了提高玉米秸秆中木质素的降解率,从腐烂的树枝和土壤中筛选木质纤维素酶高产菌株,以秸秆为唯一C源富集培养后,采用PDA-愈创木酚法进行初筛,筛选出产木质素酶真菌5株,然后以玉米秸秆为主要C源进行固态发酵和复筛。结果表明:第5号菌株在发酵玉米秸秆5 d后,使木质素的降解率达到最高(34.95%),粗纤维的降解率达到20.00%,显著高于其他4种菌株(P<0.05),其羧甲基纤维素酶比酶活达到116.35 U/g;10 d后,其木质素酶比酶活达到最高(45.64 U/g)。     

Glucoamylase production by Aspergillus niger on rice bran is improved by adding nitrogen sources

Corn steep liquor, peptone or NH _inf4 ^sup+ salts increased the yield of glucoamylase from Aspergillus niger growing in a solid-state fermentation on rice bran up to 360 IU/g dry substrate over 96 h at 30°C.The authors are with the Biotechnology Unit, Regional Research Laboratory, CSIR, Trivandrum-695 019, India;     

Degradation of natural lignins and lignocellulosic substrates by soil-inhabiting fungi imperfecti

Abstract: The most powerful lignin-degraders among the 82 microbial strains isolated during a screening of ligninolytic microorganisms from forest soil were identified as Penicillium chrysogenum, Fusarium oxysporum and Fusarium solani . These fungi imperfecti mineralized 27.4%, 23.5% and 22.6% of ¹⁴C-labelled milled wood lignin (MWL) from wheat straw after 28 days of incubation in liquid media. Degradation of MWL from pine by P. chrysogenum was 8% and 19% when it was evaluated by spectrophotometry and Klason lignin, respectively, but this substrate was hardly mineralized. All fungi were able to attack the hemicellulosic, cellulosic and also lignin fractions of wheat straw during solid-state fermentation, F. solani being capable of degrading about 25% of both carbohydrates and lignin. When the selected fungi were tested for dye decolourization, they all readily attacked the polymeric dye Remazol brilliant blue R (RBBR) and also poly R-478 to a minor extent.     

Novel bioconversion of wheat straw to bio-organic fertilizer in a solid-state bioreactor

In order to increase the eco-efficiency and overall availability of naturally renewable resource, the novel bioconversion of steam-exploded wheat straw to bio-organic fertilizer containing N₂-fixer, P and K solubilizers was investigated. The conversion was performed in solid-state fermentation (SSF) with periodic air-forced pressure oscillation (PAPO). The results showed that SSF-PAPO was competitive with the conventional solid-state fermentation (cSSF) in biomass accumulation and wheat straw digestion. With solid–liquid ratio 1:3, microbial biomass production at 72 h was high up to 2 × 10¹¹ cfu g⁻¹, nearly twice as that in cSSF. The degradation rate of cellulose, hemicellulose and lignin after fermentation in SSF-PAPO reached 48.57 ± 10.66, 84.77 ± 2.75 and 2.15 ± 10.11, respectively, which was greater than that of 29.30 ± 10.28%, 33.47 ± 4.85% and 0.53 ± 9.07% in cSSF, correspondingly. The SSF-PAPO system displayed unique advantage, by a novel gas phase control strategy on gas concentration and heat gradient, on the bioconversion of wheat straw to the bio-organic fertilizer.     

Correlation of brightening with cumulative enzyme activity related to lignin biodegradation during biobleaching of kraft pulp by white rot fungi in the solid-state fermentation system.

Biobleaching of hardwood unbleached kraft pulp (UKP) by Phanerochaete chrysosporium and Trametes versicolor was studied in the solid-state fermentation system with different culture media. In this fermentation system with low-nitrogen and high-carbon culture medium, pulp brightness increased by 15 and 30 points after 5 days of treatment with T. versicolor and P. chrysosporium, respectively, and the pulp kappa number decreased with increasing brightness. A comparison of manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase activities assayed by using fungus-treated pulp and the filtrate after homogenizing the fungus-treated pulp in buffer solution indicated that enzymes secreted from fungi were adsorbed onto the UKP and that assays of these enzyme activities should be carried out with the treated pulp. Time course studies of brightness increase and MnP activity during treatment with P. chrysosporium suggested that it was difficult to correlate them on the basis of data obtained on a certain day of incubation, because the MnP activity fluctuated dramatically during the treatment time. When brightness increase and cumulative MnP, LiP, and laccase activities were determined, a linear relationship between brightness increase and cumulative MnP activity was found in the solid-state fermentation system with both P. chrysosporium and T. versicolor. This result suggests that MnP is involved in brightening of UKP by white rot fungi.     

Progress in biopulping of non-woody materials: Chemical, enzymatic and ultrastructural aspects of wheat straw delignification with ligninolytic fungi from the genus Pleurotus

Abstract: During screening of basidiomycetes for wheat straw delignification, considerable lignin degradation with a limited attack to cellulose was attained with Pleurotus eryngii . Straw solid-state fermentation (SSF) was optimized, and the enzymatic mechanisms for lignin degradation were investigated. No lignin peroxidase was detected under liquid or SSF conditions, but high laccase and aryl-alcohol oxidase levels were found. The latter enzyme has been fully characterized in PI. eryngii and it seems to be involved in a cyclic redox system for H₂0₂ generation from aromatic compounds. Results obtained using homoveratric acid suggest that Pleurotus laccase could be involved in degradation of phenolic and non-phenolic lignin moieties. Histological and ultrastructural studies provided some general morphological characteristics of the fungal attack on wheat straw. Whereas a simultaneous degradation pattern was observed in straw treated with Phanerochaete chrysosporium , PI. eryngii caused partial degradation of middle lamella and separation of individual sclerenchymatic fibers. When these straw samples were subjected to refining tests, energy saving after biological treatment was the highest in the case of straw treated with PI. eryngii , which also produced the lowest substrate loss. From these results, a correlation between preferential removal of lignin, separation of sclerenchymatic fibers and pulping properties was provided during fungal treatment of wheat straw.