Enzymatic hydrolysis of cellulose and various pretreated wood fractions

Three strains of Trichoderma-T. reesei C30, T. reesei QM9414, and Trichoderma species E-58-were used to study the enzymatic hydrolysis of pretreated wood substrates. ach of the culture filtrates was incubated with a variety of commercially prepared cellulose substrates and pretreated wood substrates. Solka floc was the most easily degraded commercial cellulose. The enzyme accessibility of steam-exploded samples which had been alkali extracted and then stored wet decreased with the duration of the steam treatment. Air drying reduced the extent of hydrolysis of all the samples but had a greater effect on the samples which had previously shown the greatest hydrolysis. Mild pulping using 2% chlorite increased the enzymatic hydrolysis of all the samples. Steam explosion was shown to be an excellent pretreatment. The results indicate that the distribution of the lignin as well as the surface area of the cellulosic substrate are important features in enzymatic hydrolysis.     

Biotechnology report: single cell proteins from cellulosic wastes

Conventional sources of protein cannot meet the present or projected needs for human consumption. Single cell proteins from fermentation of petroleum and cellulosic wastes are likely sources of additional protein. The volume of cellulosic wastes is sufficient to supply all additional protein needs on a continuing basis for cellulose is a renewable resource. Both mesophilic and thermophilic microorganisms utilize cellulose at reasonable rates. Biodegradation of lignin and lignin–cellulose complexes constitutes a major obstacle to commercial utilization of cellulosic wastes. Thermophilic actinomyces appear to be the most effective organisms for single cell protein production from cellulosic wastes.     

Optimization of cellobiose dehydrogenase production by Schizophyllum commune and effect of the enzyme on kraft pulp bleaching by ligninases

The effects of various parameters on cellobiose dehydrogenase (CDH) production by Schizophyllum commune AS 5.391 were investigated. Among different carbon and nitrogen sources tested, dewaxed cotton powder and diammonium hydrogen phosphate produced the highest titers of CDH. S. commune AS 5.391 produced CDH only when grown on cellulosic substrates but the lignin-related compounds veratryl alcohol and guaiacol had no effect on CDH production. The optimum pH for CDH production was 4.5. Addition of succinate and Tween 80 to the medium significantly improved the enzyme yield. Optimized culture conditions were obtained and the highest level of CDH was 150 U/l. CDH could facilitate kraft pulp lignin degradation by ligninases. The influence of CDH on kraft pulp bleaching by ligninases was also studied.     

Degradation of cellulosic materials by Sporotrichum thermophile culture filtrate for sugar production

Sporotrichum thermophile Apinis, was the most active carboxymethyl-cellulose (CMC)-ase producer among seven thermophilic and four thermotolerant fungal species isolated from Egyptian soil and screened for their ability to produce extracellular cellulase in culture media containing CMC as a sole carbon source. The fungus also efficiently hydrolysed filter paper cellulose. Comparison of various untreated and alkali-treated cellulosic and lignocellulosic materials as substrates for cellulase production by S. thermophile revealed the most easily degraded substrate was sugarcane bagasse at 2% concentration. This substrate when alkali treated was the most susceptible to enzymic hydrolysis by culture filtrates of S. thermophile grown on untreated bagasse. Optimum hydrolysis was obtained after 18 h incubation with the filtrate at pH 3·5–4 and 45°C. Alkali treatment of bagasse reduced its lignin content significantly and the culture filtrate of S. thermophile grown on untreated bagasse was found to contain xylanase and polygalacturonase in addition to cellulase and cellobiase.     

Bioconversion of cellulose into ethanol by Clostridium thermocellum--product inhibition

Direct anaerobic bioconversion of cellulosic substances into ethanol by Clostridium thermocellum ATCC 27405 has been carried out at 60 degrees C and pH 7.0 (initial for 100 L) under continuous sparging of oxygen free nitrogen in a culture vessel. Raw bagasse, mild alkali-treated bagasse, and solka floc were used as substrates. The extent of conversion of raw bagasse (cellulose, 50%; hemicellulose, 25%; lignin, 19%) was observed as 52% (w/w) and 79% (w/w) in the case of mild alkali and steam-treated bagasse (cellulose, 72%; hemicellulose, 11%; lignin, 12%), respectively. Use of bagasse concentration above 10 g/L showed a decreased rate in ethanol production. An inoculum age between 28-30 h and cell mass content of 0.027-0.036 g/L (dry basis) were used. The results obtained with raw and pretreated bagasse have been compared with those of highly pure Solka Floc (hemicellulose, 10%). Studies on the product inhibition indicated a linear fall of the percent of survivors with time. An Arrhenius type correlation between the cell decay rate constant and the product concentration was predicted. Even at low levels, the inhibitory effects of products on cell viability, the specific growth rate, and extracellular cellulase enzyme were observed.     

Selective solvent delignification for fermentation enhancement

Cellulose and hemicellulose in renewable biomass resources such as cornstover and wheat straw have been examined as substrates for the production of ethanol. A mixed culture of selected strains of Clostridium thermocellum and Clostridium thermosaccharolyticum are used to accomplish both the hydrolysis and fermentation of these carbohydrates in a single step. However, lignin and related phenolic materials are shown to diminish the rate, extent, and yield at which these carbohydrates can be utilized for ethanol production. In order to overcome this problem, a selective solvent pretreatment with alkaline-ethanol-water mixtures was examined for the delignification of cellulosic biomass under conditions where very little loss of fermentable carbohyrates results. Under optimal conditions, up to 67% of the initial lignin in cornstover can be extracted while 95% of the alpha-cellulose and pentosan carbohydrates remain insoluble. Subsequent mixed culture fermentation of the treated material has shown a 400% increase in the rate of degradation and greater than 85% utilization of the substrate. The effects of various extraction parameters on delignification kinetics and subsequent fermentation performance are discussed.     

The effect of isolated lignins,obtained from a range of pretreated lignocellulosic substrates,on enzymatic hydrolysis

The influence of the residual lignin remaining in the cellulosic rich component of pretreated lignocellulosic substrates on subsequent enzymatic hydrolysis was assessed. Twelve lignin preparations were isolated by two isolation methods (protease treated lignin (PTL) and cellulolytic enzymatic lignin (CEL)) from three types of biomass (corn stover, poplar, and lodgepole pine) that had been pretreated by two processes (steam and organosolv pretreatments). Comparative analysis of the isolated lignin showed that the CEL contained lower amounts of carbohydrates and protein than did the PTL and that the isolated lignin from corn stover contained more carbohydrates than did the lignin derived from the poplar and lodgepole pine. The lower yields of acid insoluble lignin (AIL) obtained from the corn stover when using the PTL method indicated that the lignin from the corn stover had a higher hydrophilicity than did the lignin from the poplar and lodgepole pine. The isolated lignin preparations were added to the reaction mixture containing crystalline cellulose (Avicel) and their possible effects on enzymatic hydrolysis were assessed. It was apparent that the lignin isolated from lodgepole pine and steam pretreated poplar decreased the hydrolysis yields of Avicel, whereas the other isolated lignins did not appear to decrease the hydrolysis yields significantly. The hydrolysis yields of the pretreated lignocellulose and those of Avicel containing the PTL showed good correlation, indicating that the nature of the residual lignin obtained after pretreatment significantly influenced hydrolysis. Biotechnol. Bioeng. 2010;105: 871–879. © 2009 Wiley Periodicals, Inc.     

Characterization of the cellulolytic activity of a Bacillus isolate

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.     

Characterization of the cellulolytic activity of a Bacillus isolate.

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.     

N+注入选育白腐菌及其降解油菜秸秆木质纤维素的研究

以白腐菌WY01为出发菌,利用N＋注入技术选育出一株遗传性状稳定的漆酶高产诱变菌株WY02,经过60 d的发酵培养,其产酶量由出发菌的13.75 U/g增加到52.5 U/g,即产酶量提高了2.82倍;诱变菌株WY02对油菜秸秆中的木质素、半纤维素和纤维素的降解率分别为54.1%,39.1%,32.8%,用红外光谱法（IR）分析经诱变菌株降解后的油菜秸秆中木质素官能团的变化,用于阐明诱变菌株对油菜秸秆中木质素的生物降解机制。结果表明：油菜秸秆经白腐菌诱变菌株降解后,其木质素含量明显降低。木质素与苯环相连的C=O键、木质素侧链上CH2结构以及木质素单体（紫丁香基和愈创木基）被部分降解,木质素的苯环结构遭到一定程度的破坏。     

Effects of lignin on the anaerobic degradation of (ligno) cellulosic wastes by rumen microorganisms

Summary There appeared to be a clear correlation between the lignin content (% of TS) of several waste and natural materials and their degradability by rumen microorganisms. Materials with lignin contents higher than 25% were not degraded within 72 h. The effects of Kraft pine lignin and some lignin monomers on filter paper degradation, methane production and CMCase activity were tested. Testing these compounds in concentrations comparable to natural conditions showed minor effects. At higher concentrations p-coumaric acid strongly inhibited cellulose degradation and methane production in batch cultures. Influence of lignin compounds on degradation is discussed in relation to structural effects and enzyme or growth inhibition.     

Delignification of straw with ozone to enhance biodegradability

Summary Wheat straw was treated with ozone to remove the lignin and increase its biodegradability. The attack of ozone on straw is not selective. Lignin and carbohydrates are oxidized concurrently though the rate of reaction with the latter is slower. A 50% reduction of the original lignin content is optimal for enzymatic hydrolysis. After treatment, 75% of the cellulose in straw is degraded within 24 h as compared to 20% in untreated straw. During ozonation lignin is converted to soluble products which to a great extent are biodegradable and thus yield a useful byproduct. At the moment, ozonation ranks among the more expensive methods of treatment. However, the economics may be improved by reducing the cost of ozone production; this is likely to take place in the near future due to technological improvements, and by reducing the ozone consumption by optimizing the process of ozonation.     

Inhibition of trehalose breakdown increases new carbon partitioning into cellulosic biomass in Nicotiana tabacum

Validamycin A was used to inhibit in vivo trehalase activity in tobacco enabling the study of subsequent changes in new C partitioning into cellulosic biomass and lignin precursors. After 12-h exposure to treatment, plants were pulse labeled using radioactive ¹¹CO₂, and the partitioning of isotope was traced into [¹¹C]cellulose and [¹¹C]hemicellulose, as well as into [¹¹C]phenylalanine, the precursor for lignin. Over this time course of treatment, new carbon partitioning into hemicellulose and cellulose was increased, while new carbon partitioning into phenylalanine was decreased. This trend was accompanied by a decrease in phenylalanine ammonia-lyase activity. After 4 d of exposure to validamycin A, we also measured leaf protein content and key C and N metabolite pools. Extended treatment increased foliar cellulose and starch content, decreased sucrose, and total amino acid and nitrate content, and had no effect on total protein.     

Protease Ti from Escherichia coli requires ATP hydrolysis for protein breakdown but not for hydrolysis of small peptides

Protease Ti, a new ATP-dependent protease in Escherichia coli, degrades proteins and ATP in a linked process, but these two hydrolytic functions are catalyzed by distinct components of the enzyme. To clarify the enzyme's specificity and the role of ATP, a variety of fluorogenic peptides were tested as possible substrates for protease Ti or its two components. Protease Ti rapidly hydrolyzed N-succinyl(Suc)-Leu-Tyr-amidomethylcoumarin (AMC) (Km = 1.3 mM) which is not degraded by protease La, the other ATP-dependent protease in E. coli. Protease Ti also hydrolyzed, but slowly, Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. However, it showed little or no activity against basic or other hydrophobic peptides, including ones degraded rapidly by protease La. Component P, which contains the serine-active site, by itself rapidly degrades the same peptides as the intact enzyme. Addition of component A, which contains the ATP-hydrolyzing site and is necessary for protein degradation, had little or no effect on peptide hydrolysis. N-Ethylmaleimide, which inactivates the ATPase, did not inhibit peptide hydrolysis. In addition, this peptide did not stimulate the ATPase activity of component A (unlike protein substrates). Thus, although the serine-active site on component P is unable to degrade proteins, it is fully functional against small peptides in the absence of ATP. At high concentrations, Suc-Leu-Tyr-AMC caused a complete inhibition of casein breakdown, and diisopropylfluorophosphate blocked similarly the hydrolysis of both protein and peptide substrates. Thus, both substrates seem to be hydrolyzed at the same active site on component P, and ATP hydrolysis by component A either unmasks or enlarges this proteolytic site such that large proteins can gain access to it.     

Influence of steam pretreatment severity on post‐treatments used to enhance the enzymatic hydrolysis of pretreated softwoods at low enzyme loadings

It is recognized that some form of post‐treatment will usually be required if reasonable hydrolysis yields (>60%) of steam pretreated softwood are to be achieved when using low enzyme loadings (5 FPU/g cellulose). In the work reported here we modified/removed lignin from steam pretreated softwood while investigating the influence that the severity of pretreatment might have on the effectiveness of subsequent post‐treatments. Although treatment at a lower severity could provide better overall hemicellulose recovery, post‐treatment was not as effective on the cellulosic component. Pretreatment at medium severity resulted in the best compromise, providing reasonable recovery of the water soluble hemicellulose sugars and the use of post‐treatment conditions that significantly increased the enzymatic hydrolysis of the water insoluble cellulosic component. Post‐treatment with alkaline hydrogen peroxide or neutral sulfonation resulted in 62% cellulose hydrolysis at an enzyme loading of 5 FPU/g cellulose, which was four times greater than was obtained when the cellulosic fraction was not post‐treated. When the enzyme loading was increased to 15 FPU/g cellulose, the post‐treated cellulosic fraction was almost completely hydrolyzed to glucose. Despite the higher lignin content (44%) of the sulfonated substrate, similar hydrolysis yields to those achieved after alkaline peroxide post‐treatment (14% lignin content) indicated that, in addition to lignin removal, lignin modification also plays an important role in influencing the effectiveness of hydrolysis when low enzyme loadings are used. Biotechnol. Bioeng. 2011;108: 2300–2311. © 2011 Wiley Periodicals, Inc.     

Maize Stover and Cob Cell Wall Composition and Ethanol Potential as Affected by Nitrogen Fertilization

Maize (Zea mays L.) stover and cobs are potential feedstock sources for cellulosic ethanol production. Nitrogen (N) fertilization is an important management decision that influences cellulosic biomass and grain production, but its effect on cell wall composition and subsequent cellulosic ethanol production is not known. The objectives of this study were to quantify the responses of maize stover (leaves, stalks, husks, and tassel) and cob cell wall composition and theoretical ethanol yield potential to N fertilization across a range of sites. Field experiments were conducted at rainfed and irrigated sites in Minnesota, USA, over a 2-year period. Stover cell wall polysaccharides, pentose sugar concentration, and theoretical ethanol yield decreased as N fertilization increased. Stover Klason lignin increased with N fertilization at all sites. Cob cell wall composition was less sensitive to N fertilization, as only pentose and Klason lignin decreased with N fertilization at two and one site(s), respectively, and hexose increased with N fertilization at one of eight sites. Cob theoretical ethanol yield was not affected by N fertilization at any site. These results indicate variation in stover cellulosic ethanol production is possible as a result of N management. This study also demonstrated that cell wall composition and subsequent theoretical ethanol yield of maize cobs are generally more stable than those with stover because of overall less sensitivity to N management.     

Protein damage and degradation by oxygen radicals. I. general aspects

Aggregation, fragmentation, amino acid modification, and proteolytic susceptibility have been studied following exposure of 17 proteins to oxygen radicals. The hydroxyl radical (.OH) produced covalently bound protein aggregates, but few or no fragmentation products. Extensive changes in net electrical charge (both + and -) were observed. Tryptophan was rapidly lost with .OH exposure, and significant production of bityrosine biphenol occurred. When incubated with cell-free extracts of human and rabbit erythrocytes, rabbit reticulocytes, or Escherichia coli, most .OH-modified proteins were proteolytically degraded up to 50 times faster than untreated proteins. The exceptions were alpha-casein and globin, which were rapidly degraded without .OH modification. ATP did not stimulate the degradation of .OH-modified proteins, but alpha-casein was more rapidly degraded. Leupeptin had little effect under any condition, and degradation was maximal at pH 7.8. The data indicate that proteins which have been denatured by .OH can be recognized and degraded rapidly and selectively by intracellular proteolytic systems. In both red blood cells and E. coli, the degradation appears to be conducted by soluble, ATP-independent (nonlysosomal) proteolytic enzymes. In contrast with the above results, superoxide (O2-) did not cause aggregation or fragmentation, tryptophan loss, or bityrosine production. The combination of .OH + O2- (+O2), which may mimic biological exposure to oxygen radicals, induced charge changes, tryptophan loss, and bityrosine production. The pattern of such changes was similar to that seen with .OH alone, although the extent was generally less severe. In contrast with .OH alone, however, .OH + O2- (+O2) caused extensive protein fragmentation and little or no aggregation. More than 98% of the protein fragments had molecular weights greater than 5000 and formed clusters of ionic and hydrophobic bonds which could be dispersed by denaturing agents. The results indicate a general sensitivity of proteins to oxygen radicals. Oxidative modification can involve direct fragmentation or may provide denatured substrates for intracellular proteolysis.     

Fermentative production of acetic acid from various pure and natural cellulosic materials by Clostridium lentocellum SG6

Clostridium lentocellum SG6 fermented various pure crystalline cellulosic materials efficiently with maximum acetic acid yield (gram acetic acid/gram substrate) of 0.67, at low substrate (8 g l⁻¹) concentration. The strain grew poorly on crude biopolymers but fermented them easily after alkali treatment, when grown with 8 g substrate l⁻¹ concentration of alkali-extracted cotton straw (AECS), paddy straw (AEPS) and sorghum stover (AESS) etc. The acetic acid to substrate (A/S) ratios were similar to those obtained with pure cellulosic materials. An increase in substrate concentration led to a decreased A/S ratio and a decreased percentage of substrate degraded. At high substrate concentration of 75 g filter paper l⁻¹, the strain SG6 converted 63.2 g filter paper into 31.28 g acetic acid l⁻¹. At 100 g l⁻¹ concentrations, AECS and AEPS served as the best substrates for acetic acid production when compared with other biopolymers. A maximum amount of 30.98 and 30.86 g acetic acid was produced from 70.6 g AEPS and 70.1 g AESS l⁻¹ of medium by strain SG6, respectively. Acetic acid production of 0.67 g g⁻¹ pure cellulose (Whatman No. 1 filter paper), 0.63 g g⁻¹ of alkali-treated cotton straw (AECS) are the highest among the cellulolytic bacteria reported so far in mono culture fermentations with pure and native cellulosic materials. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromatographic detection of lignin-carbohydrate complexes in annual plants by derivatization in ionic liquid

The opportunity for detecting the presence and the amount of lignin-carbohydrate complexes (LCCs) in renewable feedstocks is a major issue for the complete utilization of biomass. Indeed, LCCs are known to shield cellulose from enzymatic hydrolysis, reducing the efficiency of the digestion processes needed for the production of biobased products. This study is focused on the chromatographic characterization of lignocellulose from agricultural residues (rice husk, wheat straw) and herbaceous energy crops ( Arundo donax , Miscanthus sinesis ) and their fractionation products (hemicellulose, cellulose, and lignin). Exploiting alternative chemical derivatizations on the aforementioned samples, it was possible to discern the connectivity among the various lignocellulosic components. The complete acetylation and benzoylation of the milled native substrates in ionic liquid media, and the systematic comparison between their GPC-UV chromatograms collected at different wavelengths has revealed itself as a straightforward technique in the detection of LCCs. This novel approach proved an extensive connectivity between the lignin and the hemicellulosic for all the analyzed specimens, whereas the cellulosic fraction was conceived as a substantially unbound moiety, accounting for the sample composition at higher molecular weights. Moreover, the collected lignin fractions were extensively characterized by means of (31)P NMR and 2D-HSQC techniques.     

Overproduction of lignin-degrading enzymes by an isolate of Phanerochaete chrysosporium.

Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. PSBL-1 is a mutant of this organism that generates the ligninolytic system under nonlimiting conditions during primary metabolism. Lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities for PSBL-1 under nonlimiting conditions were 4- to 10-fold higher than those of the wild type (WT) under nitrogen-limiting conditions. PSBL-1 was still in the log phase of growth while secreting the enzymes, whereas the WT had ceased to grow by this time. As in the WT, manganese(II) increased manganese peroxidase activity in the mutant. However, manganese also caused an increase in lignin peroxidase and glyoxal oxidase activities in PSBL-1. Addition of veratryl alcohol to the culture medium stimulated lignin peroxidase activity, inhibited glyoxal oxidase activity, and had little effect on manganese peroxidase activity in PSBL-1, as in the WT. Fast protein liquid chromatography (FPLC) analysis shows production of larger amounts of isozyme H2 in PSBL-1 than in the WT. These properties make PSBL-1 very useful for isolation of large amounts of all ligninolytic enzymes for biochemical study, and they open the possibility of scale-up production for pratical use.