Assessment of metal pollution and effects of physicochemical factors on soil microbial communities around a landfill.

The physicochemical properties, chemical fractions of six metals (Cu, Zn, Pb, Cd, Cr, and Mn), and microbial communities of soil around a typical sanitary landfill were analyzed. The results indicate that soils around the landfill were from neutral to weak alkalinity. The contents of organic matter (OM), total nitrogen (TN), total phosphorous (TP), and activities of catalase, cellulase, and urease were significantly higher in landfill soils than those in background soils. Negative correlations were found between pH and metals. Cr was the dominant metal. Cu, Pb, Cr, and Mn were accumulated in the nearby farmland soils. Cd had the highest percentage of exchangeable fraction (33.7%-51.8%) in landfill and farmland soils, suggesting a high bioavailability to the soil environment affected by the landfill. Pb, Cr, and Mn existed mostly in oxidable fraction, and Cu and Zn were dominant in residual fraction. There was a low risk of soil metals around the landfill based on the RI values, while according to RAC classification, Cd had high to very high environmental risk. The MisSeq sequencing results showed that Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria were the dominant phyla of bacteria, and the most abundant phylum of fungi was Ascomycota. The NMDS analysis revealed that the landfill could influence soil fungal communities more intensely than bacterial communities. TN, cellulase, and bioavailable metals (Pb-Bio and Cr-Bio) were identified to have main influences on microbial communities. Pb-Bio was the most dominant driving factor for bacterial community structures. For fungi, Pb-Bio was significantly negatively related to Olpidiomycota and Cr-Bio had a significantly negative correlation with Ascomycota. It manifests that bioavailable metals play important roles in assessing environmental risks and microbial community structures of soil around landfill.

Assessment of degradation mechanism of imidacloprid residues in grape rhizosphere soil by UHPLC-Orbitrap™-MS and its residual impact on soil enzyme activity.

Imidacloprid (IM) is a systemic insecticide persistent in the environment and possesses a negative impact on the non-targeted ecosystem. The objective of the present study was to evaluate the dissipation and degradation mechanism of IM residues in grape rhizosphere soil and to investigate its residual effect on soil enzyme activity at different IM spiking levels. The half-life of IM residue in soil was 27, 36, and 43.5 days at a spiking level of 1, 10, and 50 mg kg-1, respectively following a bi-phasic first + first-order dissipation kinetics. UHPLC-Orbitrap™-MS analysis by targeted metabolomics approach revealed that IM metabolites such as IM-amine analogue, guanidine (reduction), 5-hydroxy IM (hydroxylation), IM-Urea (oxidation), reduced NO analogue of IM (oxidation), and olefin of guanidine IM (dehydrogenation) were identified and proposed the degradation mechanism in grape rhizosphere soil. Toxicity of IM residues on five extracellular enzymes, viz., dehydrogenase, acid phosphatase, alkaline phosphatase, ß-glucosidase, and urease revealed that activity of dehydrogenase, acid phosphatase, and alkaline phosphatase remained unaffected at 60th day of sampling. The ß-glucosidase and urease were negatively affected throughout the incubation period indicating the influence of IM residues on carbon and nitrogen mineralization in soil. Thus, long-term exposure of IM to grape rhizosphere through soil drenching could affect soil enzyme activity which has a negative effect on the soil nutrient cycle and soil microbiome.

Assessment of cell wall degrading enzymes by molecular docking and dynamics simulations: Effects of novel infrared treatment.

Cell wall-degrading enzymes' activities under infrared treatment are vital for peeling; it is critical to elucidate the mechanisms of the novel infrared peeling in relation to its impact on cell wall-degrading enzymes. In this study, the activities, and gene expressions of eight degrading enzymes closely related to pectin, cellulose and hemicellulose were determined. The most influential enzyme was selected from them, and then the mechanism of its changes was revealed by molecular dynamics simulation and molecular docking. The results demonstrated that infrared had the most significant effect on ß-glucosidase among the tested enzymes (increased activity and up-regulated gene expression of 195.65 % and 7.08, respectively). It is suggested infrared crucially promotes cell wall degradation by affecting ß-glucosidase. After infrared treatment, ß-glucosidase's structure moderately transformed to a more open one and became flexible, increasing the affinity between ß-glucosidase and substrate (increasing 75 % H-bonds and shortening 15.89 % average length), thereby improving ß-glucosidase's activity. It contributed to cell wall degradation. The conclusion is that the effect of infrared on the activity, gene expression and molecular structure of ß-glucosidase causes damage to the peel, thus broadening the applicability of the new infrared dry-peeling technique, which has the potential to replace traditional wet-peeling methods.

In vitro assessment of two novel Cellulases from Trabulsiella odontotermitis for agricultural waste utilization.

BACKGROUND: The production of agricultural wastes still growing as a consequence of the population growing. However, the majority of these residues are under-utilized due their chemical composition, which is mainly composed by cellulose. Actually, the search of cellulases with high efficiency to degrade this carbohydrate remains as the challenge. In the present experiment, two genes encoding an endoglucanase (EC 3.2.1.4) and ß-glucosidase (EC 3.2.1.21) were overexpressed in Escherichia coli and their recombinant enzymes (egl-FZYE and cel-FZYE, respectively) characterized. Those genes were found in Trabulsiella odontermitis which was isolated from the gut of termite Heterotermes sp. Additionally, the capability to release sugars from agricultural wastes was evaluated in both enzymes, alone and in combination. RESULTS: The results have shown that optimal pH was 6.0 and 6.5, reaching an activity of 1051.65 ± 47.78 and 607.80 ± 10.19 U/mg at 39 °C, for egl-FZYE and cel-FZYE, respectively. The Km and Vmax for egl-FZYE using CMC as substrate were 11.25 mg/mL and 3921.57 U/mg, respectively, whereas using Avicel were 15.39 mg/mL and 2314.81 U/mg, respectively. The Km and Vmax for cel-FZYE using Avicel as substrate were 11.49 mg/mL and 2105.26 U/mg, respectively, whereas using CMC the enzyme did not had activity. Both enzymes had effect on agricultural wastes, and their effect was improved when they were combined reaching an activity of 955.1 ± 116.1, 4016.8 ± 332 and 1124.2 ± 241 U/mg on corn stover, sorghum stover and pine sawdust, respectively. CONCLUSIONS: Both enzymes were capable of degrading agricultural wastes, and their effectiveness was improved up to 60% of glucose released when combined. In summary, the results of the study demonstrate that the recombinant enzymes exhibit characteristics that indicate their value as potential feed additives and that the enzymes could be used to enhance the degradation of cellulose in the poor-quality forage generally used in ruminant feedstuffs.

Assessment of wasteland derived biomass for bioethanol production

Background: The bioethanol produced from biomass is a promising alternative fuel. The lignocellulose from marginal areas or wasteland could be a promising raw material for bioethanol production because it is present in large quantities, is cheap, renewable and has favorable environmental properties. Despite these advantages, lignocellulosic biomass is much more difficult to process than cereal grains, due to the need for intensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis. Therefore, there is a need to develop an efficient and cost-effective method for the degradation and fermentation of lignocellulosic biomass to ethanol. Results: The usefulness of lignocellulosic biomass from wasteland for the production of bioethanol using pretreatment with the aid of ionic liquids of 1-ethyl-3-methylimidazolium acetate and 1-ethyl-3-methylimidazolium chloride was evaluated in this study. The pretreatment process, enzymatic hydrolysis and alcoholic fermentation lasted a total of 10 d. The largest amounts of bioethanol were obtained from biomass originating from agricultural wasteland, in which the dominant plant was fireweed (Chamaenerion angustifolium) and from the field where the common broom (Cytisus scoparius) was the dominant. Conclusions: The plants such as fireweed, common broom, hay and goldenrod may be useful for the production of liquid biofuels and it would be necessary in the further stage of research to establish and optimize the conditions for the technology of ethyl alcohol producing from these plant species. Enzymatic hydrolysis of biomass from agricultural wastelands results in a large increase in fermentable sugars, comparable to the enzymatic hydrolysis of rye, wheat, rice or maize straw.

Microbial dynamics for lignocellulosic waste bioconversion and its importance with modern circular economy, challenges and future perspectives.

An extensive use of microbial dynamics for utilizing the lignocellulosic wastes has been attributed to their efficiency in bioenergy and bioproducts development as a cost effective high nutritional value. The integration of lignocellulosic waste into the circular economy can scaleup the sustainable bioproducts and bioenergy development. In this review paper, the aim is to describe the existing research efforts on organic lignocellulosic waste, cellulase producing microbes, their potential enzyme, modern circular economy with associated challenges and future perspectives. Presently, it has been reviewed that microbial cellulases have provided treasure bioproducts visions into industrial bioproducts marvels unveiled through lignocellulosic waste cutting-edge microbial explorations. Furthermore, the review focused on new insights of the growing circular economy of lignocellulosic waste used for many bioproducts and bioenergy dealings and explored the emergent lignocellulosic biorefinery approaches which could then be applied to review industrial-scale sustainable economic models for upgraded bioproducts and other production associated problems.

Insights into the economic viability of cellulases recycling on bioethanol production from recycled paper sludge.

The economics of Recycled Paper Sludge conversion into ethanol was here assessed with emphasis on integrating a cellulase recycling system. Without cellulases recycling this process presented positive economic outputs (payback period of 7.85 years; 10.90 Million US$ of accumulated NPV) despite the modest ethanol titers. Recycling both free and solid-bound enzymes allowed considerable savings of enzyme but also an increase on annual costs (0.88%), resulting on a superior economic output: payback period decreased to 7.25 years; accumulated NPV increased to 14.44 Million US$. Recycling exclusively the liquid fraction enabled a clear costs reduction, however, also total ethanol decreased, attenuating the abovementioned benefits. Targeting higher ethanol concentrations, superior solids consistencies were also evaluated. Despite a costs reduction, total ethanol decreased due to a higher ethanol retention on the solid. A sensitivity analysis further revealed that the cost of enzymes and ultrafiltration membrane may be critical on enzyme recycling economic feasibility.

Chloroplasts as Cellular Factories for the Cost-effective Production of Cellulases.

Chloroplasts are vital photosynthetic organelles in plant cells that carry out several important cellular functions including synthesis of amino acids, fatty acids, and lipids and metabolism of nitrogen, starch, and Sulphur to sustain the homeostasis in plants. These organelles have got their own genome, and related genetic machinery to synthesize required proteins for various plant functions. Genetic manipulations of the chloroplast genome for different biotech applications has been of great interest due to desired features including the availability of operonal mode of gene expression, high copy number, and maternal mode of inheritance (in the most field crops). Their capacity to often express transgenes at high levels make it a cost-effective platform for the production of foreign proteins, particularly high-value targets of industrial importance, at large scale. This article reviews briefly the research work carried out to produce cellulolytic enzymes in higher plant chloroplasts. The challenges and future opportunities for the same are also discussed.

Cost-effective production of biotechnologically important hydrolytic enzymes by Sporotrichum thermophile.

Economical production of xylanase and three cellulases, endo-ß-1,4-glucanase (CMCase), exo-ß-1,4-glucanase (FPase), ß-glucosidase (BGL) was studied in submerged fermentation using cane molasses medium. A statistical optimization approach involving Plackett-Burman design and response surface methodology (RSM) resulted in the production of 72,410, 36,420, 32,420 and 5180 U/l of xylanase, CMCase, FPase and ß-glucosidase, respectively. Optimization resulted in more than fourfold improvements in production of xylanolytic and cellulolytic enzymes. Scale up of enzymes production in shake flasks of varied volumes was sustainable, suggesting a good scope for large scale enzyme production. Addition of microparticles engineered fungal morphology and enhanced enzymes production. Xylanase of S. thermophile is a neutral xylanase displaying its optimal activity at 60 °C while all the cellulases are optimally active at pH 5.0 and 60 °C. The efficacy of enzyme cocktail in waste tea cup paper and rice straw hydrolysis showed that maximum sugar yield of 578.12 and 421.79 mg/g substrate for waste tea cup and rice straw, respectively, were achieved after 24 h. Therefore, concomitant production of cellulolytic and xylanolytic enzymes will be beneficial for the saccharification of lignocellulosics in generating both monomeric and oligomeric sugars for biofuels and other biotechnological applications.

Advances in improving the performance of cellulase in ionic liquids for lignocellulose biorefinery.

Ionic liquids (ILs) have been considered as a class of promising solvents that can dissolve lignocellulosic biomass and then provide enzymatic hydrolyzable holocellulose. However, most of available cellulases are completely or partially inactivated in the presence of even low concentrations of ILs. To more fully exploit the benefits of ILs to lignocellulose biorefinery, it is critical to improve the compatibility between cellulase and ILs. Various attempts have been made to screen natural IL-tolerant cellulases from different microhabitats. Several physical and chemical methods for stabilizing cellulases in ILs were also developed. Moreover, recent advances in protein engineering have greatly facilitated the rational engineering of cellulases by site-directed mutagenesis for the IL stability. This review is aimed to provide the first detailed overview of the current advances in improving the performance of cellulase in non-natural IL environments. New ideas from the most representative progresses and technical challenges will be summarized and discussed.

Exploration of extremophiles for high temperature biotechnological processes.

Industrial processes often take place under harsh conditions that are hostile to microorganisms and their biocatalysts. Microorganisms surviving at temperatures above 60°C represent a chest of biotechnological treasures for high-temperature bioprocesses by producing a large portfolio of biocatalysts (thermozymes). Due to the unique requirements to cultivate thermophilic (60-80°C) and hyperthermophilic (80-110°C) Bacteria and Archaea, less than 5% are cultivable in the laboratory. Therefore, other approaches including sequence-based screenings and metagenomics have been successful in providing novel thermozymes. In particular, polysaccharide-degrading enzymes (amylolytic enzymes, hemicellulases, cellulases, pectinases and chitinases), lipolytic enzymes and proteases from thermophiles have attracted interest due to their potential for versatile applications in pharmaceutical, chemical, food, textile, paper, leather and feed industries as well as in biorefineries.

A novel and fully scalable Agrobacterium spray-based process for manufacturing cellulases and other cost-sensitive proteins in plants.

Transient transfection of plants by vacuum infiltration of Agrobacterium vectors represents the state of the art in plant-based protein manufacturing; however, the complexity and cost of this approach restrict it to pharmaceutical proteins. We demonstrated that simple spraying of Nicotiana plants with Agrobacterium vectors in the presence of a surfactant can substitute for vacuum inoculation. When the T-DNA of Agrobacterium encodes viral replicons capable of cell-to-cell movement, up to 90% of the leaf cells can be transfected and express a recombinant protein at levels up to 50% of total soluble protein. This simple, fast and indefinitely scalable process was successfully applied to produce cellulases, one of the most volume- and cost-sensitive biotechnology products. We demonstrate here for the first time that representatives of all hydrolase classes necessary for cellulosic biomass decomposition can be expressed at high levels, stored as silage without significant loss of activity and then used directly as enzyme additives. This process enables production of cellulases, and other potential high-volume products such as noncaloric sweetener thaumatin and antiviral protein griffithsin, at commodity agricultural prices and could find broad applicability in the large-scale production of many other cost-sensitive proteins.

Assessment of the enzymatic hydrolysis profile of cellulosic substrates based on reducing sugar release.

The activity profile of a 1:0.30 mixture of Celluclast 1.5L FG and Novozym 188 (Novozymes) was investigated using Whatman #1 filter paper (W1FP) as a single substrate for hydrolysis. The procedure was based on the ability of the enzymes to release total (RS(Tot)), insoluble (RS(Insol)) and soluble (RS(Sol)) reducing sugars from W1FP. RS(Insol) was used to estimate endoglucanase (EnG) activity whereas exoglucanases (ExG) were assessed by measuring RSSol in the presence of δ-gluconolactone. Finally, the ß-glucosidase (ßG) activity was derived from the difference between RS(Sol) measurements in the presence and absence of δ-gluconolactone. When this analytical procedure was applied to W1FP using 9.64 mg mL(-1) of the enzyme mixture, the relative contributions of EnG, ExG and ßG to the total cellulase activity were 63.28%, 12.02% and 24.70%, respectively. Also, this ratio changed with changes in the enzyme loading, giving a new insight into the synergy that exists among the enzymes.

[Cost-effective production of protein by using cellulose-binding domain fusion tag in Corynebacterium glutamicum].

The CBD gene from Trichoderma reesei was cloned into the Corynebacterium glutamicum secretion expression vector pXMJ19-sp, in which green fluorescent protein was inserted to obtain pXMJ19-sp-GFP-CBD. After induced by 0.5 mmol/L IPTG, GFP-CBD was expressed in Corynebacterium glutamicum at high level of 200 mg/L. The GFP-CBD could be purified to high purity with cellulose column. The results indicated CBD can be successfully used in Corynebacterium glutamicum expression system and thus offer an extremely simple, effective and scalable way for production of recombinant proteins.

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid.

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H(2)SO(4) with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, ß-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2(3) central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19-4.81%, w/w) and loadings of enzymes (1.9-38.1 FPU/g bagasse) and Tween 20 (0.0-0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.

Assessment of saccharification efficacy in the cellulase system of the brown rot fungus Gloeophyllum trabeum.

Brown rot fungi uniquely degrade wood by creating modifications thought to aid in the selective removal of polysaccharides by an incomplete cellulase suite. This naturally successful mechanism offers potential for current bioprocessing applications. To test the efficacy of brown rot cellulases, southern yellow pine wood blocks were first degraded by the brown rot fungus Gloeophyllum trabeum for 0, 2, 4, and 6 weeks. Characterization of the pine constituents revealed brown rot decay patterns, with selective polysaccharide removal as lignin compositions increased. G. trabeum liquid and solid state cellulase extracts, as well as a commercial Trichoderma reesei extract (Celluclast 1.5 L), were used to saccharify this pretreated material, using beta-glucosidase amendment to remove limitation of cellobiose-to-glucose conversion. Conditions varied according to source and concentration of cellulase extract and to pH (3.0 vs. 4.8). Hydrolysis yields were maximized using solid state G. trabeum extracts at a pH of 4.8. However, the extent of glucose release was low and was not significantly altered when cellulase loading levels were increased threefold. Furthermore, Celluclast 1.5 L continually outperformed G. trabeum cellulase extracts, although extent of glucose release never exceeded 22.0%. Results suggest methodological advances for utilizing crude G. trabeum cellulases and imply that the suboptimal hydrolysis levels obtained with G. trabeum and Celluclast 1.5 L cellulases, even at high loading levels, may be due to brown rot modifications insufficiently distributed throughout the pretreated material.

[Industrial exploitation of renewable resources: from ethanol production to bioproducts development]. / Valorisation des ressources renouvelables : de la production d'éthanol au développement de nouveaux bioproduits.

Plants, which are one of major groups of life forms, are constituted of an amazing number of molecules such as sugars, proteins, phenolic compounds etc. These molecules display multiple and complementary properties involved in various compartments of plants (structure, storage, biological activity etc.). The first uses of plants in industry were for food and feed, paper manufacturing or combustion. In the coming decades, these renewable biological materials will be the basis of a new concept: the "biorefiner" i.e. the chemical conversion of the whole plant to various products and uses. This concept, born in the 90ies, is analogous to today's petroleum refinery, which produces multiple fuels and derivative products from petroleum. Agriculture generates lots of co-products which were most often wasted. The rational use of these wasted products, which can be considered as valuable renewable materials, is now economically interesting and will contribute to the reduction of greenhouse has emissions by partially substituting for fossil fuels. Such substructures from biological waste products and transforming them into biofuels and new industrial products named "bioproducts". These compounds, such as bioplastics or biosurfactants, can replace equivalent petroleum derivatives. Towards that goal, lots of filamentous fungi, growing on a broad range of vegetable species, are able to produce enzymes adapted to the modification of these type of substrates. The best example, at least the more industrially developed to date, is the second generation biofuel technology using cellulose as a raw material. The process includes an enzymatic hydrolysis step which requires cellulases secreted from Trichoderma fungal species. This industrial development of a renewable energy will contribute to the diversification of energy sources used to transport and to the development of green chemistry which will partially substitute petrochemicals.

Consolidated bioprocessing of cellulosic biomass: an update.

Biologically mediated processes seem promising for energy conversion, in particular for the conversion of lignocellulosic biomass into fuels. Although processes featuring a step dedicated to the production of cellulase enzymes have been the focus of most research efforts to date, consolidated bioprocessing (CBP)--featuring cellulase production, cellulose hydrolysis and fermentation in one step--is an alternative approach with outstanding potential. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer, and engineering non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase system enabling cellulose utilization. Recent studies of the fundamental principles of microbial cellulose utilization support the feasibility of CBP.

Industrial scale-up of pH-controlled liquid hot water pretreatment of corn fiber for fuel ethanol production.

The pretreatment of cellulose in corn fiber by liquid hot water at 160 degrees C and a pH above 4.0 dissolved 50% of the fiber in 20 min. The pretreatment also enabled the subsequent complete enzymatic hydrolysis of the remaining polysaccharides to monosaccharides. The carbohydrates dissolved by the pretreatment were 80% soluble oligosaccharides and 20% monosaccharides with <1% of the carbohydrates lost to degradation products. Only a minimal amount of protein was dissolved, thus enriching the protein content of the undissolved material. Replication of laboratory results in an industrial trial at 43 gallons per minute (163 L/min) of fiber slurry with a residence time of 20 min illustrates the utility and practicality of this approach for pretreating corn fiber. The added costs owing to pretreatment, fiber, and hydrolysis are equivalent to less than 0.84 dollars/gal of ethanol produced from the fiber. Minimizing monosaccharide formation during pretreatment minimized the formation of degradation products; hence, the resulting sugars were readily fermentable to ethanol by the recombinant hexose and by pentose-fermenting Saccharomyces cerevisiae 424A(LNH-ST) and ethanologenic Escherichia coli at yields >90% of theoretical based on the starting fiber. This cooperative effort and first successful trial opens the door for examining the robustness of the pretreatment system under extended run conditions as well as pretreatment of other cellulose-containing materials using water at controlled pH.