Environmental Biotechnology Research Group: Research Report.

Environmental biotechnology exploits living organisms (plant, bacteria, fungi, etc) in various applications of waste treatment and remediation of pollutants. The organisms that demonstrate the potential to utilize the wastes or pollutants as food are naturally found in the environment. Rapid development in genetic technology also assists in introducing genetically modified organisms with enhanced capability to degrade wastes or pollutants. It has been proven that environmental biotechnology can provide safer methods of cleaning hazardous wastes compared to conventional methods since it uses natural agents. Finally the utilization of biowastes and organic residues to useful value added product were also studie

Mining the secretome of the lignocellulose degrading fungus Parascedosporium putredinis NO1

The demand for sustainable and renewable alternatives to finite and environmentally damaging fossil fuel resources is growing. Lignocellulosic biomass is available in vast amounts, at low cost, and could provide fuels, chemicals, and materials if deconstructed effectively. However, its recalcitrant nature makes the cost-effective utilisation of this substrate difficult to achieve. Despite this, wood-degrading fungi have evolved an array of powerful enzymes for the deconstruction of all components of lignocellulose including the polysaccharides and the aromatic polymer lignin. In this work, the lignocellulose-degrading capacity of the ascomycete fungus Parascedosporium putredinis NO1 was explored in detail. New bioinformatic strategies were developed and employed to probe the genome of P. putredinis NO1, the first genome of its genus, and to isolate an in silico secretome to allow clearer characterisation of the biomass-degrading response. Proteomic investigations of the growth of P. putredinis NO1 on multiple industrially relevant lignocellulosic substrates demonstrated remarkable variation in the P. putredinis NO1 secretome depending on growth substrate. Molecular techniques were used to expand the demonstration of the varied secretome temporally and support the hypothesis of a tailored enzymatic response to different lignocellulosic substrates, an understanding of which will be important for the development of efficient biorefinery technology. The tailored secretome was exploited by investigating the enzymatic response of P. putredinis NO1 when grown on substrates with varying lignin contents. This allowed identification of proteins with patterns of abundance suggesting roles in the breakdown of lignin. The characterisation of lignocellulose-degrading organisms from underexplored branches of the tree of life, and the identification and characterisation of new enzymes with unclear or unknown roles in lignocellulose breakdown will be vital to improving our understanding of lignocellulose deconstruction and to achieve this efficiently in biorefineries

Identification and characterisation of lignin degrading bacteria and enzymes fromlarval guts of the African Palm Weevil (Rhynchophorus phoenicis)

As developing economies continue to grow, the world’s demand for energy which currently stands at 84MB (million barrels oil) per day is projected to rise to 116 MB per day by the year 2030. The need to meet this continuous rise in demand for energy while lowering the emission of CO2 and other greenhouse gases has necessitated a shift in focus from the exploitation of fossil fuels which are limited, to more renewable and environmentally safe biological resources such as lignocellulosic biomass, the main structural components of plant cell walls. Lignocellulose, however, is resistant to degradation, thus there is a high cost and energy requirement associated with its pretreatment in order to access the lignin bound polysaccharides for subsequent hydrolysis, fermentation and conversion to biofuels and biomaterials.Xylophagous (wood-feeding) insects such as the African palm weevil (Rhynchophorus phoenicis) have developed the ability to effectively utilize lignocellulosic substrates as an energy source due to the synergistic association with their gut microbes. This makesthem viable resources to explore for novel lignocellulose degrading enzymes.Metagenomics allows access to the entire microbiome present in a particular environment and has been adopted in recent studies, rather than culture-based methods, thereby allowing for discovery of novel genes and enzymes from both culturable and non-culturable microbes.In this study, we carried out taxonomic profiling of the bacteria in the gut of the African palm weevil’s larvae using 16S rRNA gene amplicon sequencing with particular interest in identifying lignin degrading bacteria. We also performed functional metagenomicsanalysis from whole metagenome sequencing data derived from whole gut metagenomic DNA of APW larvae to identify genes and by extension, enzymes that can deconstruct lignocellulose and degrade its lignin component. The predominant bacterial genera found across all gut segments were Enterococcus, Lactococcus,Shimwellia, Lelliotia, Klebsiella and Enterobacter, with the foregut having the mostdiverse and abundant lignin degraders mostly from the Proteobacteria phylum. Onethousand, one hundred and forty-one (1,141) annotated genes identified from the R. phoenicis larval gut bacterial metagenome aligned with genes encoding CAZymes and 249 of these belonged to the “Auxiliary Activities” class which harbours the suite of genes implicated to play different roles in lignin deconstruction. Three genes of putative lignin degraders were successfully amplified by PCR, one of the three amplified genes B-38773 (encoding a putative deferrochelatase/ peroxidase of approximately 46kDa insize that has a conserved domain match to the dye decolourising peroxidase superfamily) was produced by heterologous expression and was found to exhibit activity on the peroxidase substrate ABTS, and the anthraquinone dye RB19 but no activity was observed with kraft lignin. Specific activity of 12.9Umg-1 at optimum temperature of 40°C and pH of 4 were recorded when B-38773 enzyme was assayed against ABTS as a substrate. Kinetic parameters: Vmax, Km, Kcat, and catalytic efficiency were determined to be 3.68 μMol/min, 1.089mM, 540.9S-1 and 4.96 X 105 M-1S-1respectively.This study elucidates the lignocellulose degrading potential of the gut community associated with the African palm weevil (APW) by robustly defining the bacterial community structure of the APW gut. Also, massive data from the metagenomic library generated will serve as a storehouse from where genes with various potential functions identified by the inhabitant gut bacteria can be harvested to contribute to areas of biotechnological relevance for industrial applications

Pretreatment and Bioconversion of Crop Residues

In this book, 15 papers, covering some of the latest advances in pretreatment and bioconversion of crop residues, are presented. Research results dealing with wheat straw, corn stover, sweet sorghum bagasse, hazelnut shells, oil palm empty fruit bunch, olive tree pruning biomass, and other residues of crop harvest and processing are discussed. Pretreatment methods, such as auto-catalyzed and acid-catalyzed hydrothermal processing, steaming, alkaline methods, and different organosolv approaches, are reported. Bioconversion with enzymes and microbes for producing fermentable sugars, xylitol, and biomethane are also included

Elaeis guineensis

Palm oil biomass is constantly produced in large quantities throughout the world as a waste product of the vast palm oil plantations. Biomass from the palm oil industry has been converted into value-added products to a limited extent via thermochemical, chemical, physical, and biochemical conversion routes. However, a significant amount of biomass, such as OPF and OPT, is still left in plantations. A pragmatic approach to converting them to value-added products will not only result in a cleaner environment but also generate significant revenue for the government. It is also suggested that more attention be paid to bioproducts in order to present them in an appealing form to end-users, thereby encouraging good patronage

Biofuels Production and Processing Technology

The negative impacts of global warming and global environmental pollution due to fossil fuels mean that the main challenge of modern society is finding alternatives to conventional fuels. In this scenario, biofuels derived from renewable biomass represent the most promising renewable energy sources. Depending on the biomass used by the fermentation technologies, it is possible to obtain first-generation biofuels produced from food crops, second-generation biofuels produced from non-food feedstock, mainly starting from renewable lignocellulosic biomasses, and third-generation biofuels, represented by algae or food waste biomass.Although biofuels appear to be the closest alternative to fossil fuels, it is necessary for them to be produced in competitive quantities and costs, requiring both improvements to production technologies and the diversification of feedstock. This Special Issue is focused on technological innovations, including the utilization of different feedstocks, with a particular focus on biethanol production from food waste; different biomass pretreatments; fermentation strategies, such as simultaneous saccharification and fermentation (SSF) or separate hydrolysis and fermentation (SHF); different applied microorganisms used as a monoculture or co-culture; and different setups for biofuel fermentation processes.The manuscripts collected represent a great opportunity for adding new knowledge to the scientific community as well as industry

Tratamento hidrotérmico de bagaço de cana-de-açúcar como ferramenta para aumentar a produção de holocelulases por Aspergillus niger

Tese (doutorado)—Universidade de Brasília, Departamento de Biologia Celular, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2018.Este trabalho teve como objetivo investigar a utilização das frações líquida (licor) e sólida obtidas a partir de tratamento hidrotérmico do bagaço de cana-de-açúcar (BCA) como fontes de carbono para a produção de holocelulases pelo fungo Aspergillus niger DCFS11. Numa primeira etapa, as condições do tratamento hidrotérmico foram investigadas por meio de análise fatorial visando a utilização dos licores resultantes como substratos para indução de hemicelulases. O tratamento de baixa severidade e utilizando baixa concentração de sólidos (170°C, 30 min, 1 % m/m BCA) foi selecionado como condição ótima devido à alta e rápida indução de xilanases pelo licor resultante. Uma variedade de mono e oligossacarídeos responsáveis pela indução de hemicelulases foi identificada nos licores por métodos analíticos (cromatografia líquida e espectrometria de massas). Numa segunda etapa, secretomas de A. niger cultivado na presença de bagaço in natura (BNT), bagaço tratado (BPT), licor (LIC) e bagaço tratado + licor (BPT+LIC) foram comparados por análise bioquímica e proteômica quantitativa. Os secretomas produzidos nas condições BPT, LIC e BPT+LIC mostraram-se superiores em termos de atividades de holocelulases, termoestabilidade e eficiência na sacarificação enzimática de BCA, além de apresentarem maior abundância de dezenas de celulases, hemicelulases e pectinases em relação ao secretoma produzido na condição BNT. Este trabalho demonstrou o potencial do tratamento hidrotérmico do BCA como ferramenta para aumentar a produção de holocelulases por A. niger, uma vez que gerou substratos menos recalcitrantes para crescimento microbiano na forma de carboidratos solúveis (licor) ou polissacarídeos mais acessíveis nos sólidos tratados.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF).The goal of this work was to investigate the use of the liquid (liquor) and solid fractions arising from hydrothermal pretreatment of sugarcane bagasse (SCB) as a carbon source for the production of holocellulases by Aspergillus niger. Initially, hydrothermal pretreatment parameters were investigated by factorial design aiming the use of preteatment liquors as a substrate for hemicellulase induction. Pretreatment of low severity and low SCB loading (170 °C, 30 min, 1 % w/w SCB) was selected as the optimum condition due to the high and fast xylanase induction promoted by the resulting liquor. Several mono and oligosaccharides responsible for hemicellulase induction were identified in liquors by analytical methods (liquid chromatography and mass spectrometry). Thereafter, secretomes of A. niger cultivated in the presence of untreated SCB (BNT), pretreated bagasse (BPT), liquor (LIC) and pretreated bagasse + liquor (BPT+LIC) were compared by biochemical and quantitative proteomic analyses. Secretomes produced under conditions BPT, LIC and BPT+LIC were superior to BNT in terms of holocellulase activities, thermostability, efficiency in enzymatic saccharification of SCB. They also provided a greater abundance of dozens of cellulases, hemicellulases and pectinases in comparison to the secretome produced in the presence of BNT. This work demonstrated the potential of hydrothermal treatment of SCB as a tool to increase the production of holocellulases by A. niger, since it generated less recalcitrant substrates for microbial growth in the form of soluble carbohydrates (liquor) or more accessible polysaccharides in pretreated solids

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Editores técnicos: Alexandre Alonso Alves, Bruno dos Santos Alves Figueiredo Brasil e Lorena Costa Garcia