Exploration of Nahoon beach milieu for lignocellulose degrading bacteria and optimizing fermentation conditions for holocellulase production by selected strains

A significant trend in the modern day industrial biotechnology is the utilization and application of renewable resources, and ecofriendly approach to industrial processes and waste management. As a consequence thereof, the biotechnology of holocellulases: cellulase and xylanase and, enzymatic hydrolysis of renewable and abundant lignocellulosic biomass to energy and value added products are rapidly increasing; hence, cost effective enzyme system is imperative. In that context, exploration of microbiota for strains and enzymes with novel industrial properties is vital for efficient and commercially viable enzyme biotechnology. Consequent on the complex characteristics of high salinity, variable pressure, temperature and nutritional conditions, bacterial strains from the marine environment are equipped with enzyme machinery of industrial importance for adaptation and survival. In this study, bacterial strains were isolated form Nahoon beach and optimized for holocellulase production. Three isolates selected for lignocellulolytic potential were identified by 16S ribosomal deoxyribonucleic acid (rDNA) sequence analysis. Isolate FS1k had 98 percent similarity with Streptomyces albidoflavus strain AIH12, was designated as Streptomyces albidoflavus strain SAMRC-UFH5 and deposited in the GenBank with accession number KU171373. Similarly, isolates CS14b and CS22d with respective percentage similarity of 98 and 99 (percent) with Bacillus cereus strains and Streptomyces sp. strain WMMB251 were named Bacillus cereus strain SAMRC-UFH9 and Streptomyces sp. strain SAMRC-UFH6; and were deposited in the GenBank with accession number KX524510 and KU171374 respectively. Optimal pH, temperature and agitation speed for cellulase production by S. albidoflavus strain SAMRC-UFH5, and B. cereus strain SAMRC-UFH9 were 6 and 7; 40 and 30 (°C); and 100 and 150 (rpm) respectively; while xylanase production was optimal at pH, temperature and agitation speed of 8 and 7; 40 and 30 (°C); and 150 and 50 (rpm) respectively. Maximum cellulase activity of 0.26 and 0.061(U/mL) by S. albidoflavus strain SAMRC-UFH5 and B. cereus strain SAMRC-UFH9 were attained at 60 h respectively, while maximal xylanase activity of 18.54 and 16.6 (U/mL) was produced by S. albidoflavus strain SAMRC-UFH5 and B. cereus strain SAMRC-UFH9 at 48 h and 60 h respectively. Furthermore, xylanase production by S. albidoflavus strain SAMRC-UFH5 and B. cereus strain SAMRC-UFH9 was maximally induced by wheat straw and xylan respectively, while cellulase production was best induced by mannose and carboxymethyl cellulose respectively. On the other hand, cellulase and xylanase production by Streptomyces sp. strain SAMRC-UFH6 was optimal at pH, temperature and agitation speed of 7 and 8, 40 °C and 100 rpm, respectively. Highest production of cellulase and xylanase was attained at 84 and 60 h with respective activity of 0.065 and 6.34 (U/mL). In addition, cellulase and xylanase production by the strain was best induced by beechwood xylan. Moreover, xylanase produced by Streptomyces sp. strain SAMRC-UFH6 at optimal conditions was characterized by optimal pH and temperature of 8 and 80-90 °C respectively; retaining over 70 percent activity at pH 5-10 after 1 h and 60 percent of initial activity at 90 °C after 90 min of incubation. In all, optimization improved cellulase and xylanase production yields, being 40 and 95.5, 10.89 and 72.17, and 10 and 115- fold increase by S. albidoflavus strain SAMRC-UFH5, B. cereus strain SAMRC-UFH9 and Streptomyces sp. SAMRC-UFH6 respectively. The results of this study suggest that the marine bacterial strains are resource for holocellulase with industrial applications

Thermophilic mixed culture degradation of Miscanthus x giganteus as a guide to strategies for consolidated bioprocessing

The successful development of consolidated bioprocessing requires microorganisms capable of degrading lignocellulosic biomass and fermenting the resulting sugars. Commercial cellulases and hemicellulases are currently being used to access these sugars, adding to the cost of producing useful products from lignocellulose. This study reports the enrichment of thermophilic, miscanthus degrading bacterial cultures from a municipal composting facility. The detected and isolated bacteria were characterized by 16S rRNA gene sequence analysis and were mostly Chitinophagaceae family, Meiothermus spp. and Geobacillus spp. Other isolated species included Cohnella spp., Brevibacillus sp., Chelatococcus spp., Thermobacillus spp., Thermoanaerobacterium spp., Thermobispora bispora, Bacillus spp., Staphylococcus sp. and Micrococcus sp. After enrichment, the mixed population was able to degrade greater than 50% of an ammonium hydroxide pre-treated Miscanthus x giganteus sample (1 g) over a six week incubation period at 55oC, with a reduction in the amounts of all components, including acid soluble and acid insoluble lignin. The glycoside hydrolases and other enzymes identified in the culture supernatants included endo-1,4-β-glucanase A, glucoamylase, xylan 1,4-β-xylosidase, xylose isomerase, xylulokinase, superoxide dismutase, transaldolase, Mn-catalase, Δ-1-pyrroline-5-carboxylate dehydrogenase and endo-β-N-acetylglucoseaminidase H. The HPLC analysis showed that fermentation products formate and lactate were present in the culture supernatant. Expression of an endoglycoside hydrolase (Csac_0137 from Caldicellulosiruptor saccharolyticus) gene in Geobacillus thermoglucosidasius strains, NCIMB 11955 and DL33, improved their β-glucosidase specific activity on cellobiose, and improved glycoside hydrolase activities of recombinant DL33 strain when grown on pre-treated M. x giganteus. Co-culturing of either transformed or wild-type NCIMB 11955 and DL33 with some of the isolated strains improved their glycoside hydrolase activity and growth on pretreated M. x giganteus.Open Acces

Enhancing Cellulase Production of Aureobasidium Pullulans for Use in Converting Dried Distillers’ Grains with Solubles into a Higher Protein Feed

Main limitations of dried distillers’ grains with solubles (DDGS) as meal for monogastric animals is the presence of high fiber content, reduced fat content, and poor amino acid balance. This causes a reduced economic value of DDGS for these species due to its low inclusion rates. The goal of this theses was to create new strains of a fungus, Aureobasidium pullulans, with enhanced cellulase production and use it to optimize an enzymatic saccharification, pretreatment, and fungal conversion process to enhance the nutritional value of DDGS. Various combinations of enzymatic saccharification, physical/chemical pretreatments, and mutant strains of fungi were investigated to hydrolyze DDGS fiber into carbohydrates which are metabolized by the fungi into single cell protein. Cellulose is the most abundant renewable carbon source found in nature and is of great interest to various industries, including the food industry. Generally, microorganisms can be used to convert cellulosic materials into higher protein content, which can subsequently be used for animal feed. However, without the aid of cellulase, the cellulose structure is resistant to degradation. Genome shuffling was used to improve cellulase production from Aureobasidium pullulans Y-2311-1. One strain developed via genome shuffling (A. pullulans GS23) displayed the largest increase in total cellulase activity, which was a 6-fold increase compared to the wild type strain. One of the strains created for the starting mutant population by methyl methanesulfonate (MMS) had a 3- fold increase compared to the wild type strain. A. pullulans GS23 also had an increase in exoglucanase and β-glucosidase activity compared to the wild type strain (6.95-fold and 1.52-fold increase, respectively). The crude protein amount of A. pullulans GS23 had a 1.04-fold increase compared to the wild type strain after 5 days of fermentation. Various commercial enzymes were also tested on DDGS to help break down the fibers along with various physical/chemical pretreatments to improve the overall fiber digestibility. But the high cost associated with the use of commercial enzymes is a challenge with the industrial application of enzymes in feed application. This experiment was a two-step process where commercial enzymes were tested on untreated DDGS to down select the enzyme that performed well at low dosages. Then the DDGS was treated by various physical and/or chemical pretreatments and tested with the best enzyme with even lower dosages. Lower enzyme dosages on pretreated DDGS was tested versus the untreated DDGS because the pretreatment would have liberated more sugars, resulting in less enzymes to be used. It was found the commercial enzyme that performed well at low enzyme dosages was Viscozyme L. When this was incorporated with the best pretreated DDGS, dilute acid, with 1 mg/g of Viscozyme L, it had an increase of ~280% in total sugars as compared to untreated DDGS with a Viscozyme L dosage of 2 mg/g. A combination of enzymatic saccharification and/or dilute acid pretreatment of DDGS was conducted with the three fungal strains to evaluate what strain and treatment performed the best. The fungal strains tested was wild type A. pullulans Y-2311-1 and two mutant strains (GS and MMS) with enhanced cellulase production created from the wild type strain. GS A. pullulans on the enzyme untreated DDGS had the highest protein level with a percent increase of 13.59%. While MMS A. pullulans on enzyme untreated DDGS performed well in increasing the fat content, with a percent increase of 27.07%, and decrease the fiber, with a percent decrease of 13.89%. Overall, GS A. pullulans on the enzyme untreated DDGS performed the best in improving the raw DDGS feedstock. The GS strain still improved the fat content and decreased the fiber content. Most of the minerals had higher levels than the MMS strain and all the amino acids analyzed were also higher than the MMS strain, except for tryptophan which was the same. Lysine levels were also higher in the GS strain than the MMS strain with 0.66% and 0.43% respectively. The dilute acid pretreatment released high levels of sugars and decrease the fiber content but with the creation of inhibitors, fungal fermentation could not be used to further improve the DDGS protein levels

Solid-State Fermentation as a Novel Paradigm for Organic Waste Valorization : a Review

The abundance of organic solid waste throughout the world has become a common issue that needs complete management at every level. Also, the scarcity of fuel and the competition between food and substance as an alternative to a petroleum-based product has become a major problem that needs to be properly handled. An urge to find renewable substances for sustainable development results in a strategy to valorize organic solid waste using solid state fermentation (SSF) and to manage the issue of solid wastes in a green approach. This paper reviews management of solid wastes using SSF, with regard to its current application, advantages and challenges, downstream processing in SSF, economic viewpoint, and future perspectives

Estudo de hidrolases glicosídicas bacterianas para aplicações biotecnológicas : bioprospecção, produção e imobilização

Orientadores: Hélia Harumi Sato, Roberto RullerTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: A biomassa lignocelulósica é um importante recurso renovável que está prontamente disponível, sendo uma fonte de matéria-prima com alto potencial biotecnológico. Os polissacarídeos complexos que compõem lignocelulose podem ser convertidos em monossacarídeos fermentescíveis, com grande aplicabilidade em diversos bioprocessos industriais. A degradação dos materiais lignocelulósicos pode ser realizada por uma diversidade de vias enzimáticas complexas, onde é requerido um número considerável de enzimas ativas sobre carboidratos. Entre elas, as famílias das celulases e hemicelulases, além de atuarem na hidrólise dos materiais lignocelulósicos, possuem um uso versátil em setores industriais, tais como, nas áreas alimentícia, bebidas e de biocombustíveis. A tese teve como principais objetivos o delineamento de estratégias para a produção de enzimas e coquetéis enzimáticos eficientes para o uso na hidrólise da biomassa vegetal e, a aplicação de técnicas de imobilização para ampliar a utilização de enzimas em escala comercial. Inicialmente, a bioprospecção de novos micro-organismos secretores de enzimas atuantes na biomassa lignocelulósica foi realizada, e dentre as oitenta linhagens de Streptomyces testadas, duas linhagens (F1 e F7) se destacaram por apresentarem elevadas atividades celulolíticas e hemicelulolíticas. Uma abordagem genômica dessas linhagens possibilitou a identificação de 85 hidrolases glicosídicas (GHs) distribuídas em 33 famílias diferentes na linhagem F1, e 100 GHs dispostas em 44 famílias na linhagem F7. Além disso, os dados genômicos das linhagens F1 e F7 também indicaram a presença de genes relacionados à degradação da lignina. Ferramentas estatísticas também foram aplicadas e possibilitaram a ampliação da produção de GHs pela linhagem F1. Com a otimização, elevadas concentrações de GHs foram alcançadas com meio nutriente adicionado de 16,4 g L-1 de farelo de trigo e 10,0 g L-1 de caseína, onde se obteve 9,27 U mL -1 de xilanase e 0,22 U mL -1 de celulase. Para confirmar a diversidade de GHs expressas pela linhagem F1, uma análise por espectrometria de massa foi realizada e observou-se que quanto maior a complexidade da fonte de carbono utilizada, maior foi a gama de proteínas expressas, incluindo vários tipos de celulases e hemicelulases. A eficiência do extrato enzimático produzido pela linhagem F1 foi estudada para a sacarificação da biomassa vegetal e possibilitou um aumento significativo na liberação de açúcares quando adicionado ao extrato celulolítico comercial, indicando que as enzimas secretadas pela Streptomyces sp. F1 podem ser aplicadas para o melhoramento dos atuais coquetéis comerciais. Foi estudada também a criação de métodos de imobilização de enzimas em condições neutras de pH. Os novos suportes produzidos com a agarose foram utilizados para a imobilização de enzimas monoméricas e multiméricas de grande importância biotecnológica. Um estudo mais detalhado explorando os novos suportes e o uso de técnicas de pós-imobilização foi também proposto. O processo desenvolvido aplicando o polímero polietilenimina (PEI) possibilitou a formação de um excelente sistema para imobilizar e estabilizar a ?-glicosidase obtida de Exiguobacterium antarcticum. A ?-glicosidase imobilizada apresentou uma melhora em suas características, incluindo estabilidade térmica e de armazenamento. Além disso, a ?-glicosidase manteve sua atividade elevada mesmo após vários ciclos de hidrólise com celobiose como substratoAbstract: Lignocellulosic biomass is an important renewable resource that is readily available, being a source of raw material with high biotechnological potential. The complex polysaccharides that compose lignocellulose can be converted into fermentable monosaccharides, with great applicability in several industrial bioprocesses. The degradation of lignocellulosic materials can be accomplished by a variety of complex enzymatic pathways, where a considerable number of carbohydrate active enzymes are required. Among them, the families of cellulases and hemicellulases, besides acting in the hydrolysis of lignocellulosic materials, have a versatile use in industrial sectors, such as in food, beverages and biofuels. The main aims of this thesis were the design of efficient strategies for the production of enzymes and enzymatic cocktails for use in plant biomass hydrolysis and the application of immobilization techniques to increase the use of enzymes in a commercial scale. Initially, bioprospection of new enzyme secreting microorganisms active in lignocellulosic biomass was performed, and among the eighty Streptomyces strains tested, two strains (F1 and F7) were distinguished by their high cellulolytic and hemicellulolytic activities. A genomic approach of these strains allowed the identification of 85 glycoside hydrolases (GHs) distributed in 33 different families the strain F1, and 100 GHs arranged in 44 families the strain F7. In addition, the genomic data from strains F1 and F7 also indicated the presence of genes related to lignin degradation. Statistical tools were also applied and allowed the increase in GH production by strain F1. With the optimization, high concentrations of GHs were achieved with a nutrient medium containing 16.4 g L-1 of wheat bran and 10.0 g L-1 of casein, where 9.27 U mL-1 of xylanase and 0.22 U mL-1 of cellulase were obtained. To confirm the diversity of GHs expressed by the strain F1, an analysis using mass spectrometry technique was performed and it was observed that the greater the complexity of the carbon source used, the greater the range of proteins secreted, including several types of cellulases and hemicellulases. The efficiency of the enzymatic extract produced with strain F1 was studied for the saccharification of plant biomass and allowed a significant increase in sugar release when added to the commercial cellulolytic extract, indicating that the enzymes expressed by Streptomyces sp. F1 can be applied for the improvement of the current commercial cocktails. The creation of enzyme immobilization methods under neutral conditions of pH was also study. The new agarose supports were used for the immobilization of monomeric and multimeric enzymes of great industrial and biotechnological importance. A more detailed study exploring the new supports and the use of post-immobilization techniques with polymers and small molecules was also proposed. The process developed by applying the polymer polyethyleneimine (PEI) enabled the formation of an excellent system to stabilize the glucose-tolerant tetrameric ?-glycosidase obtained from Exiguobacterium antarcticum. The immobilized ?-glycosidase showed an improvement in its characteristics, with an increased activity, including thermal and storage stability. In addition, the ?-glycosidase maintained a high activity even after several cycles of hydrolysis applying cellobiose as substrateDoutoradoCiência de AlimentosDoutor em Ciência de Alimentos140610/2014-6CNP

Extremophilic Cellulases: A Comprehensive Review

Microbial cellulases are an important industrial enzyme having diverse applications in biotechnology, environmental challenges, industrial products and processes. Extremophiles like thermophillic bacteria are a good source of industrially important cellulases as these can withstand industrially rigorous procedures like paper deinking, fabric material softening, bio stoning, paper and pulp, biopolishing cloth material, animal feed and juice. Identification of novel cellulases or improving them through biotechnological interventions has remained a challenge for researchers. Genetic manipulation of thermophilic bacteria for increased cellulase production or synthetic biology approaches for cellulase gene/gene cluster extraction from thermophilic bacteria and expression in appropriate hosts for improved cellulase synthesis. The classic and high-throughput technologies like genomics, metagenomics and bioinformatics could be exploited to isolate cellulase genes from a variety of thermophilic bacteria and further processing. Keeping in view the ultimate requirement of extremophilic cellulases in industries, present study is a compilation of various aspects related to extremophilic cellulases their sources, production, biotechnological interventions and challenges. 

Characterization of novel cellulase and glucose isomerase-producing bacteria and optimization of the enzyme production conditions and their potential applications in environment and industry

Cellulases and glucose isomerases are vital enzymes in converting cellulose into fructose. Cellulases catalyze cellulose conversion to glucose, while glucose isomerase catalyzes the reversible isomerization of glucose to fructose. There is a growing interest lately in producing bio-based chemicals and materials from fructose. Soil bacteria produce these enzymes. The characterization of bacteria for enzyme saccharification of biomass is essential for fructose production and reducing the time and cost of current bioconversion processes. From this perspective, we characterized novel cellulase and glucose isomerase-producing bacteria from soil samples and optimize their enzyme production. Coculturing and whole-cell immobilization for glucose isomerase and bacterial resistance to environmental factors were also investigated. Six bacterial strains, Paenarthrobacter sp. MKAL1, Hymenobacter sp. MKAL2, Mycobacterium sp. MKAL3, Stenotrophomonas sp. MKAL4, Chryseobacterium sp. MKAL5 and Bacillus sp. MKAL6 were isolated from mixture soil samples collected at Kingfisher Lake and the University of Manitoba campus and identified using 16S rRNA gene sequence analysis. Using plate assay techniques, these strains were selected for cellulase and glucose isomerase production based on the clearance zone appearance. These strains displayed various morphological and biochemical characteristics. [...