Production, purification, characterization and application of two novel endoglucanases from buffalo rumen metagenome.

BACKGROUND: Lignocellulose biomass is the most abundant and renewable material in nature. The objectives of this study were to characterize two endoglucanases TrepCel3 and TrepCel4, and determine the effect of the combination of them (1.2 mg TrepCel3, 0.8 mg TrepCel4) on in vitro rumen fermentation characteristics. In this study, three nature lignocellulosic substrates (rice straw, RS; wheat straw, WS; leymus chinensis, LC) were evaluated for their in vitro digestibility, gas, NH3-N and volatile fatty acid (VFA) production, and microbial protein (MCP) synthesis by adding enzymatic combination. METHODS: Two endoglucanases' genes were successfully expressed in Escherichia coli (E. coli) BL21 (DE3), and enzymatic characteristics were further characterized. The combination of TrepCel3 and TrepCel4 was incubated with lignocellulosic substrates to evaluate its hydrolysis ability. RESULTS: The maximum enzymatic activity of TrepCel3 was determined at pH 5.0 and 40 °C, while TrepCel4 was at pH 6.0 and 50 °C. They were stable over the temperature range of 30 to 60 °C, and active within the pH range of 4.0 to 9.0. The TrepCel3 and TrepCel4 had the highest activity in lichenan 436.9 ± 8.30 and 377.6 ± 6.80 U/mg, respectively. The combination of TrepCel3 and TrepCel4 exhibited the highest efficiency at the ratio of 60:40. Compared to maximum hydrolysis of TrepCel3 or TrepCel4 separately, this combination was shown to have a superior ability to maximize the saccharification yield from lignocellulosic substrates up to 188.4% for RS, 236.7% for wheat straw WS, 222.4% for LC and 131.1% for sugar beet pulp (SBP). Supplemental this combination enhanced the dry matter digestion (DMD), gas, NH3-N and VFA production, and MCP synthesis during in vitro rumen fermentation. CONCLUSIONS: The TrepCel3 and TrepCel4 exhibited the synergistic relationship (60:40) and significantly increased the saccharification yield of lignocellulosic substrates. The combination of them stimulated in vitro rumen fermentation of lignocellulosic substrates. This combination has the potential to be a feed additive to improve agricultural residues utilization in ruminants. If possible, in the future, experiments in vivo should be carried out to fully evaluate its effect.

Control of phenotypic diversification based on serial cultivations on different carbon sources leads to improved bacterial xylanase production.

Thermobacillus xylanilyticus is a thermophilic and hemicellulolytic bacterium of interest for the production of thermostable hemicellulases. Enzymes' production by this bacterium is challenging, because the proliferation of a cheating subpopulation of cells during exponential growth impairs the production of xylanase after serial cultivations. Accordingly, a strategy of successive cultivations with cells transfers in stationary phase and the use of wheat bran and wheat straw as carbon sources were tested. The ratio between subpopulations and their corresponding metabolic activities were studied by flow cytometry and the resulting hemicellulases production (xylanase, acetyl esterase and ß-xylosidase) followed. During serial cultivations, the results pointed out an increase of the enzymatic activities. On xylan, compared to the first cultivation, the xylanase activity increases by 7.15-fold after only four cultivations. On the other hand, the debranching activities were increased by 5.88-fold and 57.2-fold on wheat straw and by 2.77-fold and 3.34-fold on wheat bran for ß-xylosidase and acetyl esterase, respectively. The different enzymatic activities then stabilized, reached a plateau and further decreased. Study of the stability and reversibility of the enzyme production revealed cell-to-cell heterogeneities in metabolic activities which could be linked to the reversibility of enzymatic activity changes. Thus, the strategy of successive transfers during the stationary phase of growth, combined with the use of complex lignocellulosic substrates as carbon sources, is an efficient strategy to optimize the hemicellulases production by T. xylanilyticus, by preventing the selection of cheaters.

Re-routing of Sugar Catabolism Provides a Better Insight Into Fungal Flexibility in Using Plant Biomass-Derived Monomers as Substrates.

The filamentous ascomycete Aspergillus niger has received increasing interest as a cell factory, being able to efficiently degrade plant cell wall polysaccharides as well as having an extensive metabolism to convert the released monosaccharides into value added compounds. The pentoses D-xylose and L-arabinose are the most abundant monosaccharides in plant biomass after the hexose D-glucose, being major constituents of xylan, pectin and xyloglucan. In this study, the influence of selected pentose catabolic pathway (PCP) deletion strains on growth on plant biomass and re-routing of sugar catabolism was addressed to gain a better understanding of the flexibility of this fungus in using plant biomass-derived monomers. The transcriptome, metabolome and proteome response of three PCP mutant strains, ΔlarAΔxyrAΔxyrB, ΔladAΔxdhAΔsdhA and ΔxkiA, grown on wheat bran (WB) and sugar beet pulp (SBP), was evaluated. Our results showed that despite the absolute impact of these PCP mutations on pure pentose sugars, they are not as critical for growth of A. niger on more complex biomass substrates, such as WB and SBP. However, significant phenotypic variation was observed between the two biomass substrates, but also between the different PCP mutants. This shows that the high sugar heterogeneity of these substrates in combination with the high complexity and adaptability of the fungal sugar metabolism allow for activation of alternative strategies to support growth.

Removal of pharmaceuticals and personal care products using native fungal enzymes extracted during the ligninolytic process.

Significant concentrations of pharmaceuticals and personal care products (PPCPs) have been detected in aquatic environment. Fungal enzymatic processes can oxidize these persistent PPCPs; thus, these processes have attracted considerable attention from the scientific community. Here, we evaluated the efficacy of the removal of PPCPs using native fungal enzymes derived from Bjerkandera spp. TBB-03 under various conditions. Among the eight lignocellulosic substrates, ash, which showed the highest laccase production, was selected as the sole enzyme inducer. TBB-03 laccase was found to exhibit remarkable stability under varied pH and temperature conditions. Acetaminophen and bisphenol A were effectively removed by TBB-03 laccase under various conditions, except at pH 8. Although TBB-03 laccase could not efficiently remove single-state sulfamethoxazole directly, a 22% of improvement in sulfamethoxazole removal was observed in the presence of acetaminophen. Overall, our proposed approach showed that Bjerkandera adusta TBB-03 can be potentially applied for further research regarding PPCP remediation.

Aqueous two-phase partitioning and characterization of xylanase produced by Streptomyces geysiriensis from low cost lignocellulosic substrates.

Microbial production of xylanase is gaining the commercial importance, due to its wide range of applications from paper and pulp to food and feed industries. Streptomyces geysiriensis was used for the production of extracellular xylanase from lignocellulosic substrates such as rice bran and saw dust, under solid-state fermentation. The influence of pH, temperature and incubation period for the maximum production of xylanase was investigated with 1:2 (w/v) of substrate to moisture ratio at 100 rpm shaking conditions. The maximum production was recorded after 5 days of fermentation with pH 8.0 at 40 °C. The scale-up was done based on the results of optimized parameters using 3 L Applikon autoclavable bioreactor with maximum yield of 186 U/ml after 4 days of fermentation. Extracellular xylanase was separated by partitioning in aqueous two-phase system consisting of 20% polyethylene glycol 6000 and 12% K2HPO4 with maximum yield of 93.97%. The investigation of the effect of pH and temperature and its incubation time showed that xylanase was retained its activity in a pH range of 6.5-8.5, with thermal stability from 20 °C to 60 °C up to 180 min. The presence of metal ions was found to inhibit the activity of xylanase especially Cu2+ and Zn2+. Xylanase was stable both at 4 °C and room temperature (35 °C) for 30 and 9 days respectively. The kinetic parameters Km (0.48 mg/ml) and Vmax (8.33 U/mg) were determined using birchwood xylan as substrate.

Biotransformation of rice and sunflower side-streams by dikaryotic and monokaryotic strains of Pleurotus sapidus: Impact on phenolic profiles and bioactive properties.

Fungi are known to modify the properties of lignocellulosic materials during solid-state fermentation (SSF). In this study, agricultural side-streams (sunflower seed hulls, rice husks and rice straw) were used as substrates for SSF with dikaryotic and monokaryotic strains of Pleurotus sapidus. The phenolic profiles of the mentioned substrates were characterized by LC-DAD/ESI-MSn pre- and post- fermentation. Moreover, antioxidant, cytotoxic and antimicrobial activities were screened against oxidizable cellular substrates, tumour and primary cell lines, and different bacteria and fungi, respectively. The concentration of phenolic compounds in the crop side-streams was reduced after fermentation with both strains of the fungus. The fermented extracts also displayed lower antioxidant and cytotoxic activities and had no hepatotoxicity. The antimicrobial activity depended upon the crop side-stream and/or SSF conditions. These results indicate that P. sapidus represent a good candidate to modify the phenolic fraction presents in crop side-streams with a consequent decrease in its bioactivities. However, the SSF with P. sapidus strains play an interesting role in the detoxification of plant materials which can be used for different applications according to the "reduce - reuse - recycle" concept contributing with the sustainable land use and circular economy.

Microbial sources of polyunsaturated fatty acids (PUFAs) and the prospect of organic residues and wastes as growth media for PUFA-producing microorganisms.

The discovery of non-fish sources of polyunsaturated fatty acids (PUFAs) is of great biotechnological importance. Although various oleaginous microalgae and fungi are able of accumulating storage lipids (single cell oils - SCOs) containing PUFAs, the industrial applications utilizing these organisms are rather limited due to the high-fermentation cost. However, combining SCO production with other biotechnological applications, including waste and by-product valorization, can overcome this difficulty. In the current review, we present the major sources of fungi (i.e. members of Mucoromycota, fungoid-like Thraustochytrids and genetically modified strains of Yarrowia lipolytica) and microalgae (e.g. Isochrysis, NannochloropsisandTetraselmis) that have come recently to the forefront due to their ability to produce PUFAs. Approaches adopted in order to increase PUFA productivity and the potential of using various residues, such as agro-industrial, food and aquaculture wastes as fermentation substrates for SCO production have been considered and discussed. We concluded that several organic residues can be utilized as feedstock in the SCO production increasing the competitiveness of oleaginous organisms against conventional PUFA producers.

Combined bioaugmentation with anaerobic ruminal fungi and fermentative bacteria to enhance biogas production from wheat straw and mushroom spent straw.

Bioaugmentation with anaerobic ruminal fungi and a pool of hydrogen-producing fermenting bacteria was tested on wheat straw (WS) and mushroom spent straw (MSS) with the aim of improving anaerobic digestion performance. Batch tests were set up to simulate a Bioaugmentation Anaerobic Digestion (BAD) treatment comparing single- (I-BAD) and two-stage (II-BAD) process configurations, at two reactor scales, 120 and 1200 ml (×10). In both cases, higher CH4 cumulative production was obtained in the II-BAD configuration on WS (65.1 ±â€¯8.9 Nml and 922 ±â€¯73.8 Nml respectively). The II-BADx10 tests allowed increasing CH4 production (≃290% and ≃330% on WS and MSS, respectively) when compared to the unaugmented condition. Final results highlighted the achievable advantages of the two stage configuration in terms of CH4 production enhancement. Microbial community investigations confirmed the efficiency of the bioaugmentation treatment and revealed that such a result was mainly related to the Methanosarcinales increase, mostly composed by Methanosaeta.

Evaluation of edible mushroom Oudemansiella canarii cultivation on different lignocellulosic substrates.

In this study, the mycelial growth rate, mycelial colonization time, yield, and biological efficiency of the edible mushroom Oudemansiella canarii were determined, and the effects of different substrate combinations on productivity, chemical contents and amino acids were evaluated. Lignocellulosic wastes, such as cottonseed hull, sawdust, corncob, and their combinations supplemented with 18% wheat bran and 2% lime, were used for the cultivation of O. canarii. The biological efficiency (BE) and essential amino acid content of treatment T1, which consisted of 80% cottonseed hull, were the highest among all the tested treatments. Mixtures that included sawdust, such as treatments T2 (80% sawdust), T4 (40% sawdust + 40% cottonseed hull), and T6 (40% sawdust + 40% corncob), exhibited lower yield and BE. Corncob was good for O. canarii production in terms of yield and BE, whereas the mycelial growth rate and colonization time were lower compared to those on other substrates. Comparing the BE, essential amino acids, and other traits of the six treatments, treatment T1 (80% cottonseed hull) was the best formula for O. canarii cultivation and should be extended in the future.

Fermentative hydrogen production from agroindustrial lignocellulosic substrates.

To achieve economically competitive biological hydrogen production, it is crucial to consider inexpensive materials such as lignocellulosic substrate residues derived from agroindustrial activities. It is possible to use (1) lignocellulosic materials without any type of pretreatment, (2) lignocellulosic materials after a pretreatment step, and (3) lignocellulosic materials hydrolysates originating from a pretreatment step followed by enzymatic hydrolysis. According to the current literature data on fermentative H2 production presented in this review, thermophilic conditions produce H2 in yields approximately 75% higher than those obtained in mesophilic conditions using untreated lignocellulosic substrates. The average H2 production from pretreated material is 3.17 ± 1.79 mmol of H2/g of substrate, which is approximately 50% higher compared with the average yield achieved using untreated materials (2.17 ± 1.84 mmol of H2/g of substrate). Biological pretreatment affords the highest average yield 4.54 ± 1.78 mmol of H2/g of substrate compared with the acid and basic pretreatment - average yields of 2.94 ± 1.85 and 2.41 ± 1.52 mmol of H2/g of substrate, respectively. The average H2 yield from hydrolysates, obtained from a pretreatment step and enzymatic hydrolysis (3.78 ± 1.92 mmol of H2/g), was lower compared with the yield of substrates pretreated by biological methods only, demonstrating that it is important to avoid the formation of inhibitors generated by chemical pretreatments. Based on this review, exploring other microorganisms and optimizing the pretreatment and hydrolysis conditions can make the use of lignocellulosic substrates a sustainable way to produce H2.

Fermentative hydrogen production from agroindustrial lignocellulosic substrates

To achieve economically competitive biological hydrogen production, it is crucial to consider inexpensive materials such as lignocellulosic substrate residues derived from agroindustrial activities. It is possible to use (1) lignocellulosic materials without any type of pretreatment, (2) lignocellulosic materials after a pretreatment step, and (3) lignocellulosic materials hydrolysates originating from a pretreatment step followed by enzymatic hydrolysis. According to the current literature data on fermentative H2 production presented in this review, thermophilic conditions produce H2 in yields approximately 75% higher than those obtained in mesophilic conditions using untreated lignocellulosic substrates. The average H2 production from pretreated material is 3.17 ± 1.79 mmol of H2/g of substrate, which is approximately 50% higher compared with the average yield achieved using untreated materials (2.17 ± 1.84 mmol of H2/g of substrate). Biological pretreatment affords the highest average yield 4.54 ± 1.78 mmol of H2/g of substrate compared with the acid and basic pretreatment - average yields of 2.94 ± 1.85 and 2.41 ± 1.52 mmol of H2/g of substrate, respectively. The average H2 yield from hydrolysates, obtained from a pretreatment step and enzymatic hydrolysis (3.78 ± 1.92 mmol of H2/g), was lower compared with the yield of substrates pretreated by biological methods only, demonstrating that it is important to avoid the formation of inhibitors generated by chemical pretreatments. Based on this review, exploring other microorganisms and optimizing the pretreatment and hydrolysis conditions can make the use of lignocellulosic substrates a sustainable way to produce H2.

Bioconversion and saccharification of some lignocellulosic wastes by aspergillus oryzae ITCC-4857.01 for fermentable sugar production

The recent interest in bioconversion of agricultural and industrial wastes to chemical feedstock has led to extensive studies on cellulolytic enzymes produced by microorganisms. In the present study three lignocellulosic substrates viz. sugarcane bagasse, sawdust and water hyacinth were pre-treated with alkali and enzyme and their effect on bioconversion has been investigated. The ability of selected substrates for induction of cellulase enzyme by A. oryzae ITCC 4857.01 and for the potentiality of the induced enzyme to saccharify the substrates were also assessed. The maximum degree of conversion of substrate (0.415 percent) and improved specific substrate consumption (0.99 g substrate/g dry biomass) was exhibited in sugarcane bagasse after alkali treatment at 96 hrs. Both alkali-treatment and enzyme-treatment, water hyacinth was the best for cellulase induction and showed maximum endoglucanase activity of 11.42 U/ml. Reducing sugar yield ranged from 1.12 mg/ml for enzyme treated sawdust at 48 hrs to 7.53 mg/ml for alkali treated sugarcane bagasse at 96 hrs. Alkali-treated sugarcane bagasse gave the highest saccharification rate of 9.03 percent after 96 hrs. The most resistant substrate was sawdust which produced 5.92 percent saccharification by alkaline treatment. The saccharification of lignocellulosic substrates by enzyme produced by A. oryzae ITCC 4857.01 indicates the enzymes specificity towards the substrates. The use of such enzyme in lingo-cellulose hydrolysis will lead to efficient conversion of cellulose materials to other important products.

Cellulolytic enzymes on lignocellulosic substrates in solid state fermentation by Aspergillus niger.

The production of cellulolytic enzymes by Aspergillus niger on lignocellulosic substrates groundnut fodder, wheat bran, rice bran and sawdust in solid state fermentation in a laboratory scale was compared. Czapek Dox liquid broth amended with cellulose (0.5%) was used to moisten lignocellulosic solid supports for cultivation of Aspergillus niger. The production of filter paperase, carboxymethyl cellulase and -glucosidase were monitored at daily intervals for 5 days. The peak production of the enzymes occurred within 3 days of incubation. Among solid supports used in the study, wheat bran was the best solid matrix followed by groundnut fodder in production of cellulolytic enzymes in solid state fermentation. Groundnut fodder supported significant production of FPase (2.09 FPU/g), CMCase (1.36 U/g) and -glucosidase activity (0.0117 U/g) in solid state fermentation. Considerable secretion of protein (5.10 mg/g) on groundnut fodder at peak time interval 1st day of incubation was recorded.