Uncovering the lignin-degrading potential of Serratia quinivorans AORB19: insights from genomic analyses and alkaline lignin degradation.

BACKGROUND: Lignin is an intricate phenolic polymer found in plant cell walls that has tremendous potential for being converted into value-added products with the possibility of significantly increasing the economics of bio-refineries. Although lignin in nature is bio-degradable, its biocatalytic conversion is challenging due to its stable complex structure and recalcitrance. In this context, an understanding of strain's genomics, enzymes, and degradation pathways can provide a solution for breaking down lignin to unlock the full potential of lignin as a dominant valuable bioresource. A gammaproteobacterial strain AORB19 has been isolated previously from decomposed wood based on its high laccase production. This work then focused on the detailed genomic and functional characterization of this strain based on whole genome sequencing, the identification of lignin degradation products, and the strain's laccase production capabilities on various agro-industrial residues. RESULTS: Lignin degrading bacterial strain AORB19 was identified as Serratia quinivorans based on whole genome sequencing and core genome phylogeny. The strain comprised a total of 123 annotated CAZyme genes, including ten cellulases, four hemicellulases, five predicted carbohydrate esterase genes, and eight lignin-degrading enzyme genes. Strain AORB19 was also found to possess genes associated with metabolic pathways such as the ß-ketoadipate, gentisate, anthranilate, homogentisic, and phenylacetate CoA pathways. LC-UV analysis demonstrated the presence of p-hydroxybenzaldehyde and vanillin in the culture media which constitutes potent biosignatures indicating the strain's capability to degrade lignin. Finally, the study evaluated the laccase production of Serratia AORB19 grown with various industrial raw materials, with the highest activity detected on flax seed meal (257.71 U/L), followed by pea hull (230.11 U/L), canola meal (209.56 U/L), okara (187.67 U/L), and barley malt sprouts (169.27 U/L). CONCLUSIONS: The whole genome analysis of Serratia quinivorans AORB19, elucidated a repertoire of genes, pathways and enzymes vital for lignin degradation that widens the understanding of ligninolytic metabolism among bacterial lignin degraders. The LC-UV analysis of the lignin degradation products coupled with the ability of S. quinivorans AORB19 to produce laccase on diverse agro-industrial residues underscores its versatility and its potential to contribute to the economic viability of bio-refineries.

A comparative study of Cellulomonas sp. and Bacillus sp. in utilizing lignocellulosic biomass as feedstocks for enzyme production.

The demand for enzymes is increasing continuously due to their applications in various avenues. The pectin-hydrolyzing bacteria, Cellulomonas sp. and Bacillus sp., isolated from forest soil have the potential to produce industrially important enzymes (pectinase, PGase, Cellulase, and xylanase). However, these bacteria have different optimal cultural conditions for pectinase production. The optimal cultural conditions for Cellulomonas sp. were room temperature (25-26℃), pH 7, 1% inoculum volume, and 1.5% citrus pectin with 8.82 ± 0.92 U/mL pectinase activity. And Bacillus sp. illustrated the highest pectinase activity (12.35 ± 0.72 U/mL) at room temperature, pH 10, 1% inoculum volume, and 1.5% pectin concentration. Among the different agro-wastes, the orange peel was found to be the best substrate for pectinase, PGase, and cellulase activity whereas barley straw for xylanase activity. Further, Cellulomonas sp. and Bacillus sp. illustrated higher pectinase activity from commercial pectin compared to orange peel showing their preference for commercial citrus pectin. In addition, the optimization by the Box-Behnken design increased pectinase activity for Cellulomonas sp., while a noticeable increase in activity was not observed in Bacillus sp. Besides, all the agro-wastes exploited in this study can be used for pectinase, PGase, and xylanase production but not cellulase. The study revealed that each bacteria has its specific optimal conditions and there is a variation in the capacity of utilizing the various lignocellulosic biomass.

Biomass-Degrading Enzyme(s) Production and Biomass Degradation by a Novel Streptomyces thermocarboxydus.

Modern society has a great challenge to decrease waste and minimize the adverse effects of wastes on the economy, environment, and individual health. Thus, this study focuses on the use of eight agro-wastes (banana peel, barley straw, canola straw, pomegranate peel, orange peel, pumpkin pulp+seeds, maple leaf, and brewer's spent grains) by a novel bacterium (Streptomyces thermocarboxydus) for enzymes production. Further, the study explored the subsequent degradation of those wastes by the bacterium. This bacterium was isolated from forest soil and identified as Streptomyces thermocarboxydus by 16S rRNA sequence analysis. The biodegrading capability of S. thermocarboxydus was determined by observing the clear zone around the colony cultured on the agar plate containing the different biomasses as sole carbon sources and calculating the substrate degradation ratios. Furthermore, scanning electron microscopy images of eight agro-wastes before and after bacterial treatment and weight loss of agro-wastes revealed the bacterium degraded the biomasses. The different trends of enzyme activities were observed for various wastes, and the maximum activity depended on the type of agro-wastes. Overall, S. thermocarboxydus was found to be a potential candidate for pectinase and xylanase production. The enzyme production varies with the concentration of the biomasses.

Surfactant-Containing Foam Effectively Enhanced the Removal of Polycyclic Aromatic Hydrocarbons from Heavily Contaminated Soil.

Surfactant remediation has an excellent record of removing polycyclic aromatic hydrocarbons (PAHs). By using simulation experiments, we investigated the properties and mechanism of a surfactant-containing foam and its effect on PAH removal. Our results suggest that the optimal conditions by foam washing are as follows: 40 mmol·L-1 of rhamnolipid and fulvic acid mixed surfactant (V: V = 3:1), with 70:3 and 20:3 foam gas-liquid ratio for naphthalene and phenanthrene, respectively (pH 6, 50°C, 2 h). Under the optimal conditions, 60.1% and 56.68% removal efficiencies were achieved against naphthalene and phenanthrene from contaminated soil, respectively. These values were lower than those from the simulated media (76.69% and 70.43% for naphthalene and phenanthrene, respectively). The strong PAH adsorption on the soil particles antagonized volatilization, the key PAH removal mechanism by foam leaching. Therefore, this research provides relevant information for using surfactant foam to remediate heavily PAH-contaminated soils.

Proteomic analysis revealed the roles of YRR1 deletion in enhancing the vanillin resistance of Saccharomyces cerevisiae.

BACKGROUND: Vanillin is one of the important phenolic inhibitors in Saccharomyces cerevisiae for bioconversion of lignocellulosic materials and has been reported to inhibit the translation process in cells. In our previous studies, it was confirmed that the deletion of the transcription factor gene YRR1 enhanced vanillin resistance by promoting some translation-related processes at the transcription level. In this work, we investigated the effects of proteomic changes upon induction of vanillin stress and deletion of YRR1 to provide unique perspectives from a transcriptome analysis for comprehending the mechanisms of YRR1 deletion in the protective response of yeast to vanillin. RESULTS: In wild-type cells, vanillin reduced two dozens of ribosomal proteins contents while upregulated proteins involved in glycolysis, oxidative phosphorylation, and the pentose phosphate pathway in cells. The ratios of NADPH/NADP+ and NADH/NAD+ were increased when cells responded to vanillin stress. The differentially expressed proteins perturbed by YRR1 deletion were much more abundant than and showed no overlaps with transcriptome changes, indicating that Yrr1 affects the synthesis of certain proteins. Forty-eight of 112 upregulated proteins were involved in the stress response, translational and transcriptional regulation. YRR1 deletion increased the expression of HAA1-encoding transcriptional activator, TMA17-encoding proteasome assembly chaperone and MBF1-encoding coactivator at the protein level, as confirmed by ELISA. Cultivation data showed that the overexpression of HAA1 and TMA17 enhanced resistance to vanillin in S. cerevisiae. CONCLUSIONS: Cells conserve energy by decreasing the content of ribosomal proteins, producing more energy and NAD(P)H for survival in response to vanillin stress. Yrr1 improved vanillin resistance by increasing the protein quantities of Haa1, Tma17 and Mbf1. These results showed the response of S. cerevisiae to vanillin and how YRR1 deletion increases vanillin resistance at the protein level. These findings may advance our knowledge of how YRR1 deletion protects yeast from vanillin stress and offer novel targets for genetic engineering of designing inhibitor-resistant ethanologenic yeast strains.

New insights in pectinase production development and industrial applications.

Pectinase, a group of pectin degrading enzymes, is one of the most influential industrial enzymes, helpful in producing a wide variety of products with good qualities. These enzymes are biocatalysts and are highly specific, non-toxic, sustainable, and eco-friendly. Consequently, both pectin and pectinase are crucially essential biomolecules with extensive applicatory perception in the biotechnological sector. The market demand and application of pectinases in new sectors are continuously increasing. However, due to the high cost of the substrate used for the growth of microbes, the production of pectinase using microorganisms is limited. Therefore, low-cost or no-cost substrates, such as various agricultural biomasses, are emphasized in producing pectinases. The importance and implications of pectinases are rising in diverse areas, including bioethanol production, extraction of DNA, and protoplast isolation from a plant. Therefore, this review briefly describes the structure of pectin, types and source of pectinases, substrates and strategies used for pectinases production, and emphasizes diverse potential applications of pectinases. The review also has included a list of pectinases producing microbes and alternative substrates for commercial production of pectinase applicable in pectinase-based industrial technology.Key points• Pectinase applications are continuously expanding.• Organic wastes can be used as low-cost sources of pectin.• Utilization of wastes helps to reduce pollution.

Dye-decolorization of a newly isolated strain Bacillus amyloliquefaciens W36.

Dye-decolorization is one of the most important steps in dye-polluted wastewater treatment. The dye-decolorization bacteria were isolated from active sludge collected from wastewater treating pond of a dyeing and printing plant using serial dilution method. Among the 44 bacteria isolates from the active sludge, the strain Bacillus amyloliquefaciens W36 was found to have strong ability in dye-decolorization. The effects of carbon source, nitrogen sources, C/N, metal ions, temperature, pH, and rotation speed for dye-decolorization were investigated. The optimum decolorization conditions were that the strain was grown in enriched mineral salt medium (EMSM) using maltose 1 g/L, (NH4)2SO4 1 g/L as carbon and nitrogen source respectively, supplemented with 100 mg/L different dyes (pH 6.0), at 30 °C, 200 rpm from 48 to 96 h. The bacteria could aerobically decolorize dyes, such as Coomassie brilliant blue (95.42%), Bromcresol purple (93.34%), Congo red (72.37%) and Sarranine (61.7%), within 96 h. The dyes decolorization products were analyzed by ultra-violet and visible (UV-vis) spectroscopy before and after decolorization, which indicated that the four dyes were significantly degraded by the strain. The results indicated that the bacteria Bacillus amyloliquefaciens W36 could be used in dye-polluted wastewater treatment.

A simplified quick microbial genomic DNA extraction via freeze-thawing cycles.

Effective isolation of high-quality genomic DNA is one of the essential steps in molecular biology, biochemistry, and genetic studies. Here we describe a simplified procedure based on repeated freeze-thawing cycles to isolate genomic DNA from different organisms of microbes (Burkholderia pyrrocinia JK-SH007, Bacillus pumilus HRl0, Botrytis cinerea) and nematodes (Bursaphelenchus xylophilus). The DNA extraction buffer includes 10% of CTAB; 4% of NaCl (W/V); 20 mM of ethylenediamine tetraacetic acid; 100 mM of Tris-HCl, pH 8.0 and 1% of polyvinylpyrrolidone. The released DNA was purified from the mixture using a phenol/chloroform mixture and precipitated in 70% ethanol to remove proteins, carbohydrates, phenols, RNA, etc. Our method is a reproducible, simple, and rapid technique for routine DNA extractions from various microorganisms and nematodes. Furthermore, the low cost of this method could be an economic benefit to large-scale studies.

Analysis of Centranthera grandiflora Benth Transcriptome Explores Genes of Catalpol, Acteoside and Azafrin Biosynthesis.

Cardiovascular diseases (CVDs) are a major cause of health loss in the world. Prevention and treatment of this disease by traditional Chinese medicine is a promising method. Centranthera grandiflora Benth is a high-value medicinal herb in the prevention and treatment of CVDs; its main medicinal components include iridoid glycosides, phenylethanoid glycosides, and azafrin in roots. However, biosynthetic pathways of these components and their regulatory mechanisms are unknown. Furthermore, there are no genomic resources of this herb. In this article, we provide sequence and transcript abundance data for the root, stem, and leaf transcriptome of C. grandiflora Benth obtained by the Illumina Hiseq2000. More than 438 million clean reads were obtained from root, stem, and leaf libraries, which produced 153,198 unigenes. Based on databases annotation, a total of 557, 213, and 161 unigenes were annotated to catalpol, acteoside, and azafrin biosynthetic pathways, respectively. Differentially expressed gene analysis identified 14,875 unigenes differentially enriched between leaf and root with 8,054 upregulated genes and 6,821 downregulated genes. Candidate MYB transcription factors involved in catalpol, acteoside, and azafrin biosynthesis were also predicated. This work is the first transcriptome analysis in C. grandiflora Benth which will aid the deciphering of biosynthesis pathways and regulatory mechanisms of active components.

Gene expression metadata analysis reveals molecular mechanisms employed by Phanerochaete chrysosporium during lignin degradation and detoxification of plant extractives.

Lignin, most complex and abundant biopolymer on the earth's surface, attains its stability from intricate polyphenolic units and non-phenolic bonds, making it difficult to depolymerize or separate from other units of biomass. Eccentric lignin degrading ability and availability of annotated genome make Phanerochaete chrysosporium ideal for studying lignin degrading mechanisms. Decoding and understanding the molecular mechanisms underlying the process of lignin degradation will significantly aid the progressing biofuel industries and lead to the production of commercially vital platform chemicals. In this study, we have performed a large-scale metadata analysis to understand the common gene expression patterns of P. chrysosporium during lignin degradation. Gene expression datasets were retrieved from NCBI GEO database and analyzed using GEO2R and Bioconductor packages. Commonly expressed statistically significant genes among different datasets were further considered to understand their involvement in lignin degradation and detoxification mechanisms. We have observed three sets of enzymes commonly expressed during ligninolytic conditions which were later classified into primary ligninolytic, aromatic compound-degrading and other necessary enzymes. Similarly, we have observed three sets of genes coding for detoxification and stress-responsive, phase I and phase II metabolic enzymes. Results obtained in this study indicate the coordinated action of enzymes involved in lignin depolymerization and detoxification-stress responses under ligninolytic conditions. We have developed tentative network of genes and enzymes involved in lignin degradation and detoxification mechanisms by P. chrysosporium based on the literature and results obtained in this study. However, ambiguity raised due to higher expression of several uncharacterized proteins necessitates for further proteomic studies in P. chrysosporium.

The ACEII recombinant Trichoderma reesei QM9414 strains with enhanced xylanase production and its applications in production of xylitol from tree barks.

BACKGROUND: ACEII transcription factor plays a significant role in regulating the expression of cellulase and hemicellulase encoding genes. Apart from ACEII, transcription factors such as XYR1, CRE1, HAP2/3/5 complex and ACEI function in a coordinated pattern for regulating the gene expression of cellulases and hemicellulases. Studies have demonstrated that ACEII gene deletion results in decreased total cellulase and xylanase activities with reduced transcript levels of lignocellulolytic enzymes. RESULTS: In this study, we have successfully transformed the ACEII transcription factor encoding gene in Trichoderma reesei to significantly improve its degrading abilities. Transformation experiments on parental strain T. reesei QM9414 has resulted in five genetically engineered strains T/Ace2-2, T/Ace2-5, T/Ace2-8, T/Ace5-4 and T/Ace10-1. Among which, T/Ace2-2 has exhibited significant increase in enzyme activity by twofolds, when compared to parental strain. The T/Ace2-2 was cultured on growth substrates containing 2% bark supplemented with (a) sugar free + MA medium (b) glucose + MA medium and (c) xylose + MA medium. The bark degradation efficiency of genetically modified T/Ace2-2 strain was assessed by analyzing the xylitol production yield using HPAEC. By 6th day, about 10.52 g/l of xylitol was produced through enzymatic conversion of bark (2% bark + MA + xylose) by the T/Ace2-2 strain and by 7th day the conversion rate was found to be 0.21 g/g. Obtained results confirmed that bark growth medium supplemented with D-xylose has profoundly increased the conversion rate of bark by T/Ace2-2 strain when compared to sugar free and glucose supplemented growth media. Results obtained from scanning electron microscopy has endorsed our current results. Bark samples inoculated with T/Ace2-2 strain has showed large number of degraded cells with clearly visible cavities and fractures, by exposing the microfibrillar interwoven complex. CONCLUSION: We propose a cost effective and ecofriendly method for the degradation of lignocellulosic biomass such as bark to produce xylitol by using genetically modified T. reesei. Efficient conversion rate and production yield obtained in our current study provides a great scope for the xylitol industries, as our method bypasses the pretreatment of bark achieving clean and low-cost xylitol production.

Colonization characteristics and surface effects of microplastic biofilms: Implications for environmental behavior of typical pollutants.

This paper summarizes the colonization dynamics of biofilms on microplastics (MPs) surfaces in aquatic environments, encompassing bacterial characteristics, environmental factors affecting biofilm formation, and matrix types and characteristics. The interaction between biofilm and MPs was also discussed. Through summarizing recent literatures, it was found that MPs surfaces offer numerous benefits to microorganisms, including nutrient enrichment and enhanced resistance to environmental stress. Biofilm colonization changes the surface physical and chemical properties as well as the transport behavior of MPs. At the same time, biofilms also play an important role in the fragmentation and degradation of MPs. In addition, we also investigated the coexistence level, adsorption mechanism, enrichment, and transformation of MPs by environmental pollutants mediated by biofilms. Moreover, an interesting aspect about the colonization of biofilms was discussed. Biofilm colonization not only had a great effect on the accumulation of heavy metals by MPs, but also affects the interaction between particles and environmental pollutants, thereby changing their toxic effects and increasing the difficulty of MPs treatment. Consequently, further attention and research are warranted to delve into the internal mechanisms, environmental risks, and the control of the coexistence of MPs and biofilms.

Low-cost cultivation of Nannochloropsis oceanica in newly designed photobioreactors and its productivity trends in semi-continuous cultivation under inland outdoor conditions.

Most marine microalgae are typically cultivated in coastal areas due to challenges in inland cultivation. In this 185 days experiment, Nannochloropsis oceanica was semi-continuously cultivated inland using different photobioreactors (PBRs). The newly designed 700-liter (L) PBR exhibited tolerance to seasonal changes compared to the 150-L PBRs. The innovative in-situ oxygen release rate (ORR) measurement method results indicated that ORR was influenced by light intensity and temperature. The optimal temperature range for N. oceanica growth was 14-25 â„ƒ, demonstrated cold tolerance and lipid accumulation at low temperatures. The maximum lipid content in 700-L and 150-L PBRs was 29 % and 28 %, respectively. Based on the average biomass productivity, the price of N. oceanica was $11.89 kg-1 (or $3.35 kg-1 based on maximum biomass productivity), which is cheaper than the current market price of $20.19 kg-1. From results, smaller PBRs at the same hydro electricity price are more cost-effective.

Lignocellulosic biomass pretreatment: Industrial oriented high-solid twin-screw extrusion method to improve biogas production from forestry biomass resources.

Forestry lignocellulosic waste is an important, largely untapped source of biomass for producing clean energy. In this study, a high-solids twin-screw extrusion approach is developed as a novel pretreatment method to effectively increase the biogas production rate to better fit commercial requirements. Multiple screw designs are progressively introduced with increasingly intensified mechanical shear. The experiments also looked at the impact of feed solids content and several cost-effective processing aids along with these screw designs. Various characterization methods were used to relate the physical state of the biomass based on its specific surface area and volatile fraction, to the rate of biomethane generation possible from a 14- and 31-day biomethane potential test. An increase in biomethane production over this period by up to 190% was possible with the optimal screw design compared to a benchmark sample. This is a promising finding for the industrialization of biomethane production from forestry lignocellulosic biomass.

Effects of nitrogen regulation on heavy metal phytoextraction efficiency (Leucaena leucocephala): Application of a nitrogen fertilizer and a fungal agent.

Lead (Pb) and cadmium (Cd) have been identified as the primary contaminants in soil, posing potential health threats. This study aimed to examine the effects of applying a nitrogen fertilizer and a fungal agent Trichoderma harzianum J2 (nitrogen alone, fungi alone, and combined use) on the phytoremediation of soils co-contaminated with Pb and Cd. The growth of Leucaena leucocephala was monitored in the seedling, differentiation, and maturity stages to fully comprehend the remediation mechanisms. In the maturity stage, the biomass of L. leucocephala significantly increased by 18% and 29% under nitrogen-alone (NCK+) and fungal agent-alone treatments (J2), respectively, compared with the control in contaminated soil (CK+). The remediation factors of Pb and Cd with NCK+ treatment significantly increased by 50% and 125%, respectively, while those with J2 treatment increased by 73% and 145%, respectively. The partial least squares path model suggested that the nitrogen-related soil properties were prominent factors affecting phytoextraction compared with biotic factors (microbial diversity and plant growth). This model explained 2.56 of the variation in Cd concentration under J2 treatment, and 2.97 and 2.82 of the variation in Pb concentration under NCK+ and J2 treatments, respectively. The redundancy analysis showed that the samples under NCK+ and J2 treatments were clustered similarly in all growth stages. Also, Chytridiomycota, Mucoromucota, and Ciliophora were the key bioindicators for coping with heavy metals. Overall, a similar remediation mechanism allowed T. harzianum J2 to replace the nitrogen fertilizer to avoid secondary pollution. In addition, their combined use further increased the remediation efficiency.

Optimal timepoint sampling in high-throughput gene expression experiments.

MOTIVATION: Determining the best sampling rates (which maximize information yield and minimize cost) for time-series high-throughput gene expression experiments is a challenging optimization problem. Although existing approaches provide insight into the design of optimal sampling rates, our ability to utilize existing differential gene expression data to discover optimal timepoints is compelling. RESULTS: We present a new data-integrative model, Optimal Timepoint Selection (OTS), to address the sampling rate problem. Three experiments were run on two different datasets in order to test the performance of OTS, including iterative-online and a top-up sampling approaches. In all of the experiments, OTS outperformed the best existing timepoint selection approaches, suggesting that it can optimize the distribution of a limited number of timepoints, potentially leading to better biological insights about the resulting gene expression patterns. AVAILABILITY: OTS is available at www.msu.edu/∼jinchen/OTS.

A high throughput screening process and quick isolation of novel lignin-degrading microbes from large number of natural biomasses.

High throughput screening approaches can significantly speed up the identification of novel enzymes from natural microbial consortiums. A two-step high throughput screening process was proposed and explored to screen lignin-degrading microorganisms. By employing this modified culture enrichment method and screening based on enzyme activity, a total of 82 bacterial and 46 fungal strains were isolated from fifty decayed wood samples (100 liquid cultures) collected from the banks of the Ottawa River in Canada. Among them, ten bacterial and five fungal strains were selected and identified based on their high laccase activities by 16S rDNA and ITS gene sequencing, respectively. The study identified bacterial strains from various genera including Serratia, Enterobacter, Raoultella, and Bacillus, along with fungal counterparts including Mucor, Trametes, Conifera and Aspergillus. Moreover, Aspergillus sydowii (AORF21), Mucor sp. (AORF43), Trametes versicolor (AORF3) and Enterobacter sp. (AORB55) exhibited xylanase and ß- glucanase activities in addition to laccase production. The proposed approach allowed for the quick identification of promising consortia and enhanced the chance of isolating desired strains based on desired enzyme activities. This method is not limited to lignocellulose and lignin-degrading microorganisms but can be applied to identify novel microbial strains and enzymes from different natural samples.

Optimization and purification of bioproducts from Bacillus velezensis PhCL fermentation and their potential on industrial application and bioremediation.

Bioproduction is considered a promising alternative way of obtaining useful and green chemicals. However, the downstream process of biomolecules has been one of the major difficulties in upscaling the application of bioproducts due to the high purification cost. Acid precipitation is the most common method for purifying biosurfactants from the fermentation broth with high purity. However, the use of strong acids and organic solvents in solvent extraction has limited its application. Hence, in this study, a new strain of Bacillus velezensis PhCL was isolated from phenolic waste, and its production of amylase had been optimized via response surface methodology. After that, amylase and biosurfactant were purified by sequential ammonium sulfate precipitation and the result suggested that even though the purified crude biosurfactant had a lower purification fold compared to the acid precipitation, the yield was higher and both enzymes and biosurfactant also could be recovered for lowering the purification cost. Moreover, the purified amylase and crude biosurfactant were characterized and the results suggested that the purified crude biosurfactant would have a higher emulsion activity and petroleum hydrocarbon removal rate compared to traditional surfactants. This study provided another approach for purifying bioactive compounds including enzymes and biosurfactants from the same fermentation broth and further explored the potential of the crude purified biosurfactant in the bioremediation of polycyclic aromatic hydrocarbons and petroleum hydrocarbons.

Characterization of Potential Virulence, Resistance to Antibiotics and Heavy Metals, and Biofilm-Forming Capabilities of Soil Lignocellulolytic Bacteria.

Soil bacteria participate in self-immobilization processes for survival, persistence, and production of virulence factors in some niches or hosts through their capacities for autoaggregation, cell surface hydrophobicity, biofilm formation, and antibiotic and heavy metal resistance. This study investigated potential virulence, antibiotic and heavy metal resistance, solvent adhesion, and biofilm-forming capabilities of six cellulolytic bacteria isolated from soil samples: Paenarthrobacter sp. MKAL1, Hymenobacter sp. MKAL2, Mycobacterium sp. MKAL3, Stenotrophomonas sp. MKAL4, Chryseobacterium sp. MKAL5, and Bacillus sp. MKAL6. Strains were subjected to phenotypic methods, including heavy metal and antibiotic susceptibility and virulence factors (protease, lipase, capsule production, autoaggregation, hydrophobicity, and biofilm formation). The effect of ciprofloxacin was also investigated on bacterial susceptibility over time, cell membrane, and biofilm formation. Strains MKAL2, MKAL5, and MKAL6 exhibited protease and lipase activities, while only MKAL6 produced capsules. All strains were capable of aggregating, forming biofilm, and adhering to solvents. Strains tolerated high amounts of chromium, lead, zinc, nickel, and manganese and were resistant to lincomycin. Ciprofloxacin exhibited bactericidal activity against these strains. Although the phenotypic evaluation of virulence factors of bacteria can indicate their pathogenic nature, an in-depth genetic study of virulence, antibiotic and heavy metal resistance genes is required.

Overexpression of an exotic thermotolerant ß-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw.

BACKGROUND: Trichoderma reesei is a widely used industrial strain for cellulase production, but its low yield of ß-glucosidase has prevented its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, which inhibits further cellulases production. This problem can be solved by adding ß-glucosidase, which hydrolyzes cellobiose to glucose for fermentation. It is, therefore, of high vvalue to construct T. reesei strains which can produce sufficient ß-glucosidase and other hydrolytic enzymes, especially when those enzymes are capable of tolerating extreme conditions such as high temperature and acidic or alkali pH. RESULTS: We successfully engineered a thermostable ß-glucosidase gene from the fungus Periconia sp. into the genome of T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg) higher ß-glucosidase activity compared to the parent strain (2.2 IU/mg) after 24 h of incubation. The transformants also showed very high total cellulase activity (about 39.0 FPU/mg) at 24 h of incubation whereas the parent strain almost did not show any total cellulase activity at 24 h of incubation. The recombinant ß-glucosidase showed to be thermotolerant and remains fully active after two-hour incubation at temperatures as high as 60°C. Additionally, it showed to be active at a wide pH range and maintains about 88% of its maximal activity after four-hour incubation at 25°C in a pH range from 3.0 to 9.0. Enzymatic hydrolysis assay using untreated, NaOH, or Organosolv pretreated barley straw as well as microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain. CONCLUSIONS: The recombinant T. reesei overexpressing Periconia sp. ß-glucosidase in this study showed higher ß-glucosidase and total cellulase activities within a shorter incubation time (24 h) as well as higher hydrolysis activity using biomass residues. These features suggest that the transformants can be used for ß-glucosidase production as well as improving the biomass conversion using cellulases.