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.

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.

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.

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.

Optimization of cultural conditions for pectinase production by Streptomyces sp. and characterization of partially purified enzymes.

The cultural parameters of Streptomyces sp. for pectinase production were optimized using the Box-Behnken design. The maximum pectinase production was obtained after 58 hours at 35℃ and pH 7 upon submerged fermentation in yeast extract-containing media. The enzymes were partially purified with acetone precipitation and the analysis by SDS-PAGE and zymogram revealed that Streptomyces sp. produced two pectinases with molecular weights of about 25 and 75 kDa. The pectinase activity was detected in a wide range of temperatures (30℃ to 80℃) and pH (3 to 9) with maximum pectinase activities observed at 70℃ and pHs 5 and 9. The enzymes retained about 30 to 40% of their activities even after incubating the enzyme at different temperatures for 120 mins. The pectinase activities of Streptomyces sp. were enhanced in the media containing 1.5% pectin, 1% casein as a nitrogen source, 0.5 mM MgSO4, and 5 mM NaCl. Further, the addition of Tween-20, amino acids, and vitamins to the media also enhanced the pectinase activity. Moreover, the bacterium illustrated the ability to decolorize crystal violet dye efficiently. The decolorization rate ranged from 39.29 to 53.75% showing the highest bacterial decolorization in the media containing 2mg/mL crystal violet at 144 hours. Therefore, the bacterium has the potential in treating wastewater produced by industries like textile industries.

Optimization of multiple enzymes production by fermentation using lipid-producing Bacillus sp.

The present study identified the pectinase-producing bacterium isolated from the contaminated broth as Bacillus sp. on 16S rDNA sequence analysis. The bacterium illustrated water-like droplets on the colony grown on the Sabouraud dextrose agar plate. It also exhibited multi-enzymes activities, such as pectinase, polygalacturonase, xylanase, and cellulase by using various agro-wastes as low-cost substrates. The orange peel was observed to be the best substrate among the agro-wastes used for maximum multi-enzymes (pectinase, polygalacturonase, xylanase, and cellulase). However, the bacterium demonstrated its capability to produce different enzymes according to the different substrates/agro-wastes used. The Plackett-Burman design was used to determine the essential influencing factors, while the Box Behnken design response surface methodology was for optimizing cultural conditions. At their optimal conditions (40°C incubation temperature, 24 h of incubation period, 1% w/v orange peel, and 2% v/v inoculum volume), the bacterium exhibited the maximum pectinase (9.49 ± 1.25 U/ml) and xylanase (16.27 ± 0.52 U/ml) activities. Furthermore, the study explored the ability of the bacterium to produce bacterial lipids and observed about 25% bacterial lipid content on a dry weight basis. Therefore, the bacterium is a good candidate for producing important multi-enzymes and subsequent agro-waste degradation controlling the environment, and facilitating waste management. Also, the bacterium can be a potential feedstock in producing renewable biofuel.

Characterization of Cellulose-Degrading Bacteria Isolated from Soil and the Optimization of Their Culture Conditions for Cellulase Production.

The characterization of bacteria with hydrolytic potential significantly contributes to the industries. Six cellulose-degrading bacteria were isolated from mixture soil samples collected at Kingfisher Lake and the University of Manitoba campus by Congo red method using carboxymethyl cellulose agar medium and identified as Paenarthrobacter sp. MKAL1, Hymenobacter sp. MKAL2, Mycobacterium sp. MKAL3, Stenotrophomonas sp. MKAL4, Chryseobacterium sp. MKAL5, and Bacillus sp. MKAL6. Their cellulase production was optimized by controlling different environmental and nutritional factors such as pH, temperature, incubation period, substrate concentration, nitrogen, and carbon sources using the dinitrosalicylic acid and response surface methods. Except for Paenarthrobacter sp. MKAL1, all strains are motile. Only Bacillus sp. MKAL6 was non-salt-tolerant and showed gelatinase activity. Sucrose enhanced higher cellulase activity of 78.87 ± 4.71 to 190.30 ± 6.42 U/mL in these strains at their optimum pH (5-6) and temperature (35-40 °C). The molecular weights of these cellulases were about 25 kDa. These bacterial strains could be promising biocatalysts for converting cellulose into glucose for industrial purposes.

Knockout of a highly GC-rich gene in Burkholderia pyrrocinia by recombineering with freeze-thawing transformation.

Genetic transformation is a valuable and essential method that provides powerful insights into the gene function of microorganisms and contributes to the construction of engineered bacteria. Here, we developed a novel genetic transformation system to easily knock out a highly GC-rich gene (74.71% GC) from Burkholderia pyrrocinia JK-SH007, a biocontrol strain of poplar canker disease. This system revealed a reliable selectable marker (trimethoprim resistance gene, Tmp) and a simplified, efficient transformation method (6,363.64 CFU/µg, pHKT2) that was developed via freeze-thawing. The knockout recombineering of B. pyrrocinia JK-SH007 was achieved through a suicide plasmid with a three-fragment mutagenesis construct. The three-fragment cassette for mutagenesis was generated by overlap extension and touchdown PCRs and composed of Tmp flanked by GC-rich upstream and downstream fragments from B. pyrrocinia JK-SH007. The mutant strain (ΔBpEG), which was verified by PCR, lost 93.3% of its ability to degrade carboxymethyl cellulose over 40 days. Overall, this system may contribute to future research on B. pyrrocinia traits.

Effect of KNO3 on Lipid Synthesis and CaCO3 Accumulation in Pleurochrysis dentata Coccoliths with a Special Focus on Morphological Characters of Coccolithophores.

Pleurochrysis genus algae are widely distributed in ocean waters. Pleurochrysis sp. algae are popularly known for its coccolithophores. Calcium carbonate (CaCO3) shells are major components of the coccolithophore, and they are key absorbers of carbondioxide. In this study, we have reported the effects of potassium nitrate (KNO3) concentration on calcium accumulation and total lipid, carbohydrate and protein contents of Pleurochrysis dentata. Results obtained from complexometric titration and scanning electron microscopy analysis showed higher rates of CaCO3 accumulation on Pleurochrysis dentata cell surface. We have also observed that overall cell size of Pleurochrysis dentata reached maximum when it was cultured at 0.75 mmol L-1 of KNO3. During 10 days of Pleurochrysis dentata culture total lipids and carbohydrate contents decreased, with slightly increased protein content. Results obtained from Fourier-Transform Infrared Spectroscopy (FTIR) also reported an increase in protein and decrease in lipids and carbohydrate contents, respectively. Similarly, Pleurochrysis dentata cultured at 1 mmol L-1 concentration of KNO3 exhibited the lowest carbohydrate (21.08%) and highest protein (32.87%) contents. Interestingly, Pleurochrysis dentata cultured without KNO3 exhibited 33.61% of total lipid content which reduced to a total lipid content of 13.67% when cultured at 1 mmol L-1 concentration of KNO3. Thus, culture medium containing higher than 1 mmol L-1 of KNO3 could inhibit the cell size of Pleurochrysis dentata and CaCO3 accumulation in shells but it could promote its cell growth. For the first time we have reported a relatively complete coccolith structure devoid of its protoplast. In this study, we have also described about the special planar structure of Pleurochrysis dentata CaCO3 shells present on its inner tube of the R unit and parallel to the outer tube of the V unit which we named it as "doornail structure". We believe that this doornail structure provides structural stability and support to the developing coccoliths in Pleurochrysis dentata. Also, we have discussed about the "double-disc" structure of coccoliths which are closely arranged and interlocked with each other. The double-disc structure ensures fixation of each coccolith and objecting its free horizontal movement and helps in attaining a complementary coccolith structure.

Efficient saccharification of agave biomass using Aspergillus niger produced low-cost enzyme cocktail with hyperactive pectinase activity.

To develop a cost-effective, time-saving and efficient saccharification system for converting biomass into mono-/oligo-saccharides for production of bioethanol or other biochemicals, a relatively low recalcitrant and widely available biomass Agave americana was selected as feedstock. During the investigation of efficient enzyme cocktail, pectinase, which usually is neglect for biomass saccharification, was confirmed that it dramatically improves the saccharification of agave biomass. A production-friendly fungal strain of Aspergillus niger Gyx086 was employed for low-cost enzyme cocktails production using wheat straw as substance. The enzyme cocktail which was with hyperactive pectinase activity of 6.29 ±â€¯0.42 U/ml could efficiently saccharify un-pretreated agave biomasses. As a result, under a mild condition at 35 °C in less than 72 h, most of the polysaccharides were completely converted into reducing sugar. The low-cost, process-simplified, and efficient biotechnology should stimulate the development of agave as feedstock for green energy and bio-based products production.

Delignification overmatches hemicellulose removal for improving hydrolysis of wheat straw using the enzyme cocktail from Aspergillus niger.

Based on a general understanding that hemicellulose removal is more efficient than delignification for biomass deconstruction, an Aspergillus niger strain producing high xylanase activity was screened out from seventeen strains by clear halo experiments. Low-cost enzyme cocktail with high xylanase activity was produced from wheat straw medium fermented by the Gyx086 strain. The enzyme cocktail with high xylanase activity could more effectively hydrolyze wheat straw than other biomasses. However, only 30% of total carbohydrates could be hydrolyzed to reducing sugar in untreated wheat straw. Further enzymatic hydrolysis and pretreated trials were carried out, the results indicated that hemicellulose removal was less effective than delignification for de-recalcitrance of wheat straw and the crystallinity is little interference with the hydrolysis process. Delignified wheat straw was near-completely hydrolyzed by the enzyme cocktail in 60 h. This study advanced the knowledge in promoting wheat straw as feedstock for bio-based industry.