A co-fermentation strategy with wood hydrolysate and crude glycerol to enhance the lipid accumulation in Rhodosporidium toruloides-1588.

Wood hydrolysate has been regarded as sustainable and renewable substrate to produce microbial lipids, a potential feedstock for the biodiesel industry. Moreover, the major by-product of biofuel industries is crude glycerol but its implementation as a carbon source is still constrained due to the presence of impurities resulting in low biomass production and low lipid titer. Thus, this study investigates the effect of different carbon ratios of hydrolysate and crude glycerol on R. toruloides-1588. Hydrolysate to crude glycerol ratio of 60:40 resulted in maximum lipid accumulation of 49% (w/w), more than 90% of sugars and glycerol consumption. Further, scale-up to bench-scale fermenter resulted in 12% higher lipid accumulation (56.3% w/w, 0.15 g/L∙h) in 50% less time than flask fermentation. Hence, the ability of R. toruloides-1588 to flourish on different carbohydrates and accumulate high lipid content will be beneficial for the further development of biorefinery industries.

Furfural degradation and its effect on Rhodosporidium toruloides-1588 during microbial growth and lipid accumulation.

The presence of furfural in the hydrolysates obtained from lignocellulosic biomass sources represents an enormous challenge during their fermentation because furfural is a toxic compound for different microorganisms. Rhodosporidium toruloides-1588 can grow and accumulate lipids using wood hydrolysate as a substrate containing up to 1 g/L of furfural. In this study, the capacity of R. toruloides-1588 to grow and accumulate lipids using furfural without glucose in the media has been observed. R. toruloides-1588 degraded up to 3 g/L of furfural into furfuryl alcohol (1.8 g/L) and 2-furoic acid (0.9 g/L). Furthermore, R. toruloides-1588 accumulated 52% and 30% of its dry weight into lipids using YM media and YM media without glucose, respectively. Fatty acids such as palmitic, stearic and oleic were the most abundant. Finally, R. toruloides-1588 could potentially utilize furfural as a carbon source.

Effect of creating a fed-batch like condition using carbon to nitrogen ratios on lipid accumulation in Rhodosporidium toruloides-1588.

Utilizing the undetoxified wood hydrolysate to accumulate maximum lipids in Rhodosporidium toruloides under optimum conditions has been regarded as a renewable and cost-effective strategy. The current investigation aims to identify the best carbon to nitrogen (C/N 20, 70, and 120) ratio for maximum lipid accumulation in R. toruloides-1588 using wood hydrolysate. Additionally, a fed-batch-like condition was employed, where C/N ratios were maintained during the fermentation that inherently decreases in batch fermentation. The C/N ratio 70 has been identified as the best condition with 3 times higher lipid accumulation (43% w/w) than the control. Additionally, >95% and 70% of glucose and xylose consumption were observed, respectively. Moreover, 50% increase in polyunsaturated fatty acids compared to the control media reinforced the potential of R. toruloides-1588 to thrive on undetoxified hydrolysate, high lipid productivity (3.8 mg/g of dry weight per hour) and produce high value monosaturated and polyunsaturated fatty acids.

Pulsed-ozonolysis assisted oxidative treatment of forestry biomass for lignin fractionation.

Lignocellulosic biomass has been used to produce biomolecules of industrial interest through thermochemical, biological, and chemical transformation. However, few works have been developed over lignin fractionation to obtain monolignols with commercial potentials, such as sinapyl, coniferyl, and p-coumaryl alcohols. This study is focused on developing a thermochemical method to delignify biomass. Additionally, an oxidative treatment with ozone was studied to increase the release of monolignol compounds. The results showed that with 30 sec of ozonation in liquid samples from softwood sawdust a total concentration of 368.50 ± 0.73 mg/kg of monolignols was released after microwave-assisted extraction (256.5 ± 0.51 mg/kg of sinapyl alcohol and 112 ± 0.22 mg/kg of coniferyl alcohol) and 629.20 ± 0.21 mg/kg was released after thermal treatment (453.70 ± 0.15 mg/kg of sinapyl alcohol and 175.5 ± 0.06 mg/kg of coniferyl alcohol). For p-coumaryl alcohol, 16.32 mg/kg was obtained only in hardwood samples. The results of the present study showed that ozonolysis improves monolignols release from forestry residues.

Physical and biological removal of Microcystin-LR and other water contaminants in a biofilter using Manganese Dioxide coated sand and Graphene sand composites.

Sand as a filter media is often challenged by the presence of organics in the form of natural organic matter, metal ions, and various micropollutants in the source water. It is mainly due to the presence of limited active adsorption sites and low surface area that governs an ineffective adsorption potential of the sand material. Herein, graphitized sand was synthesized to tackle the above limitations using two sugar solution sources: a) brewery effluent (as a low-cost solution) (GS1) and; b) sucrose solution (GS2). GS1 showed 68%, 60%, and 99% higher maximum adsorption constant (qmax) for divalent metal ions: iron, copper, and manganese, respectively as compared to raw sand (RS). Coating of MnO2 over the graphitized sand (GSMs: GS1M and GS2M) further helped in Microcystin-LR (MC-LR) removal (3%-9%) when inoculated with MC-LR-degraders, but was not as effective in removing metals, organic carbon and nitrogen when compared to just graphitized sand (GS1 or GS2). Inoculating GS and GSMs (for both sugar sources) not only helped in higher MC-LR removal (10%-15% more) but also enhanced the removal of other water contaminants including metals, organic nitrogen, and carbon. GS1 showed 20% and 50% more MC-LR removal than the sand material when tested at a low and high initial concentration of MC-LR (5 µg/L and 50 µg/L). The highest breakthrough period was obtained for GS1 filter using 1 mg/L Rhodamine-B dye, which was 12 times (48 min) more than the raw sand filter and almost 2.5 times (second best, 21 min) than GS1M. After three cycles of regeneration and reuse of GS1 filter, a decrease of just 14% in saturation adsorption capacity indicated its high reusability aspects.

Data set of green extraction of valuable chemicals from lignocellulosic biomass using microwave method.

Lignocellulosic biomass is a promising alternative for the replacement of limited fossil resources to produce various chemical compounds, such as 5-hydroxymethylfurfural, furfural, vanillin, vanillic acid, ferulic acid, syringaldehyde, and 4-aminobenzoic acid. However, the complex biomass structure is a limitation to making effective use of this naturally found feedstock. This research presents a data set of different compounds obtained directly from forest residues, with special emphasis on achieving effective utilization of the biomass. The extraction method and the catalyst are considered as the two main factors in this valorization process.

Microwave-assisted extraction of chitosan from Rhizopus oryzae NRRL 1526 biomass.

Microwave-assisted extraction (MAE) of chitosan from dried fungal biomass of Rhizopus oryzae NRRL1526, obtained by culturing on potato dextrose broth (PDB), was performed and the optimal conditions required were identified using statistical analysis for the first time in this study. This microwave-assisted extraction (MAE) was compared against the conventional autoclave assisted method of chitosan extraction. The full factorial experimental design was used to investigate the impact of operating parameters of MAE, microwave power (100 W-500 W), and duration (10 min-30 min), on alkaline insoluble material (AIM) yield, chitosan yield, and degree of deacetylation (DDA). The effect of operating conditions was then evaluated using full factorial data analysis and optimum condition for MAE of chitosan was identified using response surface methodology to be 300 W and 22 min. This optimum condition identified was then further evaluated and the chitosan obtained characterized. Higher chitosan yield of 13.43 ± 0.3% (w/w) of fungal biomass was obtained when compared to that obtained, 6.67% ± 0.3% (w/w) of dry biomass, for the conventional extraction process. MAE yielded chitosan of higher degree of deacetylation, 94.6 ± 0.9% against 90.6 ± 0.5% (conventional heating), but the molecular weight was observed to be similar to that obtained by using conventional autoclave heating. MAE of chitosan was observed to yield a higher quantity of chitosan when compared to conventional extraction process and obtained chitosan exhibited a higher degree of deacetylation as well as molecular weight. The lower energy consumption of 0.11 kW h for MAE (5 kW h for conventional process) and the concomitant reduction in the energy bill to 1.1 cents from 50 cents, in addition to the above results, show that microwave irradiation is a more efficient and environment-friendly means to obtain chitosan from fungal biomass.

Bioproduction of fumaric acid: an insight into microbial strain improvement strategies.

Fumaric acid (FA), a metabolic intermediate, has been identified as an important carbohydrate derived platform chemical. Currently, it is commercially sourced from petrochemicals by chemical conversion. The shift to biochemical synthesis has become essential for sustainable development and for the transition to a biobased economy from a petroleum-based economy. The main limitation is that the concentrations of FA achieved during bioproduction are lower than that from a chemical process. Moreover, the high cost associated with bioproduction necessitates a higher yield to improve the feasibility of the process. To this effect, genetic modification of microorganism can be considered as an important tool to improve FA yield. This review discusses various genetic modifications strategies that have been studied in order to improve FA production. These strategies include the development of recombinant strains of Rhizopus oryzae, Escherichia coli, Saccharomyces cerevisiae, and Torulopsis glabrata as well as their mutants. The transformed strains were able to accumulate fumaric acid at a higher concentration than the corresponding wild strains but the fumaric acid titers obtained were lower than that reported with native fumaric acid producing R. oryzae strains. Moreover, one plausible adoption of gene editing tools, such as Agrobacterium-mediated transformation (AMT), CRISPR CAS-9 and RNA interference (RNAi) mediated knockout and silencing, have been proposed in order to improve fumaric acid yield. Additionally, the introduction of the glyoxylate pathway in R. oryzae to improve fumaric acid yield as well as the biosynthesis of fumarate esters have been proposed to improve the economic feasibility of the bioprocess. The adoption of some of these genetic engineering strategies may be essential to enable the development of a feasible bioproduction process.

Agro-industrial residues as a unique support in a sand filter to enhance the bioactivity to remove microcystin-Leucine aRginine and organics.

In the past, the versatility of a biosand filter has been successfully checked to counter suspended solids, metals, dissolved organic carbon (DOC), coliforms and other water quality parameters (WQPs) from the drinking water sources. In this study, cyanotoxin in the form of microcystin-LR (MC-LR) along with above-mentioned WQPs including nitrate, nitrite, and ammonia are analyzed for their removal using agro-residue based biosand filters (ARSFs) for 49 days (7 cycles). Three different agro-residue materials (ARMs) viz. deinking sludge (DSF), hemp fiber (HFF) and paper-pulp dry sludge (PPF) were used as the support material (top 5 cm) along with sand (49 cm) as the primary filter media to enhance the overall bioactivity. This enhancement in bioactivity is hypothesized to remove more MC-LR, DOC, coliform along with efficient nitrification/denitrification. Native bacterial community isolated from the filtration unit of a drinking water treatment plant (Chryseobacterium sp. and Pseudomonas fragi = X) along with the MC-LR-degrader: Arthrobacter ramosus (which was screened as the best biofilm-former among two other MC-LR-degraders tested) were used to inoculate the filters (all three ARSFs). Overall, DSF performed the best among all the ARSFs when compared to the sand filter (SFI) inoculated with the same bacterial strains (A + X). An increase in the bioactivity for ARSFs, particularly DSF was evident from the DOC removal (44 ±â€¯11%, 15% more than SFI), coliform removal (92.7 ±â€¯12.8%, 24% more than SFI), MC-LR removal (87 ±â€¯14%, 13% more than SFI) and an effective nitrification/denitrification, reducing ammonia, nitrate and nitrite level to below guideline values. Toxic assessment using bioindicator (Rhizobium meliloti) revealed safe filter water only in case of DSF.

Potential of biological approaches for cyanotoxin removal from drinking water: A review.

Biological treatment of cyanotoxins has gained much importance in recent decades and holds a promise to work in coordination with various physicochemical treatments. In drinking water treatment plants (DWTPs), effective removal of cyanotoxins with reduced toxicity is a primary concern. Commonly used treatments, such as ozonation, chlorination or activated carbon, undergo significant changes in their operating conditions (mainly dosage) to counter the variation in different environmental parameters, such as pH, temperature, and high cyanotoxin concentration. Presence of metal ions, natural organic matter (NOM), and other chemicals demand higher dosage and hence affect the activation energy to efficiently break down the cyanotoxin molecule. Due to these higher dose requirements, the treatment leads to the formation of toxic metabolites at a concentration high enough to break the guideline values. Biological methods of cyanotoxin removal proceed via enzymatic pathway where the protein-encoding genes are often responsible for the compound breakdown into non-toxic metabolites. However, in contrast to the chemical treatment, the biological processes advance at a much slower kinetic rate, predominantly due to a longer onset period (high lag phase). In fact, more than 90% of the studies reported on the biological degradation of the cyanotoxins attribute the biodegradation to the bacterial suspension. This suspended growth limits the mass transfer kinetics due to the presence of metal ions, NOMs and, other oxidizable matter, which further prolongs the lag phase and makes biological process toxic-free, albeit less efficient. In this context, this review attempts to bring out the importance of the attached growth mechanism, in particular, the biofilm-based treatment approaches which can enhance the biodegradation rate.

Biodegradation of microcystin-LR using acclimatized bacteria isolated from different units of the drinking water treatment plant.

Bacterial community isolated from different units of a Drinking Water Treatment Plant (DWTP) including pre-ozonation unit (POU), the effluent-sludge mixture of the sedimentation unit (ESSU) and top-sand layer water sample from the filtration unit (TSFU) were acclimatized separately in the microcystin-leucine arginine (MC-LR)-rich environment to evaluate MC-LR biodegradation. Maximum biodegradation efficiency of 97.2 ±â€¯8.7% was achieved by the acclimatized-TSFU bacterial community followed by 72.1 ±â€¯6.4% and 86.2 ±â€¯7.3% by acclimatized-POU and acclimatized-ESSU bacterial community, respectively. Likewise, the non-acclimatized bacterial community showed similar biodegradation efficiency of 71.1 ±â€¯7.37%, 86.7 ±â€¯3.19% and 94.35 ±â€¯10.63% for TSFU, ESSU and POU, respectively, when compared to the acclimatized ones. However, the biodegradation rate increased 1.5-folds for acclimatized versus non-acclimatized conditions. The mass spectrometry studies on MC-LR degradation depicted hydrolytic linearization of cyclic MC-LR along with the formation of small peptide fragments including Adda molecule that is linked to the reduced toxicity (qualitative toxicity analysis). This was further confirmed quantitatively by using Rhizobium meliloti as a bioindicator. The acclimatized-TSFU bacterial community comprised of novel MC-LR degrading strains, Chryseobacterium sp. and Pseudomonas fragi as confirmed by 16S rRNA sequencing.

Production and characterization of novel hydrocarbon degrading enzymes from Alcanivorax borkumensis.

This study investigates the production of alkane hydroxylase, lipase and esterase by the marine hydrocarbon degrading bacteria Alcanivorax borkumensis. The focus of this study is the remediation of petroleum hydrocarbons, hexane, hexadecane and motor oil as model substrates. A. borkumensis showed an incremental growth on these substrates with a high cell count. Growth on motor oil showed highest alkane hydroxylase and lipase production of 2.62 U/ml and 71 U/ml, respectively, while growth on hexadecane showed the highest esterase production of 57.5 U/ml. The percentage of hexane, hexadecane, and motor oil degradation during A. borkumensis growth after 72 h, was around 80%, 81.5% and 75%, respectively. Zymogram showed two different bands with a molecular weight of approx. 52 and 40 kDa, respectively with lipase and esterase activity. Alkane hydroxylase reached optimum activity at pH 8.0 and 70 ±â€¯1 °C for hexane and hexadecane and 75 ±â€¯1 °C for motor oil. Lipase and esterase showed optimum activity at 35 ±â€¯1 °C and 40 ±â€¯1 °C, respectively and pH 7.0. The crude enzymes showed higher stability in a wide range of pH, but they were not thermostable at higher temperatures.

Tetracyclines metal complexation: Significance and fate of mutual existence in the environment.

Concern over tetracyclines (TCs) complexation with metals in the environment is growing as a new class of emerging contaminants. TCs exist as a different net charged species depending on their dissociation constants, pH and the surrounding environment. One of the key concerns about TCs is its strong tendency to interact with various metal ions and form metal complexes. Moreover, co-existence of TCs and metals in the environment and their interactions has shown increased antibiotic resistance. Despite extensive research on TCs complexation, investigations on their antibiotic efficiency and pharmacological profile in bacteria have been limited. In addition, the current knowledge on TCs metal complexation, their fate and risk assessment in the environment are inadequate to obtain a clear understanding of their consequences on living systems. This indicates that vital and comprehensive studies on TCs-metal complexation, especially towards growing antibiotic resistance trends are required. This review summarizes the role of TCs metal complexation on the development of antibiotic resistance. Furthermore, impact of metal complexation on degradation, toxicity and the fate of TCs in the environment are discussed and future recommendations have been made.

Genetic Engineering Strategies for Enhanced Biodiesel Production.

The focus on biodiesel research has shown a tremendous growth over the last few years. Several microbial and plant sources are being explored for the sustainable biodiesel production to replace the petroleum diesel. Conventional methods of biodiesel production have several limitations related to yield and quality, which led to development of new engineering strategies to improve the biodiesel production in plants, and microorganisms. Substantial progress in utilizing algae, yeast, and Escherichia coli for the renewable production of biodiesel feedstock via genetic engineering of fatty acid metabolic pathways has been reported in the past few years. However, in most of the cases, the successful commercialization of such engineering strategies for sustainable biodiesel production is yet to be seen. This paper systematically presents the drawbacks in the conventional methods for biodiesel production and an exhaustive review on the present status of research in genetic engineering strategies for production of biodiesel in plants, and microorganisms. Further, we summarize the technical challenges need to be tackled to make genetic engineering technology economically sustainable. Finally, the need and prospects of genetic engineering technology for the sustainable biodiesel production and the recommendations for the future research are discussed.

Structural stability and unfolding properties of cutinases from Thermobifida fusca.

A comparative analysis of the structural and functional aspects along with equilibrium unfolding of two homologous cutinases, Cut1 and Cut2, from Thermobifida fusca was carried out. The CD and fluorescence profile at different pH in the range of 6 to 9 showed no structural variations for both cutinases, indicating their stability to a wide range of pH. Tryptophan quenching studies suggested that all the four Trp residues in the protein are in inaccessible hydrophobic pockets. Further, near-UV CD analysis of tertiary structure revealed a dissimilar distribution of aromatic amino acid on the surface of these two enzymes. Denaturation profiles obtained in aqueous solutions of the guanidine hydrochloride revealed different tolerance levels for unfolding of the two cutinases, with Cut2 showing higher resistivity to unfolding in comparison to Cut1. Both cutinases retained all the structural parameters even in the presence of 8 M urea, indicating the protein to be highly resistant to urea-induced unfolding. Structural study by homology modeling revealed a high resemblance of secondary structure between the two cutinases; however, their tertiary structure, hydrophobicity, and surface electrostatic properties were very different, which contributed to the difference in the structural stability of these two cutinases.

Production optimization and characterization of recombinant cutinases from Thermobifida fusca sp. NRRL B-8184.

Two genes, cut1 and cut2, of Thermobifida fusca NRRL B-8184 with cutin-hydrolyzing activity were cloned and expressed in Escherichia coli BL21 (DE3) separately. Enhanced expression was achieved after screening of six different media, optimization of the culture conditions and medium components. Among the screened media, modified Terrific Broth was found to be the best for maximum production of recombinant cutinases in E. coli BL21 (DE3). Under optimal conditions, the production of recombinant Cut1 and Cut2 (cutinases) were found to be 318±0.73 and 316±0.90 U/ml, respectively. The production of recombinant cutinases was increased by 11-fold as compared with T. fusca NRRL B-8184 wild-type strain. Both the recombinant cutinases were purified to homogeneity. They were found to be thermostable, organic solvent, and surfactant tolerant. Both the cutinase were active in a broad range of temperature (40-80 °C) and pH (6.8-9) with optimum activity at pH 8.0 and 55 °C.