Attenuation of Las/Rhl quorum sensing regulated virulence and biofilm formation in Pseudomonas aeruginosa PAO1 by Artocarpesin.

The emergence of multidrug resistance and increased pathogenicity in microorganisms is conferred by the presence of highly synchronized cell density dependent signalling pathway known as quorum sensing (QS). The QS hierarchy is accountable for the secretion of virulence phenotypes, biofilm formation and drug resistance. Hence, targeting the QS phenomenon could be a promising strategy to counteract the bacterial virulence and drug resistance. In the present study, artocarpesin (ACN), a 6-prenylated flavone was investigated for its capability to quench the synthesis of QS regulated virulence factors. From the results, ACN showed significant inhibition of secreted virulence phenotypes such as pyocyanin (80%), rhamnolipid (79%), protease (69%), elastase (84%), alginate (88%) and biofilm formation (88%) in opportunistic pathogen, Pseudomonas aeruginosa PAO1. Further, microscopic observation of biofilm confirmed a significant reduction in biofilm matrix when P. aeruginosa PAO1 was supplemented with ACN at its sub-MIC concentration. Quantitative gene expression studies showed the promising aspects of ACN in down regulation of several QS regulatory genes associated with production of virulence phenotypes. Upon treatment with sub-MIC of ACN, the bacterial colonization in the gut of Caenorhabditis elegans was potentially reduced and the survival rate was greatly improved. The promising QS inhibition activities were further validated through in silico studies, which put an insight into the mechanism of QS inhibition. Thus, ACN could be considered as possible drug candidate targeting chronic microbial infections.

Sequential Co-Immobilization of Enzymes on Magnetic Nanoparticles for Efficient l-Xylulose Production.

Multi-enzymatic strategies have shown improvement in bioconversion during cofactor regeneration. In this study, purified l-arabinitol 4-dehydrogenase (LAD) and nicotinamide adenine dinucleotide oxidase (Nox) were immobilized via individual, mixed, and sequential co-immobilization approaches on magnetic nanoparticles, and were evaluated to enhance the conversion of l-arabinitol to l-xylulose. Initially, the immobilization of LAD or Nox on the nanoparticles resulted in a maximum immobilization yield and relative activity of 91.4% and 98.8%, respectively. The immobilized enzymes showed better pH and temperature profiles than the corresponding free enzymes. Furthermore, co-immobilization of these enzymes via mixed and sequential methods resulted in high loadings of 114 and 122 mg/g of support, respectively. Sequential co-immobilization of these enzymes proved more beneficial for higher conversion than mixed co-immobilization because of better retaining Nox residual activity. Sequentially co-immobilized enzymes showed a high relative conversion yield with broader pH, temperature, and storage stability profiles than the controls, along with high reusability. To the best of our knowledge, this is the first report on the mixed or sequential co-immobilization of LAD and Nox on magnetic nanoparticles for l-xylulose production. This finding suggests that selecting a sequential co-immobilization strategy is more beneficial than using individual or mixed co-immobilized enzymes on magnetic nanoparticles for enhancing conversion applications.

Functional analysis of a putative type III polyketide synthase from deep-sea sediment metagenome.

Type III polyketide synthases (type III PKSs) are single homodimeric enzymes that produce diverse products such as phloroglucinol, pyrones, resorcinols and chalcones which are biotechnologically important molecules. In an attempt to identify new type III PKS from extreme environments, the deep-sea sediment metagenome from Bay of Bengal was screened for type III PKS genes. BLASTX analyses of Nanopore sequence derived metagenome with the in-house created PKS database revealed a full length type III PKS from a 5 kb fragment. The annotated full length type III PKS, S9PKS showed 25-30 % sequence identity towards previously characterized enzymes. To functionally characterize the gene, it was synthesized, cloned into pET28a and pColdI vectors under T7 and csp promoters, respectively, and expressed in Escherichia coli Rosetta(DE3) pLysS. The optimized PKS (OptiPKS) was expressed as inclusion bodies under both promoters. The inclusion bodies were successfully solubilised using low concentration of urea, refolded and purified using Ni-NTA Agarose resin. The purified OptiPKS was tested for functionality using fatty acyl-CoA substrates at various temperatures. High performance liquid chromatography (HPLC) analyses revealed that OptiPKS produced tri and tetraketide pyrones using C4 to C10 acyl-CoA starter substrates. Further characterization and mutation of the enzyme would reveal its functional significance. Thus, the study could be a lead for the annotation and functional characterization of putative type III PKS from environmental metagenome data.

Manipulating Microbial Cell Morphology for the Sustainable Production of Biopolymers.

The total rate of plastic production is anticipated to surpass 1.1 billion tons per year by 2050. Plastic waste is non-biodegradable and accumulates in natural ecosystems. In 2020, the total amount of plastic waste was estimated to be 367 million metric tons, leading to unmanageable waste disposal and environmental pollution issues. Plastics are produced from petroleum and natural gases. Given the limited fossil fuel reserves and the need to circumvent pollution problems, the focus has shifted to biodegradable biopolymers, such as polyhydroxyalkanoates (PHAs), polylactic acid, and polycaprolactone. PHAs are gaining importance because diverse bacteria can produce them as intracellular inclusion bodies using biowastes as feed. A critical component in PHA production is the downstream processing procedures of recovery and purification. In this review, different bioengineering approaches targeted at modifying the cell morphology and synchronizing cell lysis with the biosynthetic cycle are presented for product separation and extraction. Complementing genetic engineering strategies with conventional downstream processes, these approaches are expected to produce PHA sustainably.

Denovo production of resveratrol by engineered Saccharomyces cerevisiae W303-1a using pretreated Gracilaria corticata extracts.

OBJECTIVE: Assembly and construction of resveratrol production pathway in Saccharomyces cerevisiae for denovo production of resveratrol using seaweed extract as fermentation medium. RESULTS: Genes involved in the production of resveratrol from tyrosine pathway, tyrosine ammonia lyase (FTAL) gene from Flavobacterium johnsoniae (FjTAL), the 4-coumarate:CoA ligase gene from Arabidopsis thaliana (4CL1) and the stilbene synthase gene from Vitis vinifera (VvSTS) were introduced into low copy, high copy and integrative vector and transformed into S. cerevisiae W303-1a. The resulting strains W303-1a/pARS-res5, W303-1a/2µ-res1 and W303-1a/IntUra-res9 produced a level of 2.39 ± 0.01, 3.33 ± 0.03 and 8.34 ± 0.03 mg resveratrol l-1 respectively. CRISPR mediated integration at the δ locus resulted in 17.13 ± 1.1 mg resveratrol l-1. Gracilaria corticata extract was tested as a substrate for the growth of transformant to produce resveratrol. The strain produced a comparable level, 13.6 ± 0.54 mg resveratrol l-1 when grown in seaweed extract medium. CONCLUSIONS: The strain W303-1a/IntδC-res1 utilized Gracillaria hydrolysate and produced 13.6 ± 0.54 mg resveratrol l-1 and further investigations are being carried out focusing on pathway engineering and optimization of process parameters to enhance resveratrol yield.

Encapsulation of Methanotrophs within a Polymeric Matrix Containing Copper- and Iron-Based Nanoparticles to Enhance Methanol Production from a Simulated Biogas.

The production of renewable energy or biochemicals is gaining more attention to minimize the emissions of greenhouse gases such as methane (CH4) and carbon dioxide for sustainable development. In the present study, the influence of copper (Cu)- and iron (Fe)-based nanoparticles (NPs), such as Cu, Fe3O4, and CuFe2O4, was evaluated during the growth of methanotrophs for inoculum preparation and on the development of a polymeric-matrix-based encapsulation system to enhance methanol production from simulated biogas (CH4 and CO2). The use of simulated biogas feed and the presence of NP-derived inoculums produce a remarkable enhancement in methanol production up to 149% and 167% for Methyloferula stellata and Methylocystis bryophila free-cells-based bioconversion, respectively, compared with the use of pure CH4 as a control feed during the growth stage. Furthermore, these methanotrophs encapsulated within a polymeric matrix and NPs-based systems exhibited high methanol production of up to 156%, with a maximum methanol accumulation of 12.8 mmol/L over free cells. Furthermore, after encapsulation, the methanotrophs improved the stability of residual methanol production and retained up to 62.5-fold higher production potential than free cells under repeated batch reusability of 10 cycles. In the presence of CH4 vectors, methanol production by M. bryophila improved up to 16.4 mmol/L and retained 20% higher recycling stability for methanol production in paraffin oil. These findings suggest that Cu and Fe NPs can be beneficially employed with a polymeric matrix to encapsulate methanotrophs and improve methanol production.

Coriolus versicolor laccase-based inorganic protein hybrid synthesis for application in biomass saccharification to enhance biological production of hydrogen and ethanol.

In this study, a bio-friendly inorganic protein hybrid-based enzyme immobilization system using partially purified Coriolus versicolor laccase (CvLac) was successfully applied to biomass hydrolysis for the enhancement of sugar production aimed at generating biofuels. After four days of incubation, the maximum CvLac production was achieved at 140 U/mg of total protein in the presence of inducers such as copper and wheat bran after four days of incubation. Crude CvLac immobilized through inorganic protein hybrids such as nanoflowers (NFs) using zinc as Zn3(PO4)2/CvLac hybrid NFs (Zn/CvLac-NFs) showed a maximum encapsulation yield of 93.4% and a relative activity of 265% compared to free laccase. The synthesized Zn/CvLac-NFs exhibited significantly improved activity profiles and stability compared to free enzymes. Furthermore, Zn/CvLac-NFs retained a significantly high residual activity of 96.2% after ten reuse cycles. The saccharification of poplar biomass improved ∼2-fold in the presence of Zn/CvLac-NFs, with an 8-fold reduction in total phenolics compared to the control. The Zn/CvLac-NFs treated biomass hydrolysate showed high biological hydrogen (H2) production and ethanol conversion efficiency of up to 2.68 mol/mol of hexose and 79.0% compared to the control values of 1.27 mol of H2/mol of hexose and 58.4%, respectively. The CvLac hybrid NFs are the first time reported for biomass hydrolysis, and a significant enhancement in the production of hydrogen and ethanol was reported. The synthesis of such NFs based on crude forms of diverse enzymes can potentially be extended to a broad range of biotechnological applications.

Bacterial biofilm inhibitors: An overview.

Bacteria that cause infectious diseases adopt biofilms as one of their most prevalent lifestyles. Biofilms enable bacteria to tolerate environmental stress and evade antibacterial agents. This bacterial defense mechanism has rendered the use of antibiotics ineffective for the treatment of infectious diseases. However, many highly drug-resistant microbes have rapidly emerged owing to such treatments. Different signaling mechanisms regulate bacterial biofilm formation, including cyclic dinucleotide (c-di-GMP), small non-coding RNAs, and quorum sensing (QS). A cell density-dependent phenomenon, QS is associated with c-di-GMP (a global messenger), which regulates gene expression related to adhesion, extracellular matrix production, the transition from the planktonic to biofilm stage, stability, pathogenicity, virulence, and acquisition of nutrients. The article aims to provide information on inhibiting biofilm formation and disintegrating mature/preformed biofilms. This treatment enables antimicrobials to target the free-living/exposed bacterial cells at lower concentrations than those needed to treat bacteria within the biofilm. Therefore, a complementary action of antibiofilm and antimicrobial agents can be a robust strategic approach to dealing with infectious diseases. Taken together, these molecules have broad implications for human health.

Exploiting Polyhydroxyalkanoates for Biomedical Applications.

Polyhydroxyalkanoates (PHA) are biodegradable plastic. Numerous bacteria produce PHAs under environmental stress conditions, such as excess carbon-rich organic matter and limitations of other nutritional elements such as potassium, magnesium, oxygen, phosphorus, and nitrogen. In addition to having physicochemical properties similar to fossil-fuel-based plastics, PHAs have unique features that make them ideal for medical devices, such as easy sterilization without damaging the material itself and easy dissolution following use. PHAs can replace traditional plastic materials used in the biomedical sector. PHAs can be used in a variety of biomedical applications, including medical devices, implants, drug delivery devices, wound dressings, artificial ligaments and tendons, and bone grafts. Unlike plastics, PHAs are not manufactured from petroleum products or fossil fuels and are, therefore, environment-friendly. In this review, a recent overview of applications of PHAs with special emphasis on biomedical sectors, including drug delivery, wound healing, tissue engineering, and biocontrols, are discussed.

Bacterial cell immobilized packed bed reactor for the elimination of dissolved organics from biologically treated post-tanning wastewater and its microbial community profile.

In conventional, the biologically treated tannery wastewaters are rich in dissolved organics and the application of reverse osmosis (RO) to biologically treated tannery wastewater was challenged with fouling and failure of RO membrane due to existence of lingering dissolved organic compounds. In present investigation the bacterial cell immobilized packed bed reactor (CIPBR) was operated to remove the dissolved organic compounds in biologically treated post-tanning wastewater to avoid membrane fouling in RO. The efficient microbial syndicate to eliminate dissolved organics in post-tanning wastewater was isolated and immobilized on to the carbon silica matrix (CSM) in the range of 2.98 ± 0.2 × 107 cells gm-1 of CSM and the same was used as a carrier matrix in the packed bed reactor. The CIPBR established the CODtot, CODdis and BOD removal efficiency by 61 ± 4%, 57 ± 4% and 87 ± 3% respectively with CODtot, CODdis and BOD remained in the treated wastewater as 236 ± 21 mg/L, 228 ± 21 mg/L, and 12 ± 3 mg/L under continuous operation. The removal of dissolved organic compounds from the post-tanning wastewater was confirmed using UV-Visible and FT-IR spectroscopic studies. Among the total microbial community, the phylum Proteobacteria played most abundant role with 48.47% of relative abundance for the removal of dissolved organics in biologically treated post-tanning wastewater. The significance of the study is to replace the tertiary treatment unit operation in the conventional ETP/CETP to remove dissolved organics in wastewater.

Laccase Immobilization on Copper-Magnetic Nanoparticles for Efficient Bisphenol Degradation.

Laccase activity is influenced by copper (Cu) as an inducer. In this study, laccase was immobilized on Cu and Cu-magnetic (Cu/Fe2O4) nanoparticles (NPs) to improve enzyme stability and potential applications. The Cu/Fe2O4 NPs functionally activated by 3-aminopropyltriethoxysilane and glutaraldehyde exhibited an immobilization yield and relative activity (RA) of 93.1 and 140%, respectively. Under optimized conditions, Cu/Fe2O4 NPs showed high loading of laccase up to 285 mg/g of support and maximum RA of 140% at a pH 5.0 after 24 h of incubation (4°C). Immobilized laccase, as Cu/Fe2O4-laccase, had a higher optimum pH (4.0) and temperature (45°C) than those of a free enzyme. The pH and temperature profiles were significantly improved through immobilization. Cu/Fe2O4-laccase exhibited 25-fold higher thermal stability at 65°C and retained residual activity of 91.8% after 10 cycles of reuse. The degradation of bisphenols was 3.9-fold higher with Cu/Fe2O4-laccase than that with the free enzyme. To the best of our knowledge, Rhus vernicifera laccase immobilization on Cu or Cu/Fe2O4 NPs has not yet been reported. This investigation revealed that laccase immobilization on Cu/Fe2O4 NPs is desirable for efficient enzyme loading and high relative activity, with remarkable bisphenol A degradation potential.

Integration of biogas derived from dark fermentation and anaerobic digestion of biowaste to enhance methanol production by methanotrophs.

Biowaste-derived sugars or greenhouse gases, such as methane (CH4) and carbon dioxide (CO2), can be used to generate eco-friendly biofuels, such as hydrogen (H2) or methanol. In the present study, enzyme-based rice straw (RS) hydrolysate was used to produce dark-fermentative (DF) biogas (H2 and CO2), which was subsequently integrated with biogas (CH4 and CO2) derived from anaerobic digestion (AD) to generate methanol via methanotrophs. First, DF of RS hydrolysate yielded 2.82 mol of H2/mol of hexose. Second, the integration of biogas derived from DF and AD in the presence of CH4 vectors yielded 13.8 mmol/L of methanol via methanotrophs. Moreover, under the repeated batch mode, 64.6 mmol/L of methanol was produced. This is the first report on the integration of biogas derived from AD and DF of biowaste to produce biomethanol. These findings may facilitate the development of a sustainable biowaste-based circular economy for producing biofuels.

Prospecting Microbial Genomes for Biomolecules and Their Applications.

Bioactive molecules of microbial origin are finding increasing biotechnological applications. Their sources range from the terrestrial, marine, and endophytic to the human microbiome. These biomolecules have unique chemical structures and related groups, which enable them to improve the efficiency of the bioprocesses. This review focuses on the applications of biomolecules in bioremediation, agriculture, food, pharmaceutical industries, and human health.

Plastic Eating Enzymes: A Step Towards Sustainability.

The large-scale usage of petro-chemical-based plastics has proved to be a significant source of environmental pollution due to their non-biodegradable nature. Microbes-based enzymes such as esterases, cutinases, and lipases have shown the ability to degrade synthetic plastic. However, the degradation of plastics by enzymes is primarily limited by the unavailability of a robust enzymatic system, i.e., low activity and stability towards plastic degradation. Recently, the machine learning strategy involved structure-based and deep neural networks show desirable potential to generate functional, active stable, and tolerant polyethylene terephthalate (PET) degrading enzyme (FAST-PETase). FAST-PETase showed the highest PET hydrolytic activity among known enzymes or their variants and degraded broad ranges of plastics. The development of a closed-loop circular economy-based system of plastic degradation to monomers by FAST-PETase followed by the re-polymerization of monomers into clean plastics can be a more sustainable approach. As an alternative to synthetic plastics, diverse microbes can produce polyhydroxyalkanoates, and their degradation by microbes has been well-established. This article discusses recent updates in the enzymatic degradation of plastics for sustainable development.

Deep-sea sediment metagenome from Bay of Bengal reveals distinct microbial diversity and functional significance.

Bay of Bengal (BoB) has immense significance with respect to ecological diversity and natural resources. Studies on microbial profiling and their functional significance at sediment level of BoB remain poorly represented. Herein, we describe the microbial diversity and metabolic potentials of BOB deep-sea sediment samples by subjecting the metagenomes to Nanopore sequencing. Taxonomic diversity ascertained at various levels revealed that bacteria belonging to phylum Proteobacteria predominantly represented in sediment samples NIOT_S7 and NIOT_S9. A comparative study with 16S datasets from similar ecological sites revealed depth as a crucial factor in determining taxonomic diversity. KEGG annotation indicated that bacterial communities possess sequence reads corresponding to carbon dioxide fixation, sulfur, nitrogen metabolism, but at varying levels. Additionally, gene sequences related to bioremediation of dyes, plastics, hydrocarbon, antibiotic resistance, secondary metabolite synthesis and metal resistance from both the samples as studied indicate BoB to represent a highly diverse environmental niche for further exploration.

Synthetic design of methanotroph co-cultures and their immobilization within polymers containing magnetic nanoparticles to enhance methanol production from wheat straw-based biogas.

In this study, various methanotroph co-cultures were designed to enhance methanol production from biogas produced through the anaerobic digestion of wheat straw (WS). Furthermore, whole-cell immobilization was performed using magnetic nanoparticle (MNP)-loaded polymers to develop an efficient bioprocess. The anaerobic digestion of WS by cattle dung yielded 219 L/kg of total solids reduced. Methanol produced was 5.08 and 6.39 mmol/L by pure- and co-cultures from biogas, respectively. The optimization of process parameters enhanced methanol production to 6.82 mmol/L by co-culturing Mithylosinus sporium and Methylocella tundrae. The immobilized co-culture within the MNP-doped polymers exhibited much higher cumulative methanol of up to 70.74 mmol/L than the production of 22.34 mmol/L by free cells after ten cycles of reuse. This study suggests that MNP-doped polymer-based immobilization of methanotrophs is a unique approach for producing renewable fuels from biomass-derived biogas, a greenhouse gas.

Characterization of a xylitol dehydrogenase from Aspergillus flavus and its application in l-xylulose production.

An NAD+-dependent xylitol dehydrogenase from A. flavus (AfXDH) was cloned and successfully expressed in Escherichia coli. AfXDH gene sequence revealed an open reading frame of 1,110 bp, encoding a polypeptide of 369 amino acids with a calculated molecular mass of 38,893 Da. Among various polyols, sorbitol and xylitol were preferred substrates of AfXDH with Km values of 16.2 and 16.9 mM, respectively. AfXDH showed the highest activity in Tris-glycine-NaOH buffer (pH 9.5) at 50°C; it required Zn2+ or Mn2+ for enzyme activity. The half-life at 40°C and half denaturation temperature (T1/2) was 200 min and 45°C, respectively. Bioinformatic analyses along with biochemical properties confirmed that AfXDH belonged to the medium-chain dehydrogenase/reductase family. AfXDH exhibits higher thermostability and k cat values than those of other XDHs. The feasibility of using AfXDH in l-xylulose production was demonstrated. AfXDH, when coupled with Streptococcus pyogenes NADH oxidase, efficiently converted xylitol to l-xylulose with 97% yield, suggesting its usefulness for the industrial l-xylulose production from xylitol.

Mild Alkaline Pretreatment of Rice Straw as a Feedstock in Microbial Fuel Cells for Generation of Bioelectricity.

The dependency on non-renewable fossil fuels as an energy source has drastically increased global temperatures. Their continuous use poses a great threat to the existing energy reserves. Therefore, the energy sector has taken a turn toward developing eco-friendly, sustainable energy generation by using sustainable lignocellulosic wastes, such as rice straw (RS). For lignocellulosic waste to be utilized as an efficient energy source, it needs to be broken down into less complex forms by pretreatment processes, such as alkaline pretreatment using NaOH. Varied NaOH concentrations (0.5%,1.0%,1.5%,2%) for alkaline pretreatment of RS were used for the holocellulose generation. Amongst the four NaOH concentrations tested, RS-1.5% exhibited higher holocellulose generation of 80.1%, whereas 0.5%, 1 5 and 2% pointed 71.9%, 73.8%, and 78.5% holocellulose generation, respectively. Further, microbial fuel cells (MFCs) were tested for voltage generation by utilizing holocellulose generated from untreated (RS-0%) and mildly alkaline pretreated RS (RS-1.5%) as a feedstock. The MFC voltage and maximum power generation using RS-0% were 194 mV and 167 mW/m2, respectively. With RS-1.5%, the voltage and maximum power generation were 556 mV and 583 mW/m2, respectively. The power density of RS-1.5% was three-fold higher than that of RS-0%. The increase in MFC power generation suggests that alkaline pretreatment plays a crucial role in enhancing the overall performance.

Nano-Structured Carbon: Its Synthesis from Renewable Agricultural Sources and Important Applications.

Carbon materials are versatile in nature due to their unique and modifiable surface and ease of production. Nanostructured carbon materials are gaining importance due to their high surface area for application in the energy, biotechnology, biomedical, and environmental fields. According to their structures, carbon allotropes are classified as carbon nanodots, carbon nanoparticles, graphene, oxide, carbon nanotubes, and fullerenes. They are synthesized via several methods, including pyrolysis, microwave method, hydrothermal synthesis, and chemical vapor deposition, and the use of renewable and cheaper agricultural feedstocks and reactants is increasing for reducing cost and simplifying production. This review explores the nanostructured carbon detailed investigation of sources and their relevant reports. Many of the renewable sources are covered as focused here, such as sugar cane waste, pineapple, its solid biomass, rise husk, date palm, nicotine tabacum stems, lapsi seed stone, rubber-seed shell, coconut shell, and orange peels. The main focus of this work is on the various methods used to synthesize these carbon materials from agricultural waste materials, and their important applications for energy storage devices, optoelectronics, biosensors, and polymer coatings.