Nonsterile Process for Biohydrogen Production: Recent Updates, Challenges, and Opportunities.

Hydrogen (H2), a clean and versatile energy carrier, has recently gained significant attention as a potential solution for reducing carbon emissions and promoting sustainable energy systems. The yield and efficiency of the biological H2 production process primarily depend on sterilization conditions. Various strategies, such as heat inactivation and membrane-based sterilization, have been used to achieve desirable yields via microbial fermentation. Almost every failed biotransformation process is linked to nonsterile conditions at any reaction stage. Therefore, the production of renewable biofuels as alternatives to fossil fuels is more attractive. Pure sugars have been widely documented as a costly feedstock for H2 production under sterile conditions. Biotransformation under nonsterile conditions is more desirable for stable and sustainable operation. Low-cost feeds, such as biowaste, are considered suitable alternatives, but they require appropriate sterilization to overcome the limitations of inherited or contaminating microbes during H2 production. This article describes the status of microbial fermentative processes for H2 production under nonsterile conditions and discusses strategies to improve such processes for sustainable, cleaner production.

Trametes versicolor Laccase-Based Magnetic Inorganic-Protein Hybrid Nanobiocatalyst for Efficient Decolorization of Dyes in the Presence of Inhibitors.

In the present investigation, an ecofriendly magnetic inorganic-protein hybrid system-based enzyme immobilization was developed using partially purified laccase from Trametes versicolor (TvLac), Fe3O4 nanoparticles, and manganese (Mn), and was successfully applied for synthetic dye decolorization in the presence of enzyme inhibitors. After the partial purification of crude TvLac, the specific enzyme activity reached 212 U∙mg total protein-1. The synthesized Fe3O4/Mn3(PO4)2-laccase (Fe3O4/Mn-TvLac) and Mn3(PO4)2-laccase (Mn-TvLac) nanoflowers (NFs) exhibited encapsulation yields of 85.5% and 90.3%, respectively, with relative activities of 245% and 260%, respectively, compared with those of free TvLac. One-pot synthesized Fe3O4/Mn-TvLac exhibited significant improvements in catalytic properties and stability compared to those of the free enzyme. Fe3O4/Mn-TvLac retained a significantly higher residual activity of 96.8% over that of Mn-TvLac (47.1%) after 10 reuse cycles. The NFs showed potential for the efficient decolorization of synthetic dyes in the presence of enzyme inhibitors. For up to five reuse cycles, Fe3O4/Mn-TvLac retained a decolorization potential of 81.1% and 86.3% for Coomassie Brilliant Blue R-250 and xylene cyanol, respectively. The synthesized Fe3O4/Mn-TvLac showed a lower acute toxicity towards Vibrio fischeri than pure Fe3O4 nanoparticles did. This is the first report of the one-pot synthesis of biofriendly magnetic protein-inorganic hybrids using partially purified TvLac and Mn.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Immobilization of Laccase Through Inorganic-Protein Hybrids Using Various Metal Ions.

In this study, the inorganic-protein hybrid strategy was employed for immobilization of laccase from Rhus vernicifera (Rvlac) using various metals calcium, cobalt, copper, and zinc (Zn). The efficient synthesis of hybrids for Rvlac immobilization was noted at 4 °C for incubation of 24 h. Among these hybrids, the maximum encapsulation yields (EY) of 90.1% and relative activity (RA) of 225% to free enzyme were recorded for Zn and Rvlac based inorganic-protein hybrids as Zn3(PO4)2-Rvlac. The upper optimum pH, and temperature values were observed of 4.0, and 45 °C after immobilization as compared to 3.5, and 40 °C for the free enzyme, respectively. After encapsulation, Rvlac showed a significant improvement up to 11.4-fold in pH and 5.7-fold in temperature the activity profiles. Free enzyme completely lost its activity at 60 °C after 2 h of incubation, whereas Zn3(PO4)2-Rvlac retained its residual activity of 56.7% under similar conditions. After ten cycles of reusability, Zn3(PO4)2-Rvlac possessed high residual activity of 90.8%. This study showed that the variation in the metal ions for immobilization of Rvlac as inorganic-protein hybrids significantly altered EY and RA. Also, Zn3(PO4)2-Rvlac proved more efficient as compared to free laccase that can be beneficially employed for biotechnological applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01000-5.

Recent Strategies for the Immobilization of Therapeutic Enzymes.

Therapeutic enzymes play important roles in modern medicine due to their high affinity and specificity. However, it is very expensive to use them in clinical medicine because of their low stability and bioavailability. To improve the stability and effectiveness of therapeutic enzymes, immobilization techniques have been employed to enhance the applications of therapeutic enzymes in the past few years. Reported immobilization techniques include entrapment, adsorption, and covalent attachment. In addition, protein engineering is often used to improve enzyme properties; however, all methods present certain advantages and limitations. For carrier-bound immobilization, the delivery and release of the immobilized enzyme depend on the properties of the carrier and enzyme. In this review, we summarize the advantages and challenges of the current strategies developed to deliver therapeutic enzymes and provide a future perspective on the immobilization technologies used for therapeutic enzyme delivery.

Profile of Neurological Manifestations in Children Presenting with Rickettsial Disease.

OBJECTIVE: To study the profile of neurological manifestation of rickettsial disease in children. METHODS: Review of hospital records was done in a tertiary care hospital for the period from January to December, 2020. Data of all the children fulfilling the inclusion criteria i.e., clinical criteria and serology were retrieved from the hospital records. RESULTS: Of the total 7974 children admitted over this period, 178 were diagnosed with rickettsial disease wherein 54 (33.3%) had neurological involvement. Convulsions (59%), altered sensorium (56%), headache (44%), meningeal signs (37%), ataxia, (11%), lateral rectus palsy (7.5%) and stroke (7.5%) were the major neurological manifestations. Cerebrospinal fluid (CSF) analysis done in 30 (55%) children showed pleocytosis [median (IQR) cells 15 (3.75, 50)] with lymphocyte predominance [median (IQR) lymphocytes 11.5 (3, 38.75)] and elevated proteins [median IQR 41.5 (29.75,61)]. Neuroimaging abnormalities noticed were cerebral edema (n=7), cerebellar hyperintensities (n=5), basal ganglia infarcts (n=2) and hippocampal hyperintensities (n=1). CONCLUSION: Early recognition of rickettsial infection as a cause of neurological manifestation would facilitate early specific management.

Emerging applications of bacteria as antitumor agents.

Bacteria are associated with the human body and colonize the gut, skin, and mucous membranes. These associations can be either symbiotic or pathogenic. In either case, bacteria derive more benefit from their host. The ability of bacteria to enter and survive within the human body can be exploited for human benefit. They can be used as a vehicle for delivering or producing bioactive molecules, such as toxins and lytic enzymes, and eventually for killing tumor cells. Clostridium and Salmonella have been shown to infect and survive within the human body, including in tumors. There is a need to develop genetic circuits, which enable bacterial cells to carry out the following activities: (i) escape the human immune system, (ii) invade tumors, (iii) multiply within the tumorous cells, (iv) produce toxins via quorum sensing at low cell densities, and (v) express suicide genes to undergo cell death or cell lysis after the tumor has been lysed. Thus, bacteria have the potential to be exploited as anticancer agents.

Biomolecules Production from Greenhouse Gases by Methanotrophs.

Harmful effects on living organisms and the environment are on the rise due to a significant increase in greenhouse gas (GHG) emissions through human activities. Therefore, various research initiatives have been carried out in several directions in relation to the utilization of GHGs via physicochemical or biological routes. An environmentally friendly approach to reduce the burden of significant emissions and their harmful effects is the bioconversion of GHGs, including methane (CH4) and carbon dioxide (CO2), into value-added products. Methanotrophs have enormous potential for the efficient biotransformation of CH4 to various bioactive molecules, including biofuels, polyhydroxyalkanoates, and fatty acids. This review highlights the recent developments in methanotroph-based systems for methanol production from GHGs and proposes future perspectives to improve process sustainability via biorefinery approaches.

Biotin and Zn2+ Increase Xylitol Production by Candida tropicalis.

In this study, the medium requirements to increase the production of xylitol by using Candida tropicalis (CT) have been investigated. The technique of single addition or omission of medium components was applied to determine the nutritional requirements. The addition of amino acids such as Asp, Glu, Gln, Asn, Thr, and Gly had no significant effect on CT growth. However, in the absence of other metal ions, there was a higher concentration of cell growth and xylitol production when only Zn2+ was present in the medium. The analysis of various vitamins unveiled that biotin and thiamine were the only vitamins required for the growth of CT. Surprisingly, when only biotin was present in the medium as a vitamin, there was less growth of CT than when the medium was complete, but the amount of xylitol released was significantly higher. Overall, this study will increase the xylitol production using the single omission or addtion technique.

Correction to: Biomethanol Production from Methane by Immobilized Co-cultures of Methanotrophs.

[This corrects the article DOI: 10.1007/s12088-020-00883-6.].