Using Fungi in Artificial Microbial Consortia to Solve Bioremediation Problems.

There is currently growing interest in the creation of artificial microbial consortia, especially in the field of developing and applying various bioremediation processes. Heavy metals, dyes, synthetic polymers (microplastics), pesticides, polycyclic aromatic hydrocarbons and pharmaceutical agents are among the pollutants that have been mainly targeted by bioremediation based on various consortia containing fungi (mycelial types and yeasts). Such consortia can be designed both for the treatment of soil and water. This review is aimed at analyzing the recent achievements in the research of the artificial microbial consortia that are useful for environmental and bioremediation technologies, where various fungal cells are applied. The main tendencies in the formation of certain microbial combinations, and preferences in their forms for usage (suspended or immobilized), are evaluated using current publications, and the place of genetically modified cells in artificial consortia with fungi is assessed. The effect of multicomponence of the artificial consortia containing various fungal cells is estimated, as well as the influence of this factor on the functioning efficiency of the consortia and the pollutant removal efficacy. The conclusions of the review can be useful for the development of new mixed microbial biocatalysts and eco-compatible remediation processes that implement fungal cells.

Enzymes with Lactonase Activity against Fungal Quorum Molecules as Effective Antifungals.

Since the growing number of fungi resistant to the fungicides used is becoming a serious threat to human health, animals, and crops, there is a need to find other effective approaches in the eco-friendly suppression of fungal growth. One of the main mechanisms of the development of resistance in fungi, as well as in bacteria, to antimicrobial agents is quorum sensing (QS), in which various lactone-containing compounds participate as signaling molecules. This work aimed to study the effectiveness of action of enzymes exhibiting lactonase activity against fungal signaling molecules. For this, the molecular docking method was used to estimate the interactions between these enzymes and different lactone-containing QS molecules of fungi. The catalytic characteristics of enzymes such as lactonase AiiA, metallo-ß-lactamase NDM-1, and organophosphate hydrolase His6-OPH, selected for wet experiments based on the results of computational modeling, were investigated. QS lactone-containing molecules (butyrolactone I and γ-heptalactone) were involved in the experiments as substrates. Further, the antifungal activity of the enzymes was evaluated against various fungal and yeast cells using bioluminescent ATP-metry. The efficient hydrolysis of γ-heptalactone by all three enzymes and butyrolactone I by His6-OPH was demonstrated for the first time. The high antifungal efficacy of action of AiiA and NDM-1 against most of the tested fungal cells was revealed.

Luminescent Bacteria as Bioindicators in Screening and Selection of Enzymes Detoxifying Various Mycotoxins.

Interest in enzymes capable of neutralizing various mycotoxins is quite high. The methods used for the screening and selection of enzymes that catalyze the detoxification of mycotoxins should be sensitive and fast. However toxic compounds can be generated under the action of such enzymes. Thus, the assessment of the overall reduction in the toxic properties of reaction media towards bioluminescent bacteria seems to be the most reasonable control method allowing a quick search for the effective enzymatic biocatalysts. The influence of a wide range of mycotoxins and glucanases, which hydrolyze toxins with different chemical structures, on the analytical characteristics of luminescent photobacteria as a biosensing element has been studied. Different glucanases (ß-glucosidase and endoglucanase) were initially selected for reactions with 10 mycotoxins based on the results of molecular docking which was performed in silico with 20 mycotoxins. Finally, the biorecognizing luminescent cells were used to estimate the residual toxicity of reaction media with mycotoxins after their interaction with enzymes. The notable non-catalytic decrease in toxicity of media containing deoxynivalenol was revealed with luminous cells for both types of tested glucanases, whereas ß-glucosidase provided a significant catalytic detoxification of media with aflatoxin B2 and zearalenone at pH 6.0.

Artificial Humic Substances as Biomimetics of Natural Analogues: Production, Characteristics and Preferences Regarding Their Use.

Various processes designed for the humification (HF) of animal husbandry wastes, primarily bird droppings, reduce their volumes, solve environmental problems, and make it possible to obtain products with artificially formed humic substances (HSs) as analogues of natural HSs, usually extracted from fossil sources (coal and peat). This review studies the main characteristics of various biological and physicochemical methods of the HF of animal wastes (composting, anaerobic digestion, pyrolysis, hydrothermal carbonation, acid or alkaline hydrolysis, and subcritical water extraction). A comparative analysis of the HF rates and HS yields in these processes, the characteristics of the resulting artificial HSs (humification index, polymerization index, degree of aromaticity, etc.) was carried out. The main factors (additives, process conditions, waste pretreatment, etc.) that can increase the efficiency of HF and affect the properties of HSs are highlighted. Based on the results of chemical composition analysis, the main trends and preferences with regard to the use of HF products as complex biomimetics are discussed.

Various Biomimetics, Including Peptides as Antifungals.

Biomimetics, which are similar to natural compounds that play an important role in the metabolism, manifestation of functional activity and reproduction of various fungi, have a pronounced attraction in the current search for new effective antifungals. Actual trends in the development of this area of research indicate that unnatural amino acids can be used as such biomimetics, including those containing halogen atoms; compounds similar to nitrogenous bases embedded in the nucleic acids synthesized by fungi; peptides imitating fungal analogs; molecules similar to natural substrates of numerous fungal enzymes and quorum-sensing signaling molecules of fungi and yeast, etc. Most parts of this review are devoted to the analysis of semi-synthetic and synthetic antifungal peptides and their targets of action. This review is aimed at combining and systematizing the current scientific information accumulating in this area of research, developing various antifungals with an assessment of the effectiveness of the created biomimetics and the possibility of combining them with other antimicrobial substances to reduce cell resistance and improve antifungal effects.

Metal Nanomaterials and Hydrolytic Enzyme-Based Formulations for Improved Antifungal Activity.

Active research of metal-containing compounds and enzymes as effective antifungal agents is currently being conducted due to the growing antifungal resistance problem. Metals are attracting special attention due to the wide variety of ligands that can be used for them, including chemically synthesized and naturally obtained variants as a result of the so-called "green synthesis". The main mechanism of the antifungal action of metals is the triggering of the generation and accumulation of reactive oxygen species (ROS). Further action of ROS on various biomolecules is nonspecific. Various hydrolytic enzymes (glucanases and proteases), in turn, exhibit antifungal properties by affecting the structural elements of fungal cells (cell walls, membranes), fungal quorum sensing molecules, fungal own protective agents (mycotoxins and antibiotics), and proteins responsible for the adhesion and formation of stable, highly concentrated populations in the form of biofilms. A wide substrate range of enzymes allows the use of various mechanisms of their antifungal actions. In this review, we discuss the prospects of combining two different types of antifungal agents (metals and enzymes) against mycelial fungi and yeast cells. Special attention is paid to the possible influence of metals on the activity of the enzymes and the possible effects of proteins on the antifungal activity of metal-containing compounds.

Quorum Sensing as a Trigger That Improves Characteristics of Microbial Biocatalysts.

Quorum sensing (QS) of various microorganisms (bacteria, fungi, microalgae) today attracts the attention of researchers mainly from the point of view of clarifying the biochemical basics of this general biological phenomenon, establishing chemical compounds that regulate it, and studying the mechanisms of its realization. Such information is primarily aimed at its use in solving environmental problems and the development of effective antimicrobial agents. This review is oriented on other aspects of the application of such knowledge; in particular, it discusses the role of QS in the elaboration of various prospective biocatalytic systems for different biotechnological processes carried out under aerobic and anaerobic conditions (synthesis of enzymes, polysaccharides, organic acids, etc.). Particular attention is paid to the biotechnological aspects of QS application and the use of biocatalysts, which have a heterogeneous microbial composition. The priorities of how to trigger a quorum response in immobilized cells to maintain their long-term productive and stable metabolic functioning are also discussed. There are several approaches that can be realized: increase in cell concentration, introduction of inductors for synthesis of QS-molecules, addition of QS-molecules, and provoking competition between the participants of heterogeneous biocatalysts, etc.).

Progressive Biocatalysts for the Treatment of Aqueous Systems Containing Pharmaceutical Pollutants.

The review focuses on the appearance of various pharmaceutical pollutants in various water sources, which dictates the need to use various methods for effective purification and biodegradation of the compounds. The use of various biological catalysts (enzymes and cells) is discussed as one of the progressive approaches to solving problems in this area. Antibiotics, hormones, pharmaceuticals containing halogen, nonsteroidal anti-inflammatory drugs, analgesics and antiepileptic drugs are among the substrates for the biocatalysts in water purification processes that can be carried out. The use of enzymes in soluble and immobilized forms as effective biocatalysts for the biodegradation of various pharmaceutical compounds (PCPs) has been analyzed. Various living cells (bacteria, fungi, microalgae) taken as separate cultures or components of natural or artificial consortia can be involved in biocatalytic processes under aerobic or anaerobic conditions. Cells as biocatalysts introduced into water treatment systems in suspended or immobilized form are used for deep biodegradation of PCPs. The potential of combinations of biocatalysts with physical-chemical methods of wastewater treatment is evaluated in relation to the effective removing of PCPs. The review analyzes recent results and the main current trends in the development of biocatalytic approaches to biodegradation of PCPs, the pros and cons of the processes and the biocatalysts used.

Destruction of Mycotoxins in Poultry Waste under Anaerobic Conditions within Methanogenesis Catalyzed by Artificial Microbial Consortia.

To reduce the toxicity of modern feeds polluted by mycotoxins, various sorbents are added to them when feeding animals. A part of the mycotoxins is excreted from the body of animals with these sorbents and remains in the manure. As a result, bulk animal wastes containing mixtures of mycotoxins are formed. It is known that it is partially possible to decrease the initial concentration of mycotoxins in the process of anaerobic digestion (AD) of contaminated methanogenic substrates. The aim of this review was to analyze the recent results in destruction of mycotoxins under the action of enzymes present in cells of anaerobic consortia catalyzing methanogenesis of wastes. The possible improvement of the functioning of the anaerobic artificial consortia during detoxification of mycotoxins in the bird droppings is discussed. Particular attention was paid to the possibility of effective functioning of microbial enzymes that catalyze the detoxification of mycotoxins, both at the stage of preparation of poultry manure for methanogenesis and directly in the anaerobic process itself. The sorbents with mycotoxins which appeared in the poultry wastes composed one of the topics of interest in this review. The preliminary alkaline treatment of poultry excreta before processing in AD was considered from the standpoint of effectively reducing the concentrations of mycotoxins in the waste.

Carrier Variety Used in Immobilization of His6-OPH Extends Its Application Areas.

Organophosphorus hydrolase, containing a genetically introduced hexahistidine sequence (His6-OPH), attracts the attention of researchers by its promiscuous activity in hydrolytic reactions with various substrates, such as organophosphorus pesticides and chemical warfare agents, mycotoxins, and N-acyl homoserine lactones. The application of various carrier materials (metal-organic frameworks, polypeptides, bacterial cellulose, polyhydroxybutyrate, succinylated gelatin, etc.) for the immobilization and stabilization of His6-OPH by various methods, enables creation of biocatalysts with various properties and potential uses, in particular, as antidotes, recognition elements of biosensors, in fibers with chemical and biological protection, dressings with antimicrobial properties, highly porous sorbents for the degradation of toxicants, including in flow systems, etc. The use of computer modeling methods in the development of immobilized His6-OPH samples provides in silico prediction of emerging interactions between the enzyme and immobilizing polymer, which may have negative effects on the catalytic properties of the enzyme, and selection of the best options for experiments in vitro and in vivo. This review is aimed at analysis of known developments with immobilized His6-OPH, which allows to recognize existing recent trends in this field of research, as well as to identify the reasons limiting the use of a number of polymer molecules for the immobilization of this enzyme.

Combination of Enzymes with Materials to Give Them Antimicrobial Features: Modern Trends and Perspectives.

Multidrug-resistant bacteria form serious problems in many areas, including medicine and the food industry. At the same time, great interest is shown in the transfer or enhancement of antimicrobial properties to various materials by modifying them with enzymes. The use of enzymes in biomaterials with antimicrobial properties is important because enzymes can be used as the main active components providing antimicrobial properties of functionalized composite biomaterials, or can serve as enhancers of the antimicrobial action of certain substances (antibiotics, antimicrobial peptides, metal nanoparticles, etc.) against cells of various microorganisms. Enzymes can simultaneously widen the spectrum of antimicrobial activity of biomaterials. This review presents the most promising enzymes recently used for the production of antibacterial materials, namely hydrolases and oxidoreductases. Computer modeling plays an important role in finding the most effective combinations between enzymes and antimicrobial compounds, revealing their possible interactions. The range of materials that can be functionalized using enzymes looks diverse. The physicochemical characteristics and functionalization methods of the materials have a significant impact on the activity of enzymes. In this context, fibrous materials are of particular interest. The purpose of this review is to analyze the current state of the art in this area.

Strategies for variable regulation of methanogenesis efficiency and velocity.

Combinations of various strategic approaches to the suppression of methanogenesis and the formation of biogas with a simultaneous decrease in the ratio of methane in its composition were investigated. Introduction of methanogenesis suppressors such as redox derivatives of humic acids, potassium persulfate (K2S2O8), possessing oxidizing and electron acceptor properties, enzyme hexahistidine-containing organophosphorus hydrolase with high lactonase activity and polypeptide antimicrobial agent bacitracin into the media with anaerobic consortia were studied. The effect of these substances was directed at various participants of the natural methanogenic consortium, as well as on the biochemical processes carried out by them. The use of K2S2O8 together with bacitracin provided maximum and almost complete suppression of CH4 production. The measured concentration of intracellular adenosine triphosphate has shown that viability of cells in the consortium remained almost the same, whereas their metabolic activity decreased. Various combinations of the above-mentioned suppressors provided different degrees of methanogenesis suppression, but redox agents played a key role in all the cases studied. Based on the accumulated data, combining suppressors in different concentrations can be used to manage the methanogenesis (efficiency and velocity of its decrease) in media with anaerobic consortia. KEY POINTS: • Various strategies for suppression of the methanogenesis were combined. • The enzyme His6-OPH was firstly used for quorum quenching in methanogenic consortium. • Velocity of methanogenesis decrease can be managed by combinations of suppressors.

Sulfur containing mixed wastes in anaerobic processing by new immobilized synthetic consortia.

Methanogenic biotransformation of unusual substrates (sulfur (S)-containing wastes: non-purified vacuum gas oil, straight-run gasoline fraction (Naphtha), gas condensate, and straight-run diesel fraction) coming from oil industry after their oxidative desulfurization was investigated. Nitrogen-containing wastes (hydrolysates of chicken manure and Chlorella vulgaris biomass) were added as co-substrates to mixture with oil industry wastes. The 100 % conversion of S-organic compounds to inorganic sulfide accumulated in the reaction liquid medium was achieved with simultaneous production of biogas containing high methane percent (greater than 70 %). Polishing of effluents from methane tank was carried out by denitrifying oxidation of ammonium (DEAMOX). The high process efficiency was due to use of original immobilized artificial consortia at the stage of methanogenesis and DEAMOX. This study reveals the real potential in the processing of very complex mixtures of large-scale wastes, usually inhibiting methanogenesis, by developing biocatalysts based on synthetic biology approaches.

"Unity and Struggle of Opposites" as a Basis for the Functioning of Synthetic Bacterial Immobilized Consortium That Continuously Degrades Organophosphorus Pesticides.

This work was aimed at the development of an immobilized artificial consortium (IMAC) based on microorganisms belonging to the Gram-positive and Gram-negative bacterial cells capable of jointly carrying out the rapid and effective degradation of different organophosphorus pesticides (OPPs): paraoxon, parathion, methyl parathion, diazinon, chlorpyrifos, malathion, dimethoate, and demeton-S-methyl. A cryogel of poly(vinyl alcohol) was applied as a carrier for the IMAC. After a selection was made between several candidates of the genera Rhodococcus and Pseudomonas, the required combination of two cultures (P. esterophilus and R. ruber) was found. A further change in the ratio between the biomass of the cells inside the granules of IMAC, increasing the packing density of cells inside the same granules and decreasing the size of the granules with IMAC, gave a 225% improvement in the degradation activity of the cell combination. The increase in the velocity and the OPP degradation degree was 4.5 and 16 times greater than the individual P. esterophilus and R. ruber cells, respectively. Multiple uses of the obtained IMAC were demonstrated. The increase in IMAC lactonase activity confirmed the role of the cell quorum in the action efficiency of the synthetic biosystem. The co-inclusion of natural strains in a carrier during immobilization strengthened the IMAC activities without the genetic enhancement of the cells.

Model Fuel Oxidation in the Presence of Molybdenum-Containing Catalysts Based on SBA-15 with Hydrophobic Properties.

We have studied for the first time the role of hydrophobicity of the mesoporous silicate SBA-15 on the activity and the service life of a catalyst in the peroxide oxidation of sulfur-containing compounds. Immobilization of the molybdate anion on the SBA-15 support via ionic bonding with triethylammonium groups allows us not only to decrease the reaction temperature to a relatively low value of 60 °C without a drop in the dibenzothiophene conversion degree but also to increase the service life of the catalyst to many times that of the known analogs. The support and catalyst structures were investigated by low-temperature nitrogen adsorption/desorption, Fourier-transform infrared spectroscopy, X-ray fluorescence analysis, and transmission electron microscopy. Immobilization of the molybdate anion on the SBA-15 support, modified with ammonium species, prevents the leaching of active sites. However, only alkyl-substituted ammonium species minimize DBT sulfone adsorption, which significantly increases the catalyst's service life. The synthesized catalyst Mo/Et3N-SBA-15 with hydrophobic properties is not sensitive to the initial sulfur content and hydrogen peroxide amount and retains its activity for at least six cycles of oxidation without regeneration. These catalysts can be efficiently used for clean fuel production.

Formation and use of anaerobic consortia for the biotransformation of sulfur-containing extracts from pre-oxidized crude oil and oil fractions.

A new solution for fossil raw materials desulfurization based on a hybrid chemical-biocatalytic scheme with biogas and sulfide production is proposed.·H2O2, formic acid and Na2MoO4 were used for petroleum or oil fractions pre-oxidation. Ethanol or dimethylformamide was used as extractant to remove sulfur-contained compounds from pre-oxidized straight-run diesel oil fraction, non-hydro treated vacuum gas oil, gas condensate or crude oil. Compositions of cells (anaerobic sludge, Desulfovibrio vulgaris, Clostridium acetobutilycum, Rhodococcus ruber, Rhodococcus erythropolis) were specially developed, immobilized in poly(vinyl alcohol) cryogel and used for methanogenic treatment of sulfur-containing extracts, diluted with phosphate buffer (pH 7.2) and hydrolysates of renewable raw materials. The sulfur coming into the reactor with the extracts was 100% converted to inorganic sulfide or cell biomass. The ratio of methane in the biogas was 68-76%. Bioluminescent express-methods were used to control the possible toxicity of media and metabolic activity of cells used as biocatalysts.

Suppression of Methane Generation during Methanogenesis by Chemically Modified Humic Compounds.

The introduction of various concentrations of chemically modified humic compounds (HC) with different redox characteristics into the media with free and immobilized anaerobic consortia accumulating landfill gases was studied as approach to their functioning management. For this purpose, quinone (hydroquinone, naphthoquinone or methylhydroquinone) derivatives of HC were synthesized, which made it possible to vary the redox and antioxidant properties of HC as terminal electron acceptors in methanogenic systems. The highest acceptor properties were obtained with potassium humate modified by naphthoquinone. To control possible negative effect of HC on the cells of natural methanogenic consortia, different bioluminescent analytical methods were used. The addition of HC derivatives, enriched with quinonones, to nutrient media at concentrations above 1 g/L decreased the energetic status of cells and the efficiency of the methanogenesis. For the first time, the significant decrease in accumulation of biogas was reached as effect of synthetic HC derivatives, whereas both notable change of biogas composition towards increase in the CO2 content and decrease in CH4 were revealed. Thus, modification with quinones makes it possible to obtain low-potential HC derivatives with strongly pronounced acceptor properties, promising for inhibition of biogas synthesis by methanogenic communities.

Metal Nanoparticles for Improving Bactericide Functionality of Usual Fibers.

A wide variety of microbiological hazards stimulates a constant development of new protective materials against them. For that, the application of some nanomaterials seems to be very promising. Modification of usual fibers with different metal nanoparticles was successfully illustrated in the work. Tantal nanoparticles have shown the highest antibacterial potency within fibrous materials against both gram-positive (Bacillus subtilis) and gram-negative (Escherichia coli) bacteria. Besides, the effect of tantal nanoparticles towards luminescent Photobacterium phosphoreum cells estimating the general sample ecotoxicity was issued for the first time.

Prospective Approach to the Anaerobic Bioconversion of Benzo- and Dibenzothiophene Sulfones to Sulfide.

Sulfur recovery from organic molecules such as toxic sulfones is an actual problem, and its solution through the use of environmentally friendly and nature-like processes looks attractive for research and application. For the first time, the possible bioconversion of organic sulfones (benzo-and dibenzothiophene sulfones) to inorganic sulfide under anaerobic conditions with simultaneous biogas production from glucose within a methanogenesis process is demonstrated. Biogas with a methane content of 50.7%-82.1% was obtained without H2S impurities. Methanogenesis with 99.7%-100% efficiency and 97.8%-100% conversion of benzo- and dibenzothiophene sulfones (up to 0.45 mM) to inorganic sulfide were obtained in eight days by using a combination of various anaerobic biocatalysts immobilized in a poly(vinyl alcohol) cryogel. Pure cell cultures of sulfate-reducing bacteria and/or H2-producing bacteria were tested as additives to the methanogenic activated sludge. The immobilized activated sludge "enhanced" by bacterial additives appeared to retain its properties and be usable multiple times for the conversion of sulfones under batch conditions.

Immobilized Luminescent Bacteria for the Detection of Mycotoxins under Discrete and Flow-Through Conditions.

A biosensitive element in the form of bacterial Photobacterium phosphoreum cells immobilized in poly(vinyl alcohol) cryogel was tested for the determination of different mycotoxins under discrete and flow-through analysis conditions. The immobilized bioluminescent cells made it possible to quantify the presence of Ochratoxin A, Sterigmatocystin, Zearalenone, and Deoxynivalenon in aqueous media in a wide range of their concentrations (0.017-56 mg/L, 0.010-33 mg/L, 0.009-14 mg/L, and 0.026-177 mg/L, respectively) via measuring the quenching of cell luminescence. The flow conditions allowed the analysis sensitivity to be improved by an order of magnitude in terms of detected concentrations. Using the immobilized luminescent bacterial cells, we have shown the possibility of evaluating the efficiency of the mycotoxins' hydrolysis under the action of enzymes. In this way, a 94 ± 4.5% efficiency of Zearalenone hydrolysis with hexahistidine-containing organophosphorus hydrolase for 1h-long treatment of the mycotoxin solution (100 mg/L) was shown.