E. Efremenko

Production of biofuels from pretreated microalgae biomass by anaerobic fermentation with immobilized Clostridium acetobutylicum cells.

The purpose of this work was to study the possible use of pretreated biomass of various microalgae and cyanobacteria as substrates for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum cells immobilized into poly(vinyl alcohol) cryogel. To this end, the biochemical composition of photosynthetic microorganisms cultivated under various conditions was studied. The most efficient technique for pretreating microalgal biomass for its subsequent conversion into biofuels appeared to be thermal decomposition at 108 °C. For the first time the maximum productivity of the ABE fermentation in terms of hydrogen (8.5 mmol/L medium/day) was obtained using pretreated biomass of Nannochloropsis sp. Maximum yields of butanol and ethanol were observed with Arthrospira platensis biomass used as the substrate. Immobilized Clostridium cells were demonstrated to be suitable for multiple reuses (for a minimum of five cycles) in ABE fermentation for producing biofuels from pretreated microalgal biomass.

Organophosphate hydrolase — an enzyme catalyzing degradation of phosphorus-containing toxins and pesticides

The available investigations on the structure and properties of the enzyme organophosphate hydrolase exhibiting catalytic activity with respect to orthophosphates are reviewed. Recent data on the mechanism of enzymatic hydrolysis of paraoxon are surveyed. The role of two metal ions involved in the enzyme active site is considered. The substrate specificity and the influence of various inhibitors on the kinetic characteristics of the enzyme are discussed.

Addition of Polybrene improves stability of organophosphate hydrolase immobilized in poly(vinyl alcohol) cryogel carrier

Organophosphate hydrolase, covalently attached to the beads of poly(vinyl alcohol) cryogel in the presence of Polybrene, was fivefold more stable in 15% (v/v) ethanol solution than the free enzyme. Immobilized biocatalyst, prepared with an addition of Polybrene, retained a half of its initial activity in 50% (v/v) aqueous ethanol solution, 90% of activity during 10 working cycles of Paraoxon hydrolysis and 85% of activity after storage in the 50 mM CHES buffer (pH 9.0) at room temperature for 2 months.

Rhizopus oryzae fungus cells producing L(+)-lactic acid: kinetic and metabolic parameters of free and PVA-cryogel-entrapped mycelium.

Spores of the filamentous fungus Rhizopus oryzae were entrapped in macroporous poly(vinyl alcohol) cryogel (PVA-cryogel). To prepare immobilised biocatalyst capable of producing L(+)-lactic acid (LA), the fungus cells were cultivated inside the carrier beads. The growth parameters and metabolic activity of the suspended (free) and immobilised cells producing LA in a batch process were comparatively investigated. The immobilised cells possessed increased resistance to high concentrations of accumulated product and gave much higher yields of LA in the iterative working cycle than the free cells did. Detailed kinetic analysis of the changes in the intracellular adenosine triphosphate concentration, specific rate of growth, substrate consumption and LA production showed that the fungus cells entrapped in PVA-cryogel are more attractive for biotechnological applications compared to the free cells.

A simple and highly effective catalytic nanozyme scavenger for organophosphorus neurotoxins

ABSTRACT A simple and highly efficient catalytic scavenger of poisonous organophosphorus compounds, based on organophosphorus hydrolase (OPH, EC 3.1.8.1), is produced in aqueous solution by electrostatic coupling of the hexahistidine tagged OPH (His6‐OPH) and poly(ethylene glycol)‐b‐poly(l‐glutamic acid) diblock copolymer. The resulting polyion complex, termed nano‐OPH, has a spherical morphology and a diameter from 25 nm to 100 nm. Incorporation of His6‐OPH in nano‐OPH preserves catalytic activity and increases stability of the enzyme allowing its storage in aqueous solution for over a year. It also decreases the immune and inflammatory responses to His6‐OPH in vivo as determined by anti‐OPH IgG and cytokines formation in Sprague Dawley rats and Balb/c mice, respectively. The nano‐OPH pharmacokinetic parameters are improved compared to the naked enzyme suggesting longer blood circulation after intravenous (iv) administrations in rats. Moreover, nano‐OPH is bioavailable after intramuscular (im), intraperitoneal (ip) and even transbuccal (tb) administration, and has shown ability to protect animals from exposure to a pesticide, paraoxon and a warfare agent, VX. In particular, a complete protection against the lethal doses of paraoxon was observed with nano‐OPH administered iv and ip as much as 17 h, im 5.5 h and tb 2 h before the intoxication. Further evaluation of nano‐OPH as a catalytic bioscavenger countermeasure against organophosphorus chemical warfare agents and pesticides is warranted.

Chemical and biological safety: Biosensors and nanotechnological methods for the detection and monitoring of chemical and biological agents

The elaboration of highly sensitive and express methods for quantitative and qualitative detection and monitoring of chemical warfare agents (CWA), organophosphate and carbamate pesticides, compounds with delayed neurotoxicity, and pathogenic microorganisms and viruses is discussed. The application of potentiometric and amperometric biosensors, automatic biosensors discriminating the neurotoxins of different classes, is performed. The information about biosensors detecting the compounds with delayed neurotoxicity through the evaluation of “neurotoxic esterase”activity in the blood is presented. The use of immunochip technology for the detection of pathogenic microorganisms and viruses is demonstrated. The enzymatic methods of destruction of organophosphorus neurotoxins are considered as the base of new defense technology.

Determination of Minimal Concentrations of Biocorrosion Inhibitors by a Bioluminescence Method

By using a bioluminescence ATP assay, we have determined the minimal concentrations of some biocorrosion inhibitors (Kathon, Khazar, VFIKS-82, Nitro-1, Caspii-2, and Caspii-4) suppressing most common microbial corrosion inducers: Desulfovibrio desulfuricans, Desulfovibrio vulgaris, Pseudomonas putida, Pseudomonas fluorescens, and Acidithiobacillus ferrooxidans. The cell titers determined by the bioluminescence method, including not only fissiparous cells but also their dormant living counterparts, are two-to sixfold greater than the values determined microbiologically. It is shown that the bioluminescence method can be applied to determination of cell titers in samples of oil-field waters in the presence of iron ions (up to 260 mM) and iron sulfide (to 186 mg/l) and in the absence or presence of biocidal corrosion inhibitors.

Degradation of thiodiglycol, the hydrolysis product of sulfur mustard, with bacteria immobilized within poly(vinyl) alcohol cryogels

Alcaligenes xylosoxidans subsp. xylosoxidans (SH91) capable of biodegradation of thiodiglycol (TDG) were immobilized in poly(vinyl) alcohol (PVA) cryogels. Cryoimmobilized biocatalyst was formed as spherical granules with a diameter of 0.5 mm; the biomass concentration inside the gel matrix was as high as 10% (w/w). The immobilized cells were capable of rapid degradation of TDG in tap water or potassium phosphate buffer (100 mM, pH 8.0) containing only (NH4)2 SO4. The immobilized biocatalyst did not show any substrate inhibition up to 200 mM TDG, and retained 100% activity during three months of continuous use in a repeated-batch bioreactor.

Polyhistidine-containing organophosphorus hydrolase with outstanding properties

The catalytic and physical–chemical properties of organophosphorus hydrolase (OPH) modified by the addition of an N-terminal dodecahistidine tag (His12-OPH) have been investigated. Introduction of the His12-tag caused a 30- and 74-fold increase in catalytic efficiency of the enzyme with parathion and methyl parathion, respectively, compared to OPH. Concurrently, the His12-OPH had a more alkaline pH-optimum and extended temperature range than OPH and OPH modified with a hexahistidine tag. A study of His12-OPH thermostability showed that the enzyme had a tendency to oligomerise. This resulted in a decrease in the enzymatic activity of His12-OPH at temperatures <50°C, but provided the enzyme with much higher thermostability at temperatures >50°C, compared to OPH.

Biocatalysts Based on Immobilized Cells of Microorganisms in the Production of Bioethanol and Biobutanol

In this work, we discuss the processes for the production of bioethanol and biobutanol, which are promising alternative fuels, using biocatalysts based on cells of various microorganisms immobilized in poly(vinyl alcohol) cryogel. Biocatalysts based on immobilized cells reliably allow ethanol production from a variety of industrial and agricultural wastes (wheat straw, beet and sugarcane bagasse, parchment, corn cobs, soybean processing waste) with a high degree of conversion of consumed substrates to the target product. Ethanol concentrations are appreciably higher in media with biocatalysts than in free cells of the same microorganisms. It is found that immobilized cells of filamentous fungi can convert a wider range of the sugars contained in processed media to ethanol than commonly used yeasts. It is shown that the immobilization of the genus Clostridium cells that produce butanol enables us to reliably change the ratio of solvents that accumulate in the medium during acetone-butanol-ethanol fermentation in the direction of a greater amount of butanol, thereby improving the process’s characteristics relative to present-day technologies based on free bacterial cells.