Интенсификация биоремедиации нефтезагрязненных почв с использованием природного консорциума микроорганизмов

Currently biological methods of cleaning oily soils significantly developed as the most accessible and effective. In their basis – the use of hydrocarbon-oxidizing microorganisms (HOM) metabolic potential. At the same time, as practice shows, existing preparations containing HOM are not effective enough in extreme soil-climatic conditions, in this connection the search for indigenous strains of oil-oxidizing bacteria and the development of new biological preparations are of current interest. The aim of this work is to study the effectiveness of HOM strains for restoring the biological activity of oil-contaminated soil. A study of the microflora of unpolluted and oil-contaminated soil taken from the experimental site of the “Shymkent” oil pumping station of “KazTransOil” JSC was carried out: the total number of microorganisms was 210·106cells/g and 130·106 cells/g, respectively. From 16 НOM strains isolated from oil-contaminated soil, 3 bacterial strains were selected to create an association and use as a biological product-oil destructor – P12, P16, A8. Based on the results of genotyping, strains P12 and P16 correspond to the species Pseudomonas aeruginosa, strain A8 belongs to the species Alcaligenes denitrificans. The associations of Pseudomonas sp. P16, Pseudomonas sp. P12 and Alcaligenes sp. A8 showed better growth in comparison with monocultures on a nutrient medium with hydrocarbons. Bioremediation of artificially oil polluted site process, which carried out for 120 days using a biological product created on the basis of the association of strains P12, P16 and A8, decreased the content of hydrocarbons 3.4 times, and with additional application of mineral fertilizers (ammophos) – 4.6 times. Thus, proved the expediency of using HOM strains P12, P16, A8 for the purpose of bioremediation of oil-contaminated soils. The results of the study can be used to improve economical and environmental efficiency of work when carrying out measures to eliminate the consequences of oil spills on the ground. Биологические методы очистки нефтесодержащих почв в настоящее время имеют существенное развитие как наиболее доступные и эффективные. В их основе – использование метаболического потенциала углеводородокисляющих микроорганизмов (УОМ). При этом, как показывает практика, существующие препараты, содержащие УОМ, недостаточно эффективны в экстремальных почвенно-климатических условиях, в связи с чем актуальными являются вопросы поиска аборигенных штаммов нефтеокисляющих бактерий и разработка новых биопрепаратов. Цель работы – исследование эффективности штаммов УОМ для восстановления биологической активности нефтезагрязненного почвогрунта. Проведено изучение микрофлоры незагрязненной и нефтезагрязненной почвы, отобранной с экспериментального участка ГНПС «Шымкент» АО «КазТрансОйл»: общее количество микроорганизмов составило 210·106 кл/г и 130·106кл/г, соответственно. Из выделенных из нефтезагрязненной почвы 16 штаммов УОМ были отобраны три штамма бактерий для создания ассоциации и применения в качестве биопрепарата-нефтедеструктора – Р12, Р16, А8. По итогам генотипирования установлено, что штаммы Р12 и Р16 соответствуют виду Pseudomonas aeruginosa, штамм А8 относится к виду Alcaligenes denitrificans. Установлено, что в составе ассоциаций штаммы Pseudomonas sp. P16, Pseudomonas sp. P12 и Alcaligenes sp. A8. показали лучший по сравнению с монокультурами рост на питательной среде с углеводородами. В процессе биоремедиации участка с искусственным нефтезагрязнением, проводимой в течение 120 суток с использованием биопрепарата, созданного на основе ассоциации штаммов P12, P16 и A8, содержание углеводородов снизилось в 3,4 раза, а при дополнительном внесении минеральных удобрений (аммофоса) – в 4,6 раза. Таким образом, доказана целесообразность использования штаммов УОМ Р12, Р16, А8 с целью биоремедиации нефтезагрязненных почв. Результаты исследования могут быть использованы для повышения экономической и экологической эффективности работ при проведении мероприятий по ликвидации последствий разливов нефти на почве.

Optimization of Expression of Nitrilase from Alcaligenes denitrificans in Rhodococcus rhodochrous to Increase the Efficiency of Biocatalytic Synthesis of Ammonium Acrylate

The Rhodococcus rhodochrous M33-2nit strain containing in its chromosome two copies of a gene for A. denitrificans В-9582 NitC1 nitrilase under the control of a promoter region of genes of R. rhodochrous M8 nitrile hydratase has been constructed. The culturing of the strain was optimized and it was shown that using a two-substrate cultivation scheme on glucose and acetate, it was possible to obtain up to 17 g cdw/L of cells with the specific activity of 7 units/mg cdw. A capacity of synthesizing ammonium acrylate from acrylonitrile by the cells of A. denitrificans В-9582 and A. rhodochrous M33-2nit under the conditions simulating the industrial process was compared. It was shown that the cells of R. rhodochrous M33-2nit were able to produce ammonium acrylate at higher rates of acrylonitrile feeding, than the cells of A. denitrificans В-9582. Using the R. rhodochrous M33-2nit cells, high-concentration solutions of ammonium acrylate (450 g/L) were obtained with the conversion rate of 99.5%. Rhodococcus rhodochrous, nitrilase, nitrile hydratase promoter, ammonium acrylate, acrylonitrile, biocatalysis.

Applications of submerged fermentation for biodegradation and decolourisation of melanoidins by an isolate Alcaligenes denitrificans SAG5

In laboratory conditions a bacterium was isolated, which was identified as Alcaligenes denitrificans SAG5.. The optimum decolourisation (72.6%) of melanoidin was achieved at pH 7.5 and temperature 37 °C within 4-6 days fermentation. The toxicity evaluation of distillery effluent with mung bean (Vigna radiata) revealed that the raw effluent is highly toxic as compared to treated effluent. This indicated that the effluent after bacterial treatment is ecofriendly.

ω-Amino Acid:Pyruvate Transaminase from Alcaligenes denitrificans Y2k-2: a New Catalyst for Kinetic Resolution of β-Amino Acids and Amines

ABSTRACT Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed ω-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic β-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for l-β-amino-n-butyric acid (l-β-ABA). The enzyme converts various β-amino acids and amines to the corresponding β-keto acids and ketones by using pyruvate as an amine acceptor. The apparent Km and V max for l-β-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM l-β-ABA, the apparent Km and V max for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM d,l-β-ABA, producing optically pure d-β-ABA (99% enantiomeric excess) with 53% conversion.