Анотація
Гриценко Н.А. Розробка технології поверхнево-активних речовин Nocardia vaccinii K-8. – На правах рукопису.
Дисертація на здобуття наукового ступеня кандидата технічних наук за спеціальністю 03.00.20 – біотехнологія. − Національний університет харчових технологій МОНМС України, Київ, 2012.
Дисертаційна робота присвячена розробці технологій поверхнево-активних речовин (ПАР) Nocardia vaccinii K-8 за використання відходів виробництва біодизелю та можливості практичного використання ПАР в природоохоронних технологіях.
За допомогою математичних методів планування експерименту оптимізовано поживне середовище для культивування N. vaccinii K-8 і показано можливість збільшення у 2–4 рази (до 12,6 г/дм3) концентрації синтезованих ПАР на гліцерині.
Встановлено, що за хімічним складом поверхнево-активні речовини штаму К-8 є комплексом нейтральних, аміно- та гліколіпідів. Показано можливість підвищення на 40 % умовної концентрації ПАР у разі внесення на початку стаціонарної фази росту у середовище з гліцерином 0,1 % фумарату і 0,1 % цитрату.
За використання суспензії клітин N. vaccinii K-8 і препаратів ПАР у вигляді постферментаційної культуральної рідини максимальний ступінь деструкції нафтових забруднень у воді (2,6 г/дм3) і ґрунті (20 г/кг) становив 85–95 %.
Ключові слова: бактерії роду Nocardia, поверхнево-активні речовини, інтенсифікація біосинтезу, відходи виробництва біодизелю.
Аннотация
Гриценко Н.А. Разработка технологии поверхностно-активных веществ Nocardia vaccinii K-8. – На правах рукописи.
Диссертация на соискание научной степени кандидата технических наук по специальности – 03.00.20. Национальный университет пищевых технологий МОНМС, Киев, 2012.
Диссертация посвящена разработке технологии получения поверхностно-активных веществ (ПАВ) Nocardia vaccinii K-8 и исследованию возможности их практического применения в природоохранных технологиях.
В составе ПАВ штамма К-8 выявлены глико-, амино- и нейтральные липиды. Экспериментально установлена возможность повышения на 40 % условной концентрации ПАВ в случае внесения в начале стационарной фазы роста N. vaccinii K-8 на среде с глицерином 0,1 % фумарата і 0,1 % цитрата.
С помощью математических методов планирования эксперимента оптимизирована питательная среда для культивирования N. vaccinii K-8 и показана способность увеличения в 4 раза (до 12,6 г/дм3) концентрации синтезированных штаммом на глицерине ПАВ по сравнению с показателями до оптимизации.
Показана возможность использования суспензии клеток N. vaccinii K-8 и препаратов ПАР в виде постферментационной культуральной жидкости для эффективной (до 98 %) деструкции нефтяных загрязнений в воде (2,6 г/дм3) и почве (20 г/кг).
Ключевые слова: бактерии рода Nocardia, поверхностно-активные вещества, интенсификация биосинтеза, отходы производства биодизеля.
SUMMARY
Grytsenko N.A. Development of technology of surface-active substances of Nocardia vaccinii K-8. – Manuscript.
Dissertation for the degree of candidate of technical sciences in specialty 03.00.20. National University of Food Technologies MESYS of Ukraine, Kyiv, 2012.
The dissertation thesis is devoted to a developing technologies of surface-active substances (SAS, surfactants) of Nocardia vaccinii K-8 with the use of different carbon substrates and studding possibilities of their practical use in environmental technologies.
In recent years, microbial surfactants are the subject of intense theoretical and applied research, because of their possible practical use in various industries, as well as for the remediation of environment. The advantages of microbial surfactants compared to chemical analogs are the biodegradability, non-toxicity, stability of physical and chemical properties over a wide range of pH and temperature.
The strain of oil oxidizing bacteria, identified as Nocardia vaccinii K-8 from was isolated from oily soil samples earlier. The possibility of remediation of oil contaminated water (100 mg/cm3) by immobilized cells of N. vaccinii K-8 was shown. It is known that the ability to assimilate hydrocarbon substrates by microorganisms is often caused by synthesis of surfactants.
The ability of N. vaccinii K-8 to the synthesize surfactants on a various hydrophobic (hexadecane, liquid paraffin) and hydrophilic (ethanol, glucose, glycerol) substrates was determined. The surfactants’ synthesis increased with substrate concentration of 1 %, the duration of cultivation 120–168 h, and addition of iron and yeast extract into the medium. The possibility of synthesis of surfactants by bacteria of Nocardia genus using ethanol and glycerol as a carbon and energy sources was shown for the first time.
It was shown that the highest indexes of surfactants’ synthesis by N. vaccinii K-8 grown on glycerol were observed when sodium nitrate was used a source of carbon nutrition and inoculum was grown to mid-exponential phase in a medium containing yeast extract and iron sulphate.
It was determined that the chemical composition of surfactants synthesized by N. vacсinii K-8 on glycerol was a complex of neutral, glyco- and aminolipids. Neutral lipids are represented by mycolic and n-alkanoic acids, glycolipids – by trehalosediacelates and trehalosemycolates.
Simultaneous addition of 0,1 % of fumarate (the precursor of gluconeogenesis), and 0.1 % of citrate (regulator of lipid synthesis) into a medium with glycerol in the early stationary growth phase of strain K-8 was accompanied by increase of conditional surfactant concentration by 40 % compared with the cultivation of bacteria in the medoum without organic acids.
The composition of nutrient medium for the cultivation of N. vaccinii K-8 was optimized with the use of mathematical methods of experimental planning, which provided maximal indexes of surfactants’ synthesis by N. vaccinii K-8 (0,5 g/dm3 NaNO3, 0,3 g/dm3 yeast extract and 1,5 % glycerol). It was shown that the concentration of surfactants synthesized by strain K-8 on glycerol increased by 4 fold (up to 12,6 g/dm3) compared to the indexes before optimization.
The ability of microorganisms of different taxonomic groups to assimilate surface-active substances (surfactants) of N. vaccinii K-8 as the sole source of carbon and energy was determined. It was shown that N. vaccinii K-8 does not use its own surfactants as a source of carbon nutrition. The use of formalin in a concentration of 0,5 % can provide the increase of storage time of N. vaccinii K-8 surfactants to 30 days without significant loss of their surface-active properties.
Our experiments have shown that effectiveness of remediation of oil polluted water (2,6 g/dm3) after a single treatment with a suspension of N. vaccinii K-8 cells (9,8 107 CFU/cm3) was 95 % on the 30th day of exposition.
10 % of oil degraded as the result of treatment of sterile sand by surfactant preparations. This confirms the fact that N. vaccinii K-8 does not assimilate oil, but stimulates the development of oil-oxidizing microflora.
The degree of oil degradation in contaminated soil (20 g of oil per kg of soil) was 85 % at 30 day after treatment with surfactants’ preparation of N. vaccinii K-8 as native culture liquid (300 cm3 per 1 kg of soil).
The oil detachment after sand (0,1 cm3 of oil / 1 g of sand) treatment with surfactants of N. vaccinii K-8 as native cultural liquid (30 cm3) was 90 %.
The antimicrobial activity of exocellular metabolites of N. vaccinii K-8 against exocellular metabolites was studied, confirming the promise of their use as environmentally friendly products for the control of plant pathogens. It was determined that exocellular metabolites of N. vaccinii K-8 exhibit antimicrobial properties against some phytopathogenic bacteria. In the presence of a surfactants’ preparations (0,085-0,85 mg/cm3) and other metabolites the quantity of cells of the most studied pathogenic bacteria was reduced by 95–100 %.
The results of experiments were used for the developing of process flow diagram of biosynthesis of N. vaccinii K-8 surfactants from glycerol in presence of organic acids (citrate, fumarate) as biosynthesis precursors, which consists of auxiliary and main works.
Developed technology of synthesis of N. vaccinii K-8 surfactantы in comparison with the known in the world on the basis of glycerol has the following advantages: firstly, higher concentration of synthesized extracellular metabolites, secondly, higher yield from the initial substrate (strain K-8 synthesizes 12,6 g/cm3 of surfactants from 1,5 % of glycerol, while other producers – from 3 to 8 g/dm3 of surfactants from 2–10 % of substrate).
Keywords: bacteria of Nocardia genus, surfactants, intensification of biosynthesis, waste of biodiesel production.