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12
UDC 577.115:663.12 https://doi.org/10.15407/biotech11.01.064
USING OF LOWLAND PEAT SUBSTRATE FOR CULTIVATION OF Pichia anomala IMB Y-5067 AND Rhodotorula gracilis IMB Y-5075 YEAST STRAINS
T. S. Kalandyrets1
V. O. Krasinko1 1National University of Food Technologies, Kyiv, Ukraine
H. S. Andriiash2
N. Ye. Beiko2 2SE "Institute of Food Biotechnology and Genomics
О. О. Tigunova2 of the National Academy of Sciences of Ukraine", Kyiv
S. M. Shulga2
E-mail: Shulga5@i.ua
Received 28.12.2017
The aim of the research was to study the accumulation of yeast lipids in the process of cultivation of strains Pichia anomala IMB Y-5067 and Rhodotorula gracilis IMB Y-5075 with the use of peat as growing substrate. The object of researches was strains P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 from "Collection of strains of microorganisms and plant lines for food and agricultural biotechnology" of SE "Institute of Food Biotechnology and Genomics of the National Academy of Sciences of Ukraine". As a raw material, lowland peat which was preliminarily processed with the help of cavitation or explosive autohydrolysis was used. The accumulation of lipids over the course of cultivation P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 on non-food substrate — lowland peat was shown. The effect of pulp explosive autohydrolysis and pulp cavitation processing on biomass accumulation and lipids synthesis by strains P. аnomala IMB Y-5067 and R. gracilis IMB Y-5075 was researched. It was found that the maximum lipids accumulation by strains P. аnomala IMB Y-5067 (9.7 g/dm3) and R. gracilis IMB Y-5075 (8.9 g/dm3) was over the course of cavitation processing of peat pulp and additional application of salts and yeast extract into cultivation environment.
Key words: growth substrate, peat, yeast lipids, Pichia anomala and Rhodotorula gracilis strains.
One of the possibilities of solving the problem of energy independence of the country can be using the alternative energy resources, in particular, biofuel. Biodiesel is a perspective alternative energy resource. Biodiesel is mainly received from agricultural oils or animal fat and used for substituting oil diesel fuel [1, 2].
Fats and oils of agricultural, animal and microbiological origin can be a raw material for biodiesel. The traditional technology of receiving biofuel for diesel engines is based on receiving methylic (ethanol) ethers by means of the reaction of oil etherification by methyl (ethanol) alcohol during which the process of oil triglycerides etherification by methanol (ethanol) with the use of base or acid catalysts is taking place [1].
Microbiological synthesis is one of the ways of receiving lipids as a potential raw material for biodiesel. The lipids of microbiological origin received in commercial scales will be able to substitute agricultural oils and animal
fats. In order to receive microbiological lipids in industrial scales, it is necessary to have productive strains-producers and relatively cheap non-food substrate. Peat can be one of the kinds of such a substrate [2].
The aim of the work was to research the accumulation of yeast lipids in the process of cultivation of strains P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 with the use of peat as growing substrate.
Materials and Methods
The subject of researches was yeast P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 from "Collection of strains of microorganisms and plant lines for food and agricultural biotechnology" of SE "Institute of Food Biotechnology and Genomics of the National Academy of Sciences of Ukraine".
Inoculation material was received on malt wort with 8.0% dry substances content. In
order to receive inoculation material, the culture was reseeded from canted malt agar, yeast cells in quantity of 0.05 dm3 were carried to 0.25 dm3 volume flask with sterile fermentation medium and cultivated in shaker-incubator BIOSAN ES-20 (Lithuania) with 240 r/min mixing rate at temperature 31 ± 1 °C during 24 hours. Inoculation material in quantity 10% was brought into enzymatic medium.
Lowland and highmoor peat (Dnipro-petrovsk region) in quantity of 50.0 g/dm3 with the addition of nutritional and growth substances pursuant to relevant variants (g/dm3): I — KH2PO4 — 2.0; MgSO4 — 0.12; (NH4)2PO4 — 10.0; yeast extract — 2.0; II — KH2PO4 — 3.0; MgSO4 — 0.18; (NH4)2PO4 — 15.0; yeast extract — 3.0; III — KH2PO4 — 4.0; MgSO4 — 0.24; (NH4)2PO4 — 20.0; yeast extract — 4.0, were used as a resource of carbon in enzymatic medium.
Explosive autohydrolysis [3, 4] and cavitation [5] were used for peat treatment processing. The medium acidity was defined with the help of pH-meter "pH-150M" (Belarus). The yeast was cultivated by deep-seated periodic way on shakers (240 r/min) at temperature 31 ± 1 °C during 72 hours.
The quantity of lipids was defined pursuant to [6]. The quantity of dry biomass was defined by weight method [7]. The concentration of sugar in the medium and cultural liquid was defined by spectrochemical method [8].
All the researches were held in three repetitions, statistical processing of results was held pursuant to [9] with the help of Microsoft Excel program. The difference between two average values was considered to be true at Р < 0.05.
Results and Discussion
Peat is a rather compound substrate for proximate consumption by microorganisms, and that is why it requires treatment (processing) [10]. In order to learn the accessibility of peat pulp macrocomponents for microorganisms without treatment, the strains R. gracilis 1MB Y-5075 and P. anomala 1MB Y-5067 were cultivated on fermentation medium — pulp with lowland and highmoor peat. The cultivation of strains R. gracilis 1MB Y-5075 and P. anomala IMB Y-5067 on the pulp of lowland peat is presented on the Fig. 1.
The Fig. 1 shows that strains P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 have the ability to accumulate reducing substances during cultivation. It is shown that the most accumulation of reducing substances was during the third day of cultivation for both cultures, herewith strain P. anomala IMB Y-5067 accumulated 1.5 g/dm3 of reducing substances and strain R. gracilis 1MB Y-5075 — accumulated 1.6 g/dm3, further cultivation led to decrease of reducing substances levels.
In order to compare accumulation of reducing substances on the pulp of highmoor peat, the cultivation of strains R. gracilis IMB Y-5075 and P. anomala 1MB Y-5067 was held. The results of the research were presented on the Fig. 2.
The Fig. 2 shows that accumulation of reducing substances in cultural liquid over the course of cultivation of strains P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 on fermentation medium with highmoor peat is less than the one over the course of cultivation on breeding environment with lowland peat. The source of energy necessary for life-sustaining activity of yeast-producers of lipids
lis ''ill
I1» mM MM
Fig. 1. Using the pulp of lowland peat for P. anomala 1MB Y-5067 and R. gracilis 1MB Y-5075 cultivation
Hereinafter: as control was a peat without culture
can be a different carbohydrate containing raw material in which the main component is carbonhydrates. Accumulation of reducing substances (monosugars) in fermentative mixture says about the capability of yeast to peat polysaccharides hydrolysis.
In further researches, lowland peat was chosen as fermentation medium, and the cultivation of strains P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 on fermentation medium with lowland peat was held. The received results are presented on the Fig. 3.
The Fig. 3 shows that strains P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 accumulated lipids on the substrate with lowland peat, although the concentration of lipids was less than 2.0 g/dm3. This can be conditioned by the fact that peat consists of inhibitors of microorganisms growth and development — humic acids and humic substances [10].
In order to decrease their influence and improve the accessibility of substrate, preliminary processing of peat pulp by explosive autohydrolysis was held. The received product after explosive autohydrolysis was used as strains cultivation substrate (Fig. 4).
The Fig. 4 shows that after explosive explosive autohydrolysis on the third cultivation day accumulation of reducing substances increased in comparison with accumulation of reducing substances onmedium with unprocessed peat pulp to 2.5 g/dm3 for P. anomala IMB Y-5067 and to 1.5 g/dm3 for R. gracilis IMB Y-5075. Accumulation of lipids and biomass after explosive autohydrolysis of lowland peat pulp on the third cultivation day is shown on the Fig. 5.
The Fig. 5 shows that after explosive autohydrolysis accumulation of biomass by strain R. gracilis IMB Y-5075 in comparison
Fig. 2. Using the pulp of highmoor peat for P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 cultivation
Fig. 3. Lipids and biomass accumulation by stains P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 during
cultivation on non-treatment lowland peat
Hereinafter: * — P < 0.05 as compared to control; in Fig. 3, 5, 7 there is no control, because control is a native peat pulp, where there is no biomass and lipids. For the remaining Figs. control is the amount of reducible pulp
materials (water-soluble sugars)
with accumulation of reducing substances on the medium of unprocessed peat pulp increased from 2.6 to 7.0 g/dm3 and accumulation of lipids increased from 0.5 to 1.0 g/dm3' Accumulation of biomass by strain P. anomala 1MB Y-5067 over the course of cultivation on the medium with lowland peat pulp after explosive autohydrolysis was 2 times higher than the one on the medium with unprocessed pulp. Accumulation of lipids by strain P. anomala 1MB Y-5067 with the use of lowland peat pulp before explosive autohydrolysis and after it was the same (2.0 g/dm3).
In order to raise accumulation of reducing substances in cultivating medium, strains P. anomala 1MB Y-5067 and R. gracilis 1MB Y-5075 were cultivated on medium with a different content of salts, growth factors and peat pulp which went through cavitation treatment (Fig. 6).
You can see from the Fig. 6 that addition of salts and yeast extract influenced over accumulation of reducing substances in e medium depending on the strain. The culture
R. gracilis 1MB Y-5075 (2.2 g/dm3) over the course of using medium pursuant to the variant I accumulated the most quantity of reducing substances. In return, the culture P. anomala 1MB Y-5067 accumulated 1.3 g/dm3 over the course of using medium pursuant to the variant II. Accumulation of lipids by strains P. anomala 1MB Y-5067 and R. gracilis 1MB Y-5075 on medium with a different content of salts, yeast extract and peat pulp which went through cavitation processing was studied (Fig. 7).
After peat cavitation processing and addition of salts and yeast extract accumulation of lipids was intensified on the 72-nd cultivation hour. The most accumulation of lipids over the course of cultivation of P. anomala 1MB Y-5067 (9.7 g/dm3) and R. gracilis 1MB Y-5075 (8.9 g/dm3) was received over the course of using processed pulp of lowland peat by cavitation and addition of salts and yeast extract in concentrations pursuant to the variant III. Over the course of using other concentrations of salts and yeast extract accumulation of lipids was less.
conti'ol K R gracilis * P. anomala Fig. 4. Using of pulp of lowland peat as a substrate after explosive autohydrolysis
10
g
li
P. anómala R. gracilis
¡Lipids : Biomass
Fig. 5. Lipids and biomass accumulation by stains P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 during cultivation on lowland peat after explosive autohydrolysis
Varios of salt concentration
control OR. gracilis *P anomala
Fig. 6. P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 cultivation using different salt concentration
and yeast extract
12
10
bL 8
5 6
5
I +
m
i
¡i
hi
Varios of salt conceutratiou
*P anomala nfi. gracilis
Fig. 7. Lipids accumulation by yeasts P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 using cavitation peat as a medium
Therefore, the results of the research testify about the opportunity of using lowland peat as a resource of carbon for receiving microbic lipids. Using hydro-dynamic cavitation as a way of preliminary processing of water-peat mixture and
addition of salts and yeast extract led to increase of accumulation of lipids by strains P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 more than in 5 times. The conducted researches confirmed one more time that the advantage of the lipids received by means of microbiological synthesis [11-13] is comparatively fast capacity of their content change by means of producer directed cultivation, use of cheap raw material and processability of producer cultivation process.
Thus, accumulation of lipids over the course of cultivation of strains P. anomala IMB Y-5067 and R. gracilis IMB Y-5075 at non-food substrate — lowland peat is shown. The influence of treatment of peat pulp by explosive autohydrolysis and cavitation to accumulation of biomass and lipids by strains P. аnomala IMB Y-5067 and R. gracilis IMB Y-5075 was researched. It is stated that the maximum accumulation of lipids by strains P. аnomala IMB Y-5067 (9.7 g/dm3) and R. gracilis IMB Y-5075 (8.9 g/dm3) was in case of cavitation processing of peat pulp and additional application of salts and yeast extract to cultivation medium.
references
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5. Saranchuk V. I., Ilyashov M. O., Oshovskii V. V., Biiletskii V. S. Fundamentals of Chemistry and Physics of Combustible Minerals. Donetsk: Shid-nyi vydavnychyi dim, 2008, 640p. (In Ukrainian).
6. Baümler E. R. Solvent extraction: kinetic study of major and minor compounds. JAOCS. 2010, V.87, P.1489-1495.
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8. Veshnyakov V.A., Habarov Yu. G, Kamakina N. D. Comparison of methods for the determination of reducing substances: the Bertrand method, ebulliostatic and photometric methods. Khimiya rastitelnogo syrya. 2008, 4, 47-50. (In Russian).
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використання субстрату низинного торфу для культивування штам1в др1ждж1в Pichia anomala imb y-5067 та Rhodotorula gracilis imb y-5075
Т. С. Каландирець1, В. О. Красшько1, Г. С. Андрiяш2, Н. G. Бейко2, О. О. Трунова2, С. М. Шульга2
Нащональний ушверситет харчових технологш, Кшв, Укра1на
2ДУ «1нститут xap40B0Ï бмтехнологп i геномши НАН Украши», Кшв
E-mail: Shulga5@i.ua
Метою роботи було дослщити накопичення дрiжджовиx лшЩв штамами Pichia anomala IMB Y-5067 та Rhodotorula gracilis IMB Y-5075 з використанням торфу як ростового субстрату. Об'ектом дослщжень були штами P. anomala IMB Y-5067 i R. gracilis IMB Y-5075 i3 «Колек-цп штaмiв мiкрооргaнiзмiв та лшш рослин для xaрчовоï i сiльськогосподaрськоï бiотеxнологiï» ДУ «1нститут xaрчовоï бiотеxнологiï та геномь ки НАН Украши». Як сировину використову-вали низинний торф, який попередньо обро-бляли за допомогою кавггацп або вибуxового aвтогiдролiзу. Було показано накопичення ль пiдiв за культивування P. anomala IMB Y-5067 та R. gracilis IMB Y-5075 на неxaрчовому субстрат — низинному торфь Дослщжено вплив вибуxового aвтогiдролiзу пульпи та кав^аць йного оброблення пульпи на накопичення б^-маси i синтез лшЩв штамами P. аnomala IMB Y-5067 та R. gracilis IMB Y-5075. Встановлено, що максимальне накопичення лшЩв штамами P. аnomala IMB Y-5067 (9,7 г/дм3) та R. gracilis IMB Y-5075 (8,9 г/дм3) вщбувалося за кавггаць йного оброблення пульпи торфу i додаткового внесення солей та дрiжджового екстракту в се-редовище культивування.
Ключовi слова: ростовий субстрат, торф, лшь ди дрiжджiв, штами Pichia anomala, Rhodotorula gracilis.
11. Tkachenko A. F., Tigunova E. A., Shulga S. M. Microbial lipids — an alternative source of
raw materials for biofuels. Mikrobiologiya i biotekhnologiya. 2012, V. 3, P. 17-33. (In Russian).
12. Tkachenko A. F., Tigunova E. A., Shulga S. M. Lipids of microorganisms as a source of biofuel. Tsitologiya i genetika. 2013, 6 (47), 22-29. (In Russian).
13. Shulga S. M,, Tkachenko A. F., Beyko N. E., Ho-menko A. I., Andriyash A. S.. Biosynthesis of lipids by yeast Rhodotorula gracilis. "Biotekh-nolohiia". 2010, 3 (3), 58-65. (In Russian).
использование субстрата низового торфа для культивирования штаммов Дрожжей Pichia anomala imb y-5067 и Rhodotorula gracilis imb y-5075
Т. С. Каландирец1, В. О. Красинько1, А. С. Андрияш2, Н. Е. Бейко2, Е. А. Тигунова2, С. М. Шульга2
Национальный университет пищевыга теxнологий, Киев, Украина
2ГО «Институт пищевой биотеxнологии и геномики НАН Украины», Киев
E-mail: Shulga5@i.ua
Целью работы было исследовать накопление липидов штаммами дрожжей Pichia anomala IMB Y-5067 и Rhodotorula gracilis IMB Y-5075 с использованием в качестве ростового субстрата торфа. Объектом исследований были дрожжи P. anomala IMB Y-5067 и R. gracilis IMB Y-5075 из «Коллекции штаммов микроорганизмов и линий растений для пищевой и сельскоxозяй-ственной биотеxнологии» ГО «Институт пищевой биотеxнологии и геномики НАН Украины». В качестве сырья использовали низовой торф, который предварительно обрабатывали с помощью кавитации или взрывного автогидролиза. Было показано накопление липидов при культивировании P. anomala IMB Y-5067 и R. gracilis IMB Y-5075 на непищевом субстрате — низинном торфе. Исследовано влияние взрывного автогидролиза и кавитационной обработки пульпы на накопление биомассы и синтез липидов дрожжами штаммов P. anomala IMB Y-5067 и R. gracilis IMB Y-5075. Установлено, что максимальное накопление липидов P. аnomala IMB Y-5067 (9,7 г/дм3) и R. gracilis IMB Y-5075 (8,9 г/дм3) происxодило при кавитационной обработке торфа и дополнительном внесении солей и дрожжевого экстракта в среду культивирования.
Ключевые слова: ростовой субстрат, торф, липиды дрожжей, штаммы Pichia anomala, Rhodotorula gracilis.
