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103
UDC 577.112:616 https://doi.org/10.15407/biotech10.05.036
BIOBUTANOL ACCUMULATION USING ALTERNATIVE SUBSTRATES BY CULTIVATION OF Clostridium acetobutylicum STRAINS
0. O. Tigunova1 1State organization "Institute of Food Biotechnology H. S. Andriiash1 and Genomics" of the National Academy of Sciences N. Ye. Beiko1 of Ukraine, Kyiv
1. V. Melnyk2 2National University of Food Technologies, Kyiv, Ukraine S. M. Shulga1
E-mail: Shulga5@i.ua
Received 02.07.2017
The aim of the research was to investigate the accumulation of biobutanol by strains Clostridium acetobutylicum using alternative substrates (Jerusalem artichoke juice, technical glycerol, crude glycerol, shredded biomass of soy, rape, wheat and switchgrass). In order to increase the accumulation of butanol in the process of cultivation, the cells of C. acetobutylicum were statically immobilized on carriers (belting strips, ferrite rings and Raschig rings) by the method of adsorption immobilization. The cells were precipitated by centrifugation, the supernatant was distilled and then fermentation products were determined. Gas chromatography was used to determine the presence of solvents in the culture fluid. The biggest accumulation of butanol (2 g/dm3) was at concentration of crude glycerol 16 g/dm3 in the medium, and complete inhibition of culture development — at glycerol concentration 25 g/dm3. The accumulation of butanol by the strain C. acetobutylicum 1MB B-7407 using fill and draw method depended on the amount of sequestered and infused medium. Immobilization of the culture using the Raschig rings allowed increasing the bioconversion to butanol twice. So it is shown the possibility to use non-traditional substrates for the production of biobutanol. The most accumulation of butanol was achieved using glycerol (11 g/dm3) as water-soluble substrate, and using switchgrass as lignocelluloses substrate (2,6 g/dm3). Immobilization of C. acetobutylicum culture cells on carriers increased the accumulation of butanol. The use of Raschig rings, as carriers for immobilization, allowed increasing the accumulation of butanol twice.
Key words: biobutanol, Clostridium acetobutylicum 1MB B-7407, immobilization.
Immobilization of the producer cells is one of the ways to increase the accumulation of the target product in the process of cultivation [1]. Both organic and nonorganic carriers [2] are used to produce immobilized cells. The materials used as carriers should have the following properties: chemical and biological stability, mechanical integrity (primarily resistance to rubbing), ensure the interaction of enzymes of microorganisms with the substrate; significant specific surface, capacity and porosity; the possibility of obtaining technologically convenient forms (granules, membranes, pipes, sheets, etc.); easy transfer to the reaction form (activation); high hydrophilicity, which ensures the possibility of carrying out cytoadherence reactions with carrier in aqueous medium; low cost. The lack of carriers that meet all these requirements at once and the diversity of tasks
stimulate the search of suitable materials for the immobilization of specific producer cells
[2, 3].
The objective of this work was to investigate the accumulation of biobutanol by immobilized cells of strains C. acetobutylicum using alternative substrates.
Materials and Methods
For the research we used: butanol producer strains Clostridium acetobutylicum IMB B-7407 (IFBG C6H), Clostridium sp. IMB B-7570 (IFBG C6H 5M) from the "Collection of microorganisms' strains and plant lines for agricultural and industrial biotechnology", Government Entity "Institute of Food Biotechnology and Genomics of the National Academy of Sciences of Ukraine"; Jerusalem artichoke juice Helianthus tuberosus (Florium,
Ukraine), technical glycerol and crude glycerol (Pharma, Belgium), shredded green biomass of soy Glycine max, rape Brassica napus, wheat Triticum sp. (all from National Research Center "Institute of Mechanization and Electrification of Agriculture" of the National Academy of Sciences of Ukraine) and switchgrass Panicum virgatum L. (Kyiv National Botanical Garden named after M.M. Hryshko). The following medium composition (g/dm3) was used to determine the accumulation of butanol while using glycerol: crude glycerol (from 10.0 to 20.0), yeast extract — 1.0; (NH4)2SO4 — 0.6; (NH4)2HPO4 — 1.6; pH 6.5. The medium was sterilized for 30 min and at pressure of 1 atm. To determine the accumulation of butanol while Jerusalem artichoke juice (Helianthus tuberosus) the root tubers were cleaned, peeled, grinded and juiced. The juice was diluted to 19.6% of dry matter and added to (g/dm3) (NH4)2SO4 — 0.6; (NH4)2HPO4 — 1.6; the pH was adjusted to 6.68.
Vynohradski medium and slices of chalk rubbed potatoes [4] were used as activation medium. The cultivation of Clostridia was carried out according to the procedure [4] in the anaerobic jar "AE 01" (RF) in nitrogen atmosphere. Anaerobic jar was placed in the thermostat at the temperature of 35 ± 1 °C.
Biomass of switchgrass, soy, rape, wheat was dried at the temperature of 30±1 °C during 48 h. The dried biomass (7% humidity) was shredded up to 200 mesh with the help of mill "Tsyklon MSH 1" (Ukraine). The moisture was determined by moisture analyzer RADWAG MA 50/C/1 (Poland). Lignin content was determined according to the procedure [4].
To immobilize cells the following carriers were used: belting strips (Promfiltr, Ukraine) with a surface area of 35.4 cm2, ferrite rings (Epcos, FRG) — 2.1 cm2, and Rushig rings (Antey, RF) — 0.5 cm2 (Fig. 1).
Fig. 1. Carriers for immobilization (from up to down):
belting strips, ferrite rings, Rushig rings
Immobilization of cells on carriers was carried out for 12 h by static method of adsorption immobilization. Cultivation of immobilized culture cells on carriers was carried out according to the procedure [4]. After 5 days cultivation was stopped, the cells were precipitated using an ultracentrifuge "Labofuge 400R" (Germany), the supernatant was distilled at 100 °C with a refrigerator until there is no output volume (100 cm3) in the receiving flask and the fermentation products were determined.
Presence of ethanol, acetone and butanol in culture liquid was determined using gas chromatograph ("Kristall-5000 lux", RF) with flame-ionization detector and packed column 3 m in length, phase Carbowax 1500 on chromaton N-A-W-DMSC (0.20-0.25 mm). The column temperature was 60 ± 2 °C, the evaporator' temperature was 160 ± 5 °C, nitrogen-hydrogen-air ratio was 1:1:10 [5].
Statistical data analysis was performed using Microsoft Excel program. All experiments were done in three replicates. The difference between two averages was considered probable at Р < 0.05 (these results are marked with *). In addition to Fig. 1-5, 7, the enzymatic medium without seed was as control (zero value), and in Fig. 6 for control was inimmobilized cells, so the control and rendered on the drawing.
Results and Discussions
In the process of biodiesel producing from renewable biomass, glycerol is produced in large quantities as a by-product of vegetable oils during esterification. Purification of glycerol is quite expensive energy-consuming process, although refined glycerol is widely used in pharmacology, cosmetology, food and other industries. To reduce the cost of microbial synthesis and increase the profitability of bio-butanol technology, the crude glycerol was used as a substrate — an unpurified waste of biodiesel production. Investigation of butanol accumulation by the strain C. acetobutylicum IMB-7407 was carried out at different concentrations of crude glycerol (Fig. 2).
From the results shown in Fig. 2 it is evident, that bioconversion to butanol takes place when crude glycerol concentration is in range from 10 to 20 g/dm3. The most accumulation of butanol (2 g/dm3) took place when crude glycerol concentration in medium was equal to 16 g/dm3, and complete inhibition of culture development at a concentration of 25 g/dm3.
Fig. 2. Butanol accumulation on different concentration of crude glycerol
Note: in addition to Figure 6, the fermentation (enzymatic) medium without seed was as control, and in Figure No. 6 for control (zero value) was inimmobilized cells, so the control and rendered on the drawing.
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0 1 2 3 4 5 6 Numbersof sequestered and infused of medium Fig. 3. Accumulation of butanol in strain C. acetobutylicum IMB B-7407 using over-fill method
In order to increase the accumulation of butanol at the minimum concentration of crude glycerol in the medium, we cultivated C. acetobutylicum IMB B-7407 by fill and draw method. Each period of sequestered and infused occurred after 96 h. The results are shown in (Fig. 3).
Accumulation of butanol in the culture liquid by strain C. acetobutylicum IMB B-7407 did not change in the process of cultivating during the first period of medium sequestering and infusing and from the second up to the fourth period the accumulation of butanol was reduced by half. The subsequent use of fill and draw method lead to decrease of butanol accumulation by 8 times and final stop in the sixth period [6-8].
To increase the accumulation of butanol by stain C. acetobutylicum IMB B-7407 using crude glycerol as a substrate, we carried out
immobilization of cell cultures on different carriers, which had high adhesion activity relative to the culture. After immobilization, biomass of cells was attached to all carriers, however, according to cytological studies the morphology of the culture has changed (Fig. 4).
Figure 4 shows that cells of immobilized culture were attached to the carriers and formed long chains of about 20 cells. The initial non-immobilized culture did not form such chains [4]. To determine the number of cells on the carrier, we carried out washing of the carriers and drying the resulting microbial biomass to a completely dry weight (CDW). The obtained results are presented in Fig. 5.
Figure 5 shows that the largest number of cells was absorbed on the fibers of the belting strips. With the use of fabrics, the
CDW of strain C. atsotobutylicum IMB-7407 exceeded other carriers by almost five times. The previous studies [4] showed that technical glycerol was the best source of carbohydrate for the culture, therefore cultivation of immobilized cells was carried out using technical glycerol as a substrate and accumulation of butanol was determined. The results obtained are shown in Fig. 6.
Figure 6 shows that the accumulation of butanol has increased due to the use of immobilized cultures on Raschig rings and belting strips. Therefore, culture immobilized on ferrite rings lost its properties to the synthesis of solvents and accumulated only acids. According to the results of the study, we can conclude that the immobilization of stain C. acetobutylicum IMB B-7407 on the Raschig rings has allowed to double the accumulation of butanol.
Jerusalem artichoke juice [9] may be one of the promising substrates for obtaining biofuels. A study was conducted with the use of Jerusalem artichoke juice as a substrate for cultivating strains C. acetobutylicum IMB B-7407 and Clostridium sp. IMV B-7570. The results obtained are shown in Fig. 7.
Fig. 7 shows that the accumulation of butanol in the culture liquid of stain C. ace-tobutylicum IMB-7407 was 3.5 g/dm3, ethanol was 0.2 g/dm3, acetone was not produced. Alternatively, the strain Clostridium sp. IMB B-7570 almost did not convert the Jerusalem artichoke juice into alcohol.
We performed cultivation of strain Clostridium sp. 1MB B-7570 using different lignocellulosic substrates — biomass of soy, rape, switchgrass and wheat, and determined accumulation of butanol. The results are presented in Fig. 8.
Fig. 4. Microphoto (increase 900) of immobilized culture (colored brawn) on belting strips
Rushig ring* limiting strips fertile rings
Camel's for immobilisation
Fig. 5. Concentration of biomass of C. atsotobutylicum IMB-7407 strain on different carriers
Carriers for im mobilization
Fig. 6. Accumulation of butanol by immobilized C. acetobutylicum IMB B-7407 stain on different carriers
I Ethanol ■ butanol ■ acetone Fig. 7. Accumulation of butanol using Jerusalem artichoke juice as substrate
I acetone ■ butanol ■ cthano! Fig. 8. Accumulation of butanol on different substrates
Fig. 8 shows that the biggest accumulation of butanol (2.6 g/dm3) was when using switchgrass biomass as a substrate and the smallest when using wheat (0.2 g/dm3).
Studies have shown that non-traditional substrates (shredded biomass of soy, rapeseed, switchgrass, wheat; Jerusalem artichoke juice, technical glycerol, crude glycerol) are converted into biobutanol. While using the fill and draw method the accumulation of butanol by the culture C. acetobutylicum IMB B-7407 on crude
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glycerol did not change during the first period of fill and draw of the medium, but during the second it decreased. Immobilization of cells C. acetobutylicum IMB B-7407 on Raschig rings, belting rings and ferrite rings as carriers showed that all carriers had high adhesion activity relative to culture. It was determined that accumulation of butanol doubled when using Raschig rings as a carrier for the immobilization of cells C. acetobutylicum of IMB B-7407.
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7. Xin F., Chen T., Jiang Y., Lu J., Dong W, Zhang W., Ma J., Zhang M, Jiang M. Enhanced biobutanol production with high yield from crude glycerol by acetone uncoupled Clostridium sp. strain CT7. Biores. Technol. 2017, 244(1), 9-11. doi: 10.1016/j. biortech.2017.08.002.
8. Liu D, Chen Y, Li A., Ding F, Zhou T, He Y., Li B., Niu H., Lin X., Xie J., Chen X., Wu J., Ying H. Enhanced butanol production by modulation of electron flow in Clostridium acetobutylicum B3 immobilized by surface adsorption. Biores. Technol. 2013, 25(2), 322232. doi: 10.1016/j.biortech.2012.11.090.
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ВИКОРИСТАННЯ АЛЬТЕРНАТИВНИХ
СУБСТРАТ1В ДЛЯ НАКОПИЧЕННЯ Б1ОБУТАНОЛУ ЗА КУЛЬТИВУВАННЯ ШТАМ1В Clostridium acetobutylicum
О. О. ТЬгунова1, Г. С. АндрЬяш1, Н. Е. Бейко1, I. В. Мельник2, С. М. Шульга1
1ДУ «1нститут xap40B0Ï бiотехнологiï
та геномши» НАН Укра1ни, Киïв 2Нацiональний унiверситет харчових технологш, Киïв
E-mail: Shulga5@i.ua
Метою роботи було досл^ити накопи-чення бшбутанолу штамами Clostridium acetobutylicum з використанням альтернатив-них субстратiв (соку топшамбуру, технiчного глiцеролу, глiцеролу сирцю, подрiбненоï зе-леноï бiомаси œï, рiпаку, дротоподiбного проса, пшенищ). Для пiдвищення накопичення бутанолу в процем культивування проводили iммобiлiзацiю клггин С. acetobutylicum на но-мях (смужках бельтингу, феритових кiльцях та шльцях Рашига) статичним способом — методом адсорбцiйноï iммобiлiзацiï. Клiтини осаджували за допомогою центрифугування, супернатант переганяли та визначали продук-ти бродшня. Наявнiсть розчинникiв у культу-ральнiй рiдинi визначали за допомогою газо-воï хроматографiï. Максимальне накопичення бутанолу (2 г/дм3) спостериали за концентра-цiï у середовищi глщеролу-сирцю 16 г/дм3, а повне шпбування розвитку культури — за 25 г/дм3. Накопичення бутанолу штамом C. acetobutylicum 1MB В-7407 за культивування вщ'емно-доливним методом залежало вщ кiлькостi вилучень та доливань середовища. Iммобiлiзацiя культури з використанням кь лець Рашига дала змогу шдвищити б^конвер-сiю до бутанолу в 2 рази. Таким чином, було показано можлив^ть використання нетради-цшних субстратiв для отримання б^бутано-лу. Найб^ьшого накопичення було досягнено за використання глщеролу (11 г/дм3) як во-дорозчинного субстрату та линоцелюлозно-го — дротоподiбного проса(2,6 г/дм3). 1ммобь лiзацiя клiтин культури С. acetobutylicum на ноиях пiдвищувала накопичення бутанолу. Використання шлець Рашига як носив для iммобiлiзацiï дало змогу шдвищити накопи-чення бутанолу в 2 рази.
Ключовi слова: бмбутанол, Clostridium acetobutylicum 1MB B-7407, iммобiлiзацiя.
ИСПОЛЬЗОВАНИЕ АЛЬТЕРНАТИВНЫХ
СУБСТРАТОВ ДЛЯ НАКОПЛЕНИЯ БИОБУТАНОЛА ПРИ КУЛЬТИВИРОВАНИИ ШТАММОВ Clostridium acetobutylicum
Е. А. Тигунова1, А. С. Андрияш1, Н. Е. Бейко1, И. В. Мельник2, С. М. Шульга1
1«Институт пищевой биотехнологии и геномики» НАН Украины, Киев 2Национальный университет пищевых технологий, Киев
E-mail: Shulga5@i.ua
Целью работы было исследовать накопление биобутанола штаммами Clostridium acetobutylicum с использованием альтернативных субстратов (сока топинамбура, технического глицерола, глицерола-сырца, измельченной зелёной биомасы сои, рапса, прутевидного проса, пшеницы). Для повышения накопления бутанола в процессе культивирования проводили иммобилизацию клеток С. acetobutylicum на носителях (полосках бель-тинга, ферритовых кольцах, кольцах Рашига) статическим способом — методом адсорбционной иммобилизации. Клетки осаждали с помощью центрифугирования, супернатант перегоняли и определяли продукты брожения. Наличие растворителей в культураль-ной жидкости определяли с помощью газовой хроматографии. Максимальное накопление бутанола (2 г/дм3) наблюдали при концентрации в среде глицерола-сырца 16 г/дм3, а полное ингибирование развития культуры — при 25 г/дм3. Накопление бутанола штаммом C. acetobutylicum 1MB В-7407 при культивировании отъемно-доливным методом зависит от количества отъема и долива среды. Иммобилизация культуры с использованием колец Рашига дала возможность повысить биоконверсию до бутанола в 2 раза. Таким образом, для получения бутанола была показана возможность использования нетрадиционных субстратов. Наибольшее накопление бутанола было достигнуто при использовании глицеро-ла (11 г/дм3) как водорастворимого субстрата и как лигноцеллюлозного — прутевидно-го проса (2,6 г/дм3). Иммобилизация клеток культуры С. acetobutylicum на носителях повышала накопление бутанола. Использование колец Рашига в качестве носителя для иммобилизации позволило повысить накопление бутанола в 2 раза.
Ключевые слова: биобутанол, Clostridium acetobutylicum 1MB В-7407, иммобилизация.
