Альтернативный вид топлива – биобутанол
Бутанол – альтернативное топливо. На фоне истощающихся мировых (доступных) запасов нефти рассматривается как потенциальный источник энергии. В промышленных масштабах бутанол получают химическим синтезом, хотя первоначально производство бутанола было связано с микробиологическим синтезом. Для экономи...
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Інститут клітинної біології та генетичної інженерії НАН України
2013
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| Zitieren: | Альтернативный вид топлива – биобутанол / Е.А. Тигунова, С.М. Шульга, Я.Б. Блюм // Цитология и генетика. — 2013. — Т. 47, № 6. — С. 51-71. — Бібліогр.: 118 назв. — рос. |
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irk-123456789-1266032017-11-29T03:02:40Z Альтернативный вид топлива – биобутанол Тигунова, Е.А. Шульга, С.М. Блюм, Я.Б. Оригинальные работы Бутанол – альтернативное топливо. На фоне истощающихся мировых (доступных) запасов нефти рассматривается как потенциальный источник энергии. В промышленных масштабах бутанол получают химическим синтезом, хотя первоначально производство бутанола было связано с микробиологическим синтезом. Для экономически выгодного производства штаммы микроорганизмов должны обладать способностью сверхсинтеза бутанола. В обзоре рассмотрены пути синтеза бутанола микроорганизмами и их регуляция, наиболее перспективные штаммы-продуценты для промышленного производства и методы повышения их продуктивности. Бутанол – альтернативне паливо, на тлі світових (доступних) запасів нафти, які виснажуються, є потенційним джерелом енергії. У промислових масштабах бутанол одержують хімічним синтезом, хоча спочатку виробництво бутанолу було пов’язано з мікробіологічним синтезом. Для економічно вигідного виробництва штами мікроорганізмів повинні мати здатність до надсинтезу бутанолу. В огляді розглянуто шляхи синтезу бутанолу мікроорганізмами і їх регуляція, найбільш перспективні штами-продуценти для промислового виробництва та методи підвищення продуктивності штамів. Butanol is an alternative type of fuel. Amid the depletion of the world’s (accessible) petroleum reserves, butanol is considered to be a potential energy source. Although butanol production was initially associated with microbiological synthesis, on an industrial scale, butanol is produced via chemical synthesis. For its production to become economically viable, strains of microorganisms must have a potential for excessive synthesis of butanol. The pathways of butanol synthesis via microorganisms and their regulation, as well as the most promising producer strains for industrial production and methods for increasing their productivity, are discussed in the survey. 2013 Article Альтернативный вид топлива – биобутанол / Е.А. Тигунова, С.М. Шульга, Я.Б. Блюм // Цитология и генетика. — 2013. — Т. 47, № 6. — С. 51-71. — Бібліогр.: 118 назв. — рос. 0564-3783 DOI: 10.3103/S0095452713060042 http://dspace.nbuv.gov.ua/handle/123456789/126603 579.222 ru Цитология и генетика Інститут клітинної біології та генетичної інженерії НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
Russian |
| topic |
Оригинальные работы Оригинальные работы |
| spellingShingle |
Оригинальные работы Оригинальные работы Тигунова, Е.А. Шульга, С.М. Блюм, Я.Б. Альтернативный вид топлива – биобутанол Цитология и генетика |
| description |
Бутанол – альтернативное топливо. На фоне истощающихся мировых (доступных) запасов нефти рассматривается как потенциальный источник энергии. В промышленных масштабах бутанол получают химическим синтезом, хотя первоначально производство бутанола было связано с микробиологическим синтезом. Для экономически выгодного производства штаммы микроорганизмов должны обладать способностью сверхсинтеза бутанола. В обзоре рассмотрены пути синтеза бутанола микроорганизмами и их регуляция, наиболее перспективные штаммы-продуценты для промышленного производства и методы повышения их продуктивности. |
| format |
Article |
| author |
Тигунова, Е.А. Шульга, С.М. Блюм, Я.Б. |
| author_facet |
Тигунова, Е.А. Шульга, С.М. Блюм, Я.Б. |
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Тигунова, Е.А. |
| title |
Альтернативный вид топлива – биобутанол |
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Альтернативный вид топлива – биобутанол |
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Альтернативный вид топлива – биобутанол |
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Альтернативный вид топлива – биобутанол |
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Альтернативный вид топлива – биобутанол |
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альтернативный вид топлива – биобутанол |
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Інститут клітинної біології та генетичної інженерії НАН України |
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2013 |
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Оригинальные работы |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/126603 |
| citation_txt |
Альтернативный вид топлива – биобутанол / Е.А. Тигунова, С.М. Шульга, Я.Б. Блюм // Цитология и генетика. — 2013. — Т. 47, № 6. — С. 51-71. — Бібліогр.: 118 назв. — рос. |
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Цитология и генетика |
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AT tigunovaea alʹternativnyjvidtoplivabiobutanol AT šulʹgasm alʹternativnyjvidtoplivabiobutanol AT blûmâb alʹternativnyjvidtoplivabiobutanol |
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2025-07-09T05:21:05Z |
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2025-07-09T05:21:05Z |
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| fulltext |
51ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Áóòàíîë – àëüòåðíàòèâíîå òîïëèâî. Íà ôîíå èñòî-
ùàþùèõñÿ ìèðîâûõ (äîñòóïíûõ) çàïàñîâ íåôòè ðàñ-
ñìàòðèâàåòñÿ êàê ïîòåíöèàëüíûé èñòî÷íèê ýíåðãèè.
 ïðîìûøëåííûõ ìàñøòàáàõ áóòàíîë ïîëó÷àþò õè-
ìè÷åñêèì ñèíòåçîì, õîòÿ ïåðâîíà÷àëüíî ïðîèçâîä-
ñòâî áóòàíîëà áûëî ñâÿçàíî ñ ìèêðîáèîëîãè÷åñêèì
ñèíòåçîì. Äëÿ ýêîíîìè÷åñêè âûãîäíîãî ïðîèçâîäñòâà
øòàììû ìèêðîîðãàíèçìîâ äîëæíû îáëàäàòü ñïîñî-
áíîñòüþ ñâåðõñèíòåçà áóòàíîëà. Â îáçîðå ðàññìîòðåíû
ïóòè ñèíòåçà áóòàíîëà ìèêðîîðãàíèçìàìè è èõ ðåãó-
ëÿöèÿ, íàèáîëåå ïåðñïåêòèâíûå øòàììû-ïðîäóöåíòû
äëÿ ïðîìûøëåííîãî ïðîèçâîäñòâà è ìåòîäû ïîâûøåíèÿ
èõ ïðîäóêòèâíîñòè.
Введение
 ïîñëåäíèå ãîäû âíèìàíèå èññëåäîâàòåëåé
âñå áîëüøå àêöåíòèðóåòñÿ íà ïðîèçâîäñòâå
æèäêèõ îðãàíè÷åñêèõ ïðîäóêòîâ è òîïëèâà, â
ïåðâóþ î÷åðåäü ýòàíîëà è áóòàíîëà, èç âîçîá-
íîâëÿåìûõ èñòî÷íèêîâ ñûðüÿ íà îñíîâå áèî-
ìàññû.
Ïðîìûøëåííîå ïðîèçâîäñòâî áóòàíîëà â
íà÷àëå ÕÕ âåêà áûëî îñíîâàíî íà ôåðìåí-
òàöèè óãëåâîäíîãî ñûðüÿ (êóêóðóçíîé ìóêè)
áàêòåðèÿìè Clostridium acetobutylicum. Óâåëè÷å-
íèå ñïðîñà íà áóòàíîë è ðåçêèé ðîñò íåôòåõè-
ìè÷åñêîãî ïðîèçâîäñòâà ïðèâåëè ê òîìó, ÷òî
áèîòåõíîëîãè÷åñêèé ïðîöåññ ïîëó÷åíèÿ áóòà-
íîëà îêàçàëñÿ äîðîãîñòîÿùèì è áûë çàìåíåí
áîëåå ýôôåêòèâíûì õèìè÷åñêèì ñèíòåçîì. Â
ïîñëåäíèå ãîäû èç-çà ãëîáàëüíîãî ïîòåïëåíèÿ
è çàãðÿçíåíèÿ àòìîñôåðû, à òàêæå ïîâûøåíèÿ
öåí íà íåôòü âîçîáíîâèëñÿ èíòåðåñ ê ìèêðî-
áèîëîãè÷åñêîìó ïðîöåññó ïîëó÷åíèÿ áóòàíîëà
íå òîëüêî â êà÷åñòâå ñûðüÿ äëÿ õèìè÷åñêîé
ïðîìûøëåííîñòè, íî è êàê àëüòåðíàòèâíîãî
òîïëèâà.
Îäíèì èç ïîäõîäîâ ê ïðîâåäåíèþ íåïðå-
ðûâíîé áèîêîíâåðñèè áèîìàññû, ÿâëÿåòñÿ èí-
òåãðèðîâàíèå ñòàäèè âûäåëåíèÿ ñïèðòà ñ ïðî-
öåññîì ôåðìåíòàöèè – òàê íàçûâàåìàÿ ýêñ-
òðàêòèâíàÿ ôåðìåíòàöèÿ.
Ïðè àöåòîí-áóòàíîë-ýòàíîëüíîé (ÀÁÝ) ôåð-
ìåíòàöèè (íàçâàíèå ïî îñíîâíûì ïðîäóêòàì
áðîæåíèÿ â ïðîèçâîäñòâå áóòèëîâîãî ñïèðòà)
áàêòåðèè C. acetobutylicum íà ïåðâîì ýòàïå âû-
ðàáàòûâàþò ìàñëÿíóþ, ïðîïèîíîâóþ, ìîëî÷-
íóþ è óêñóñíóþ êèñëîòû (ñòàäèÿ ïðîèçâîäñòâà
êèñëîò), çàòåì âîäîðîäíûé ïîêàçàòåëü ñíèæà-
åòñÿ è íà÷èíàåòñÿ ñòàäèÿ ñèíòåçà ðàñòâîðèòå-
ëåé áóòàíîëà, àöåòîíà, ýòàíîëà è èçîïðîïàíîëà.
Ýòà ñòàäèÿ èíèöèèðóåòñÿ ïîâûøåíèåì êîí-
öåíòðàöèè ìàñëÿíîé êèñëîòû (áîëüøå 2 ã/ë)
è ñíèæåíèåì âîäîðîäíîãî ïîêàçàòåëÿ ðÍ < 5.
Ïðè èñïîëüçîâàíèè îáû÷íîé ôåðìåíòàöèè
ÀÁÝ âûõîä áóòàíîëà èç ãëþêîçû îòíîñèòåëü-
íî íèçêèé è ñîñòàâëÿåò ïðèáëèçèòåëüíî 15 %,
ðåäêî ïðåâûøàåò 25 %. Ïðîèçâîäñòâî áóòàíî-
ëà ëèìèòèðóåòñÿ òåì, ÷òî ïðè êîíöåíòðàöèè
áóòàíîëà 1–2 % îùóòèìî áëîêèðóåòñÿ ðîñò
ìèêðîîðãàíèçìîâ, ÷òî ïðèâîäèò ê îñòàíîâêå
ôåðìåíòàöèè. Ïî ýòîé ïðè÷èíå êîíöåíòðàöèÿ
áóòàíîëà ïðè îáû÷íîì ïðîöåññå ÀÁÝ íå ïðå-
âûøàåò 1,5 % îáùåãî îáúåìà ïðè ïðîäóêòèâ-
íîñòè 4,5 ã/ë·÷–1 è âûõîä ñîñòàâëÿåò ìåíüøå
25 % îò ìàññû ãëþêîçû [1].
Ïèðîã è äð. [2], èñïîëüçîâàâ ðÿä èíæåíåð-
íûõ ðåøåíèé, àíîíñèðîâàëè ñîçäàíèå ïîëíî-
öåííîé ïðîìûøëåííîé òåõíîëîãèè ïîëó÷åíèÿ
áèîáóòàíîëà. Â ïðîöåññå ôåðìåíòàöèè èñïîëü-
çîâàëè ñäâîåííûå áèîðåàêòîðû íåïðåðûâíîãî
äåéñòâèÿ ñ èììîáèëèçèðîâàííûìè ìèêðîîð-
ãàíèçìàìè, ïðîâåäåíà îïòèìèçàöèÿ ïðîöåññà
ôåðìåíòàöèè ÀÁÝ, ïðè ýòîì ñèíòåç áóòàíîëà
ïðîõîäèë â äâà ýòàïà. Íà ïåðâîì ýòàïå ñèí-
òåçèðîâàëàñü ìàñëÿíàÿ êèñëîòà, íà âòîðîì –
áóòàíîë. Òàêîé ïðîöåññ ïîçâîëèë çíà÷èòåëüíî
óâåëè÷èòü âûõîä, ñêîðîñòü íàêîïëåíèÿ è êîí-
öåíòðàöèþ áóòàíîëà.
Ïî ñðàâíåíèþ ñ îáû÷íûì ïðîöåññîì ÀÁÝ
íîâàÿ òåõíîëîãèÿ èñêëþ÷àëà ïðîèçâîäñòâî íå-
æåëàòåëüíûõ ïðîäóêòîâ (óêñóñíàÿ, ìîëî÷íàÿ è
ïðîïèîíîâàÿ êèñëîòû, àöåòîí, èçîïðîïàíîë,
ýòàíîë).  òàêîì ïðîöåññå íà âûõîäå ïîëó÷àëè
ìàñëÿíóþ êèñëîòó, áóòàíîë, óãëåêèñëûé ãàç è
âîäîðîä. Íåîáõîäèìî îòìåòèòü, ÷òî íîâàÿ òåõ-
УДК 579.222
Е.А. ТИГУНОВА, С.М. ШУЛЬГА, Я.Б. БЛЮМ
ГУ «Институт пищевой биотехнологии и геномики» НАН Украины, Киев
E-mail: Shulga5@i.ua
АЛЬТЕРНАТИВНЫЙ ВИД ТОПЛИВА – БИОБУТАНОЛ
© Å.À. ÒÈÃÓÍÎÂÀ, Ñ.Ì. ØÓËÜÃÀ, ß.Á. ÁËÞÌ, 2013
52 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Å.À. Òèãóíîâà, Ñ.Ì. Øóëüãà, ß.Á. Áëþì
íîëîãèÿ ïîçâîëÿëà íàðÿäó ñ îñíîâíûì ïðîäóê-
òîì ïîëó÷àòü åùå îäèí âèä àëüòåðíàòèâíîãî
òîïëèâà – âîäîðîä.
Ïðîâåäåíèå ïðîöåññà ñ ïîìîùüþ òàêîé òåõ-
íîëîãèè ïîçâîëèëî ìèíèìèçèðîâàòü èíãèáè-
ðóþùåå äåéñòâèå ñïèðòîâ íà ìèêðîîðãàíèçìû,
óâåëè÷èòü ïðîäóêòèâíîñòü ðåàêòîðà è óìåíü-
øèòü ýíåðãîçàòðàòû íà êîíöåíòðèðîâàíèå è
âûäåëåíèå öåëåâîãî ïðîäóêòà.
1. Физические и химические характеристики
Áóòàíîë – íîðìàëüíûé áóòèëîâûé ñïèðò
(n-áóòàíîë, ìåòèëîëïðîïàí), áåñöâåòíàÿ æèä-
êîñòü ñ îò÷åòëèâûì ñèâóøíûì çàïàõîì, ïðåä-
ñòàâëÿåò ñîáîé ëèíåéíûé ÷åòûðåõêàðáîíîâûé
àëèôàòè÷åñêèé ñïèðò ñ ìîëåêóëÿðíîé ôîðìóëîé
C4H9OH èëè ÑÍ3(ÑÍ2)3ÎÍ. Ñóùåñòâóþò èçîìå-
ðû áóòàíîëà – èçîáóòàíîë, âòîðáóòàíîë è òðåò-
áóòàíîë. Áóòàíîë ñìåøèâàåòñÿ ñ îðãàíè÷åñêè-
ìè ðàñòâîðèòåëÿìè è ÷àñòè÷íî ñ âîäîé, îáðàçóÿ
àçåîòðîï. Òîêñè÷íîñòü áóòàíîëà îòíîñèòåëüíî
íåâåëèêà, LD50 = 2290–4360 ìã/êã, õîòÿ è ÿâ-
ëÿåòñÿ íàèâûñøåé ñðåäè ìëàäøèõ ñïèðòîâ [3–
15]. Îñíîâíûå õàðàêòåðèñòèêè áóòàíîëà ïðåä-
ñòàâëåíû â òàáë. 1.
Áóòàíîë èìååò ðÿä ïðåèìóùåñòâ ïî ñðàâíå-
íèþ ñ ýòàíîëîì, êîòîðûé â íàñòîÿùåå âðåìÿ
èñïîëüçóåòñÿ êàê îñíîâíîå áèîòîïëèâî, è îá-
ëàäàåò áîëåå âûñîêîé ýíåðãîåìêîñòüþ, ãèäðî-
ôîáíîñòüþ è ñðàâíèòåëüíî íåáîëüøîé êîððî-
çèîííîé àêòèâíîñòüþ.
 ïðîìûøëåííûõ ìàñøòàáàõ áóòàíîë ïîëó-
÷àþò èç ïðîïèëåíà èëè àöåòàëüäåãèäà ñ ïîìî-
ùüþ ðàçëè÷íûõ ñõåì õèìè÷åñêîãî ñèíòåçà –
ñõåìà Îêñî, ñõåìà Ðåïïå è êðîòîíèëäåãèäðî
ãèäðîãåíàöèåé (ðèñ.1).
Òàáëèöà 1. Îñíîâíûå õàðàêòåðèñòèêè áóòàíîëà [16]
*LogP ÿâëÿåòñÿ ìåðîé ãèäðîôîáíîñòè (ëèïîôèëüíîñòè) è ïîõîæ íà ïîëÿðíîñòü.
Ñâîéñòâà Áóòàíîë Õèìè÷åñêàÿ ñòðóêòóðà
Òåìïåðàòóðà ïëàâëåíèÿ, ºÑ
Ìîëÿðíàÿ ìàññà, ã/ìîëü
Òåìïåðàòóðà âîñïëàìåíåíèÿ, ºÑ
Òåìïåðàòóðà ñàìîâîñïëàìåíåíèÿ, ºÑ
Òåìïåðàòóðà âñïûøêè, ºÑ
Îòíîñèòåëüíàÿ ïëîòíîñòü (âîäà : 1,0)
Êðèòè÷åñêîå äàâëåíèå (ãÏà)
Êðèòè÷åñêàÿ òåìïåðàòóðà, ºÑ
Ïðåäåëû âçðûâàåìîñòè, % â âîçäóõå
Ðàñòâîðèìîñòü â âîäå
Îòíîñèòåëüíàÿ ïëîòíîñòü ïàðà (âîçäóõ : 1,0)
Äàâëåíèå ïàðîâ (êÏà ïðè 20 ºÑ)
Òî÷êà êèïåíèÿ, ºÑ
Ïëîòíîñòü ïðè 20 ºÑ, ã/ìë
Ðàñòâîðèìîñòü â 100 ã âîäû
Ïëîòíîñòü ýíåðãèè, ÌÄæ·ë–1
Ñîäåðæàíèå ýíåðãèè/çíà÷åíèå (ÁÒÅ/ãàëëîí)
Ñîîòíîøåíèå âîçäóõ–òîïëèâî
Òåïëîòà ïàðîîáðàçîâàíèÿ (ÌÄæ/êã)
Æèäêèå òåïëîåìêîñòè (Ñð) íà ÑÒÏ, êÄæ/êìîëü ºK
Òåîðåòè÷åñêîå îêòàíîâîå ÷èñëî
Ìîòîðíîå îêòàíîâîå ÷èñëî
Êîýôôèöèåíò ðàñïðåäåëåíèÿ îêòàíîë/âîäà (êàê
logPo/â)*
Äèïîëüíûé ìîìåíò (ïîëÿðíîñòü)
Âÿçêîñòü, 10–3 Ïà
–89,3
74,12
35–37
343–345
25–29
0,81
48,4
287
1,4–11,3
9,0 ìë íà 100 ìë
(7,7 ã/100 ìë ïðè 20 ºÑ)
2,6
0,58
Áóòàíîë
117–118
0,8098
–
27–29,2
110000
11,2
0,43
178
96
78
0,88
1,66
2,593
Áåíçèí
27–221
0,7–0,8
–
32
115000
14,6
0,36
160-300
91–99
81–89
3,52 ± 0,62
–
0,24–0,32
Ýòàíîë
78
0,7851
+
19,6
84000
9
0,92
112,3
129
102
–0,31
1,7
1,078
Ìåòàíîë
64,7
0,7866
+
16
76000
6,5
1,2
81,14
136
104
–0,77
1,6
0,5445
53ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Àëüòåðíàòèâíûé âèä òîïëèâà – áèîáóòàíîë
 Îêñî-ñèíòåçå (ãèäðîôîðìèëèðîâàíèå),
ìîíîîêñèä óãëåðîäà è âîäîðîä äîáàâëÿþòñÿ ê
äâîéíîé Ñ=Ñ ñâÿçè ñ èñïîëüçîâàíèåì êàòàëè-
çàòîðîâ, òàêèõ êàê Co, Rh èëè Ru. Â çàâèñè-
ìîñòè îò óñëîâèé ðåàêöèè (äàâëåíèÿ, òåìïå-
ðàòóðû), à òàêæå òèïà êàòàëèçàòîðà ïîëó÷àþò
ðàçëè÷íûå èçîìåðíûå ñîîòíîøåíèÿ áóòàíîëà.
Ïðîöåññ Ðåïïå – ïðîèçâîäñòâî áóòàíî-
ëà ïðè íèçêîé òåìïåðàòóðå è äàâëåíèè. Ýòîò
ïðîöåññ íå áûë êîììåð÷åñêè óñïåøíûì, ñêî-
ðåå âñåãî, èç-çà äîðîãîñòîÿùåé òåõíîëîãèè. Äî
íåäàâíåãî âðåìåíè ñàìûì ðàñïðîñòðàíåííûì
íàïðàâëåíèåì äëÿ ñèíòåçà áóòàíîëà áûëî èñ-
ïîëüçîâàíèå êðîòîíàëüäåãèä ãèäðîãåíèçàöèè
àöåòàëüäåãèäà. Ïðîöåññ ñîñòîèò èç àëüäîëü-
íîé êîíäåíñàöèè, îáåçâîæèâàíèÿ è ãèäðîãå-
íèçàöèè. Â íàñòîÿùåå âðåìÿ îí èñïîëüçóåòñÿ
âñå ðåæå, õîòÿ â áóäóùåì ìîæåò âåðíóòü ñåáå
óòðà÷åííûå ïîçèöèè. Ýòî ñâÿçàíî ñ òåì, ÷òî
îñòàëüíûå ïóòè õèìè÷åñêîãî ñèíòåçà ñâÿçàíû
ñ èñïîëüçîâàíèåì íåôòè, à ïðîöåññ êðîòîíàëü-
äåãèä ãèäðîãåíèçàöèè îáåñïå÷èâàåò àëüòåðíà-
òèâíûé ìàðøðóò ñ èñïîëüçîâàíèåì â êà÷åñòâå
èñõîäíîãî ñûðüÿ ýòàíîëà, êîòîðûé ìîæíî ïî-
ëó÷àòü èç áèîìàññû.  ýòîì ñëó÷àå ýòàíîë äå-
ãèäðèðóþò â àöåòàëüäåãèä, èç êîòîðîãî ìîæíî
ïåðåéòè ê äàëüíåéøåìó ñèíòåçó áóòàíîëà [3]. Â
2010 ã. ìèðîâîå ïðîèçâîäñòâî áóòàíîëà ñîñòà-
âèëî áîëåå 3 ìëí òîíí.
2. Ферментация АБЭ
 êëàññè÷åñêîé ôåðìåíòàöèè ÀÁÝ ïîëó-
÷àþò êîíå÷íûå ïðîäóêòû – àöåòîí, áóòàíîë è
ýòàíîë â ñîîòíîøåíèè 3:6:1 ñîîòâåòñòâåííî.
Ïðîöåññ ôåðìåíòàöèè ðàçäåëÿåòñÿ íà äâà
ýòàïà – êèñëîòîîáðàçîâàíèå è ïîëó÷åíèå ñïèð-
òîâ. Ïðè íàêîïëåíèè äîñòàòî÷íîãî êîëè÷åñòâà
àöåòàòà (óêñóñíîé êèñëîòû) è áóòèðàòà (ìàñ-
ëÿíîé êèñëîòû) ïåðâàÿ ôàçà ñèíòåçà çàïóñêàåò
ïîñëåäóþùèé ïðîöåññ ñïèðòîîáðàçîâàíèÿ.
Ïðè èñïîëüçîâàíèè â êà÷åñòâå èñõîäíîãî
ñóáñòðàòà ãëþêîçû ïîëó÷åíèå ïèðóâàòà ïðîèñ-
õîäèò ïî ñõåìå Ýìáäåíà–Ìåéåãîôà–Ïàðíàñà
(ðèñ. 2).
Ðèñ. 1. Ñõåìà ïðîìûøëåííîãî ñèíòåçà áóòàíîëà: à – ñõåìà Îêñî, á – ñõåìà Ðåïïå, â – êðîòîíèëäåãèäðî
ãèäðîãåíàöèåé [16]
54 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Å.À. Òèãóíîâà, Ñ.Ì. Øóëüãà, ß.Á. Áëþì
Ðèñ. 2. Ñõåìà áèîõèìè÷åñêîãî ïóòè ÀÁÝ ôåðìåíòàöèè. Ýíçèìû îáîçíà÷åíû áóêâàìè: A – ãëèöåðàëüäåãèä-3-
ôîñôàò äåãèäðîãåíàçà, B – ïèðóâàò-ôåððåäîêñèí îêñèäîðåäóêòàçà, C – ÍÀÄÍ-ôåððåäîêñèí îêñèäîðåäóêòà-
çà, D – ÍÀÄÔÍ-ôåððåäîêñèí îêñèäîðåäóêòàçà, E – ÍÀÄÍ ðóáðåäîêñèí îêñèäîðåäóêòàçà, F – ãèäðîãåíàçà,
G – ôîñôîòðàíñàöåòèëàçà, H – àöåòàò êèíàçà, I – òèîëàçà, J – 3-ãèäðîêñèáóòèðèë-ÊîÀ äåãèäðîãåíàçà, K –
êðîòîíàçà, L – áóòèðèë-ÊîÀ äåãèäðîãåíàçà, M – ôîñôàò áóòèòðàíñôåðàçà (ôîñôîòðàíñáóòèðèëàçà), N –
áóòèðàò êèíàçà, O – àöåòîàëüäåãèä äåãèäðîãåíàçà, P – ýòàíîë äåãèäðîãåíàçà, Q – áóòèðàëüäåãèä äåãèäðîãå-
íàçà, R – áóòàíîë äåãèäðîãåíàçà, S – àöåòîàöåòèë-ÊîÀ : àöåòàò/áóòèðàò : ÊîÀ òðàíñôåðàçà, T – àöåòîàöåòàò
äåêàðáîêñèëàçà, U – èçîïðîïàíîë äåãèäðîãåíàçà, V – ëàêòàò äåãèäðîãåíàçà; CoA – êîýíçèì À, P – ôîñôàò,
Rh – ðóáðåäîêñèí, Fd – ôåððåäîêñèí [17]
55ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Àëüòåðíàòèâíûé âèä òîïëèâà – áèîáóòàíîë
 ýòîì ïðîöåññå äâå ìîëåêóëû ÀÒÔ (àäå-
íîçèíòðèôîñôàò) è ÍÀÄÍ (íèêîòèíàìèääè-
íóêëåîòèä) èñïîëüçóþòñÿ íà 1 ìîëü ãëþêîçû.
Ñóáñòðàòû, ñîäåðæàùèå ïåíòîçû, ïåðåõîäÿò â
ïèðóâàò ïî ïåíòîçîôîñôàòíîé ñõåìå. Â ýòîé
ñõåìå íà òðè ìîëåêóëû ïåíòîçû òðàòèòñÿ ïÿòü
ìîëåêóë ÀÒÔ è ÍÀÄÍ.
Èç ïèðóâàòà ìîæåò òàêæå ôîðìèðîâàòüñÿ
ëàêòàò.  ýòîì ïðîöåññå ïðèíèìàþò ó÷àñòèå
äâà ýíçèìà. Ëàêòàòäåãèäðîãåíàçà êàòàëèçèðóåò
ôîðìèðîâàíèå ëàêòàòà èç ïèðóâàòà ñ ïîìîùüþ
ðåãåíåðàöèè ÍÀÄ+, à âòîðîé ýíçèì – ÍÀÄ-
íåçàâèñèìàÿ ëàêòàòäåãèäðîãåíàçà – ïðèíèìàåò
ó÷àñòèå â óòèëèçàöèè ëàêòàòà è ïðîäóöèðóåò èç
íåãî ïèðóâàò.
 îáû÷íûõ óñëîâèÿõ ïèðóâàò ïðåâðàùàåòñÿ
â àöåòèë-ÊîÀ ñ ïîìîùüþ ïèðóâàò-ôåððåäîêñèí
îêñèäîðåäóêòàçû ñ âûäåëåíèåì ÑÎ2 è îêèñëå-
íèåì ôåððåäîêñèíà [18], ïðè ýòîì àöåòèë-ÊîÀ
ÿâëÿåòñÿ ñðåäíèì ðàçâåòâëåíèåì îñíîâíîãî ìå-
òàáîëè÷åñêîãî ïóòè. Òàêèì îáðàçîì, äâà íà-
ïðàâëåíèÿ ñèíòåçà ïðèâîäÿò ê îáðàçîâàíèþ
àöåòàòà è áóòèðàòà, à òðåòüå íàïðàâëåíèå – ê
îáðàçîâàíèþ ýòàíîëà.  ýòîì ñëó÷àå àöåòî-
àëüäåãèä äåãèäðîãåíàçà êàòàëèçèðóåò ðåàêöèþ
ïðåâðàùåíèÿ àöåòèë-ÊîÀ â àöåòîàëüäåãèä ñ
èñïîëüçîâàíèåì ÍÀÄÍ è ïîëó÷åíèåì ÍÀÄ+
è ÊîÀ. Ïðåâðàùåíèå àöåòîàëüäåãèäà â ýòàíîë
ïðîèñõîäèò ïðè ïîìîùè ýòàíîë äåãèäðîãåíàçû
ñ ó÷àñòèåì ÍÀÄÍ.
Ïðîöåññ ïðåâðàùåíèÿ àöåòèë-ÊîÀ â àöå-
òàò êàòàëèçèðóåòñÿ ôîñôàò àöåòèëòðàíñôåðà-
çîé (ôîñôîòðàíñàöåòèëàçîé) ñ èñïîëüçîâàíèåì
ôîñôîðà, ïîëó÷åíèåì ÊîÀ è îáðàçîâàíèåì àöå-
òèëôîñôàòà. Äàëüíåéøåå ïðåîáðàçîâàíèå àöå-
òèëôîñôàòà ïðîèñõîäèò ñ ïîìîùüþ àöåòàòêè-
íàçû â àöåòàò. Àöåòàò, â ñâîþ î÷åðåäü ìîæåò
ïðåâðàùàòüñÿ â àöåòèë-ÊîÀ ñ ïîìîùüþ àöå-
òîàöåòèë-ÊîÀ : àöåòàò/áóòèðàò : ÊîÀ òðàíñôå-
ðàçû. Òðåòèé ïóòü èçìåíåíèé àöåòèë-ÊîÀ ñâÿ-
çàí ñ òèîëàçîé (àöåòèë-ÊîÀ àöåòèëòðàíñôåðà-
çîé), êîòîðàÿ ïðåîáðàçóåò àöåòèë-ÊîÀ â àöåòî-
àöåòèë-ÊîÀ ñ ïîëó÷åíèåì ÊîÀ. Àöåòîàöåòèë-
ÊîÀ ÿâëÿåòñÿ åùå îäíîé òî÷êîé ðàçâåòâëåíèÿ
îñíîâíîãî ïóòè ñèíòåçà. Â ñïèðòîâîé ôàçå àöåòî-
àöåòèë-ÊîÀ ñ ó÷àñòèåì àöåòîàöåòèë-ÊîÀ : àöå-
òàò/áóòèðàò : ÊîÀ òðàíñôåðàçû ïðåâðàùàåòñÿ â
àöåòîàöåòàò, êîòîðûé â ñâîþ î÷åðåäü ïåðåõîäèò
â àöåòîí. Ýòà ðåàêöèÿ êàòàëèçèðóåòñÿ àöåòîà-
öåòàò äåêàðáîêñèëàçîé è ñîïðîâîæäàåòñÿ âû-
äåëåíèåì ÑÎ2. Â ïðîöåññå êèñëîòîîáðàçîâàíèÿ
àöåòîàöåòèë-ÊîÀ ñ ïîìîùüþ 3-ãèäðîêñèáóòè-
ðèë-ÊîÀ äåãèäðîãåíàçû è ÍÀÄÍ ïðåîáðàçó-
åòñÿ â 3-ãèäðîêñèáóòèðèë-ÊîÀ. Ïðåâðàùåíèå
3-ãèäðîêñèáóòèðèë-ÊîÀ â êðîòîíèë êàòàëèçè-
ðóåòñÿ êðîòîíàçîé ñ ïîëó÷åíèåì âîäû. Äàëü-
íåéøåå ïðåîáðàçîâàíèå êðîòîíèëà ïðîèñõîäèò
ïðè ïîìîùè áóòèðèë-ÊîÀ äåãèäðîãåíàçû è
ÍÀÄÍ ñ ïîëó÷åíèåì áóòèðèë-ÊîÀ. Áóòèðèë-
ÊîÀ ÿâëÿåòñÿ ïîñëåäíåé òî÷êîé ðàçâåòâëåíèÿ
îñíîâíîé ñõåìû.
Ïðåîáðàçîâàíèå áóòèðèë-ÊîÀ â çàâèñèìîñ-
òè îò ýòàïà (ñïèðòîîáðàçîâàíèÿ èëè êèñëîòî-
îáðàçîâàíèÿ) ïðîõîäèò ïî íàïðàâëåíèþ ñèí-
òåçà áóòàíîëà èëè áóòèðàòà.  ïåðâîì ñëó÷àå
áóòèðàëüäåãèä äåãèäðîãåíàçà êàòàëèçóåò ðå-
àêöèþ ñ èñïîëüçîâàíèåì ÍÀÄÍ, ïîëó÷åíè-
åì ÊîÀ è ïðåîáðàçîâàíèåì â áóòèðàëüäåãèä,
êîòîðûé â äàëüíåéøåì ñ ó÷àñòèåì áóòàíîë
äåãèäðîãåíàçû è ÍÀÄÔÍ ïðåîáðàçóåòñÿ â áó-
òàíîë. Ïðåîáðàçîâàíèå áóòèðèë-ÊîÀ íà ýòàïå
êèñëîòîîáðàçîâàíèÿ êàòàëèçèðóåòñÿ ôîñôàòáó-
òèëòðàíñôåðàçîé (ôîñôîòðàíñáóòèðèëàçîé) ñ
èñïîëüçîâàíèåì ôîñôîðà, ïîëó÷åíèåì ÊîÀ è
îáðàçîâàíèåì áóòèðèëôîñôàòà. Áóòèðèëôîñôàò
ïðè ó÷àñòèè áóòèðèëêèíàçû è ÀÄÔ ïðåîá-
ðàçóåòñÿ â áóòèðàò.  ñâîþ î÷åðåäü áóòèðàò
ìîæåò ïðåâðàùàòüñÿ â áóòèðèë-ÊîÀ ñ ïîìî-
ùüþ àöåòîàöåòèë-ÊîÀ : àöåòàò/áóòèðàò : ÊîÀ
òðàíñôåðàçû. Íåîáõîäèìî îòìåòèòü, ÷òî ïðè
ñïèðòîîáðàçîâàíèè â êà÷åñòâå àëüòåðíàòèâû
àöåòîíó ìîæåò îáðàçîâûâàòüñÿ èçîïðîïèëîâûé
ñïèðò.  ýòîì ñëó÷àå ðåàêöèÿ êàòàëèçèðóåòñÿ
èçîïðîïàíîë äåãèäðîãåíàçîé è ÍÀÄÍ.
 êëàññè÷åñêîé ñõåìå ÀÁÅ ôåðìåíòàöèè
ïðè êèñëîòîîáðàçîâàíèè îäíà ìîëåêóëà ãëþ-
êîçû ïðåîáðàçóåòñÿ â 0,8 ìîëåêóëû áóòèðàòà +
+ 0,4 ìîëåêóëû àöåòàòà + 2 ìîëåêóëû ÑÎ2+
+ 2,4 ìîëåêóëû Í2 [19]. Ïðè ñïèðòîîáðàçîâà-
íèè îäíà ìîëåêóëà ãëþêîçû ïðåâðàùàåòñÿ â 0,3
ìîëåêóëû àöåòîíà + 0,65 ìîëåêóëû áóòàíîëà +
+ 1,4 ìîëåêóëû Í2 + 2,3 ìîëåêóëû ÑÎ2 [19].
Äëèòåëüíîñòü êèñëîòîîáðàçóþùåãî è ñïèð-
òîâîîáðàçóþùåãî ýòàïîâ ìîæåò áûòü èçìåíåíà
ñ ïîìîùüþ âàðüèðîâàíèÿ êîíöåíòðàöèé Í2,
ÑÎ2, îðãàíè÷åñêèõ êèñëîò è ìèíåðàëüíûõ äî-
áàâîê. Ïîñêîëüêó ïðåîáðàçîâàíèå ïèðóâàòà â
àöåòèë-ÊîÀ âêëþ÷àåò èñïîëüçîâàíèå æåëåçî-
ñåðíûõ áåëêîâ (ôåððåäîêñèí îêñèäîðåäóêòàç),
òî, êàê ïðèìåð, âàæíîé ìèíåðàëüíîé äîáàâêîé
56 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Å.À. Òèãóíîâà, Ñ.Ì. Øóëüãà, ß.Á. Áëþì
ÿâëÿåòñÿ æåëåçî. Èçìåíåíèå êîíöåíòðàöèè
æåëåçà ñóùåñòâåííî âëèÿåò íà ïðîöåññ ñèíòåçà
êîíå÷íûõ ïðîäóêòîâ [20–30].
Äëèòåëüíîñòü ïðîöåññà ôåðìåíòàöèè ÀÁÝ
ñîñòàâëÿëà 2–6 äíåé â çàâèñèìîñòè îò âûáîðà
ñóáñòðàòà è óñëîâèé êóëüòèâèðîâàíèÿ. Ïðè çà-
âåðøåíèè ïðîöåññà ïåðèîäè÷åñêîé ôåðìåíòà-
öèè êîíöåíòðàöèÿ ðàñòâîðèòåëåé íàõîäèëàñü â
ïðåäåëàõ 12–20 ã/ë.  ýòîì ñëó÷àå ðàñòâîðèòåëè
âûäåëÿëè èç ñðåäû ñ ïîìîùüþ äèñòèëëÿöèè.
Ïðè èñïîëüçîâàíèè ïðîöåññà íåïðåðûâíîãî
êóëüòèâèðîâàíèÿ â êëàññè÷åñêîé ôåðìåíòàöèè
ÀÁÝ ïîëó÷èòü êîììåð÷åñêè âûãîäíûå êîíå÷-
íûå ïðîäóêòû íåâîçìîæíî èç-çà òîêñè÷íîñòè
ðàñòâîðèòåëåé è áèôàçíîñòè ôåðìåíòàöèè. Äëÿ
ðåøåíèÿ ýòîé ïðîáëåìû ïðîöåññ íåïðåðûâíî-
ãî êóëüòèâèðîâàíèÿ áûë ñîåäèíåí ñ ïðîöåññîì
âîññòàíîâëåíèÿ in situ è ïðîâåäåíî ìíîãîñòó-
ïåí÷àòîå íåïðåðûâíîå áðîæåíèå [3].
3. Продуценты бутирата и бутанола
Ñóùåñòâóþò íåñêîëüêî âèäîâ áàêòåðèé, êî-
òîðûì ñâîéñòâåííî ìàñëÿíîêèñëîå áðîæåíèå
è åãî ðàçíîâèäíîñòü – àöåòîíîáóòèëîâîå áðî-
æåíèå. Ïðîöåññ àöåòîíîáóòèëîâîãî áðîæåíèÿ
ÿâëÿåòñÿ àíàýðîáíûì è îñóùåñòâëÿåòñÿ ìèêðî-
îðãàíèçìàìè, êîòîðûå ïðèíàäëåæàò ê ðîäàì
Clostridium, Butyrvibrio, Butyribacterium, Sarcina,
Eubacterium, Fusobacterium è Megasphera, èç
êîòîðûõ íàèáîëåå èçó÷åíû ðîäû Clostridium,
Butyrvibrio è Butyribacterium [31, 32] (òàáë. 2).
Äëÿ ïðîèçâîäñòâà áóòàíîëà â ïðîìûøëåííûõ
ìàñøòàáàõ èñïîëüçóþò ìèêðîîðãàíèçìû ðîäà
Clostridium: Ñ. acetobutylicum, Ñ. beijerinckii, Ñ. sac-
charobutylicum è Ñ. saccharoperbutylacetonicum.
Áàêòåðèè Clostridia ïàëî÷êîâèäíûå, ñïîðî-
îáðàçóþùèå ãðàìïîëîæèòåëüíûå è, êàê ïðà-
âèëî, ñòðîãèå àíàýðîáû. Øòàììû êëîñòðèäèé
ìîãóò áûòü èçîëèðîâàíû èç ïî÷âû, ñòî÷íûõ
âîä, îñòàòêîâ ïðîöåññà æèçíåäåÿòåëüíîñòè æè-
âîòíûõ, êàðòîôåëÿ, êîðíåé àçîòîñâÿçûâàþùèõ
çåðíîáîáîâûõ êóëüòóð, ìîëîêà, ñûðà è ò.ä.
Áîëüøèíñòâî ìàñëÿíî-êèñëûõ áàêòåðèé ñèí-
òåçèðóþò ìàñëÿíóþ è óêñóñíóþ êèñëîòû íà
ýòàïå êèñëîòîîáðàçîâàíèÿ, à íà ýòàïå ñïèðòî-
îáðàçîâàíèÿ – áóòàíîë è àöåòîí. Øòàììû –
ïðîäóöåíòû àöåòîíà è áóòàíîëà, ñåé÷àñ áîëü-
øåé ÷àñòüþ êëàññèôèöèðîâàíû êàê C. acetobu-
tylicum. Íåêîòîðûå âèäû ïðîäóöåíòîâ ñèíòåçè-
ðóþò òàêæå è äîïîëíèòåëüíûå ïðîäóêòû, òàêèå
êàê ýòàíîë, ëàêòàò, èçîïðîïàíîë.
C. butyricum, C. tyrobutyricum, C. thermobuty-
ricum, C. beijerinckii è C. ðopuleti – îñíîâíûå
øòàììû äëÿ ïîëó÷åíèÿ ìàñëÿíîé êèñëîòû è áó-
òàíîëà. C. beijerinckii ñèíòåçèðóåò ðàñòâîðèòåëè
ïðèáëèçèòåëüíî ñ òàêèì æå ïîêàçàòåëåì, êàê
C. acetobutylicum, îäíàêî âìåñòî àöåòîíà ñèí-
òåçèðóåò èçîïðîïàíîë. C. aurantibutyricum êðî-
ìå áóòàíîëà è àöåòîíà, ñèíòåçèðóåò åùå è èçî-
ïðîïàíîë. Îñíîâíûì èñòî÷íèêîì óãëåðîäà äëÿ
ðîñòà áàêòåðèé C. tyrobutyricum ñëóæèò ëàê-
òîçà, à ïðîäóêòàìè ôåðìåíòàöèè – ìàñëÿíàÿ
êèñëîòà, âîäîðîä è óãëåêèñëûé ãàç [32, 33]. C.
tetanomorphum ÿâëÿåòñÿ îòíîñèòåëüíî íîâûì
ïîëó÷åííûì ïðîäóöåíòîì, êîòîðûé ñèíòåçè-
ðóåò ïî÷òè ýêâèìîëÿðíîå êîëè÷åñòâî áóòàíîëà
è ýòàíîëà è íå ñèíòåçèðóåò äðóãèõ ðàñòâîðèòå-
ëåé [20].
Ðàçëè÷íûå âèäû Clostridium èìåþò ðàçíóþ
ïðîèçâîäèòåëüíîñòü è âûõîä ñîîòâåòñòâóþùèõ
êèñëîò, ðàñòâîðèòåëåé è èõ ïðåäøåñòâåííè-
êîâ. Âûõîä ñîîòâåòñòâóþùèõ ïðîäóêòîâ ñèí-
òåçà çàâèñèò êàê îò ñðåäû êóëüòèâèðîâàíèÿ,
ôàêòîðîâ ðîñòà, ðÍ, òåìïåðàòóðû, òàê è îò ñà-
ìîãî øòàììà [20]. Â òàáë. 3 ïðèâåäåíû ïðî-
äóêòû ñèíòåçà è ïðîäóêòèâíîñòü íåêîòîðûõ îñ-
íîâíûõ øòàììîâ.
 ïðîöåññå áèîñèíòåçà áóòàíîëà ïðè åãî
êîíöåíòðàöèè 1,0–2,0 % ñóùåñòâåííî áëîêè-
ðóåòñÿ ðîñò êëåòîê, ÷òî ïðèâîäèò ê ïðåêðàùå-
íèþ ôåðìåíòàöèè [13]. Áóòàíîë, ñêîðåå âñåãî,
âëèÿåò íà îáîëî÷êè êëåòîê, èçìåíÿÿ èõ ôóíê-
öèè. Ïðè ýòîì çàìåäëÿåòñÿ òðàíñïîðò àíàëîãîâ
ñàõàðà â êëåòêó, ðàçðóøàþòñÿ ôîñôîëèïèä-
íûå êîìïîíåíòû, ÷òî ïðèâîäèò ê óâåëè÷åíèþ
òåêó÷åñòè êëåòî÷íûõ ìåìáðàí. Äîáàâëåíèå
7–13 ã/ë áóòàíîëà â ñðåäó ïðèâîäèò ê èíãèáè-
ðîâàíèþ ðîñòà ó 50 % ïðîäóöåíòîâ, à ïðè óâå-
ëè÷åíèè êîíöåíòðàöèé àöåòîíà è ýòàíîëà (äî
40 ã/ë) òåìïû ðîñòà ñíèæàþòñÿ âñåãî ëèøü íà
50 % [3, 36, 37].
Îäíèì èç ïóòåé ðåøåíèÿ ýòîé ïðîáëåìû ÿâ-
ëÿåòñÿ íàïðàâëåííàÿ ñåëåêöèÿ øòàììîâ, òîëå-
ðàíòíûõ ê áóòàíîëó.
Ñîçäàíèå íà îñíîâå C. beijerinckii NCIMB
8052 ñ ïîìîùüþ ìóòàãåíåçà íîâîãî øòàììà Ñ.
beijerinckii BA 101, êîòîðûé áîëåå òåðïèì ê ðàñ-
òâîðèòåëÿì, ñëóæèò îäíèì èç òàêèõ ïðèìåðîâ
[3]. Åùå îäíèì ïðèìåðîì ìîæåò ñëóæèòü ïî-
ëó÷åíèå èçìåíåííîãî øòàììà C. acetobutylicum
57ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Àëüòåðíàòèâíûé âèä òîïëèâà – áèîáóòàíîë
ÀÒÑÑ 824 ñ ìóòàöèåé â ãåíå lyt–1. Ýòîò øòàìì
ñïîñîáåí ê äàëüíåéøåìó ðîñòó ïðè êîíöåíòðà-
öèè áóòàíîëà 15 ã/ë.
Ïðè îáðàáîòêå N-ìåòèë-N’-íèòðî-N-íèòðî-
çîãóàíèäèíîì øòàììà C. acetobutylicum ÀÒÑÑ
824 áûëè ïîëó÷åíû ìóòàíòû C. acetobutylicum
903 è C. acetobutylicum 904, ðåçèñòåíòíûå ê áó-
òàíîëó è èìåþùèå ïîâûøåííîå êèñëîòîîáðà-
çîâàíèå [20].
Äëÿ óâåëè÷åíèÿ òîëåðàíòíîñòè ê áóòàíîëó
èñïîëüçîâàëñÿ è ìåòîä siÐÍÊ. Òîëåðàíòíûé ê
áóòàíîëó ìóòàíò øòàììà C. beijerinckii NCIMB
8052 ñêîíñòðóèðîâàí ñ ïîìîùüþ siÐÍÊ, êîòî-
ðûå áûëè íàöåëåíû íà èçìåíåíèå ãåíà ãëèöå-
ðèí äåãèäðîãåíàçû (gldA) [3].
4. Субстраты
 ïðîöåññå ôåðìåíòàöèè ÀÁÝ ñòîèìîñòü ñóá-
ñòðàòà ÿâëÿåòñÿ êëþ÷åâûì ôàêòîðîì. Êàê ïðà-
âèëî, äî íåäàâíåãî âðåìåíè â ÀÁÝ ïðîöåññå èñ-
ïîëüçîâàëè êðàõìàëîñîäåðæàùèå (êóêóðóçà, êàð-
òîôåëü, ïøåíèöà, ìàíèîêà è ò.ä.) èëè ñàõàðîñî-
äåðæàùèå (ñàõàðíûé òðîñòíèê, ñâåêëà, ìåëàññû)
ñóáñòðàòû. Âîçìîæíîñòü ñàõàðîëèòè÷åñêèõ êëî-
ñòðèäèé ñáðàæèâàòü ðàçíîãî ðîäà óãëåâîäû,
âêëþ÷àÿ ìîíî- è äèñàõàðèäû, êñèëîçó è öåëëî-
áèîçó, è ïîëèìåðû, íàïðèìåð, êðàõìàë è êñè-
ëàí [38], ñòèìóëèðîâàëî ïîèñêè àëüòåðíàòèâíûõ,
áîëåå äåøåâûõ ñóáñòðàòîâ [39]. Òàêèìè ñóáñòðà-
òàìè â äàëüíåéøåì ìîãóò âûñòóïàòü ëèãíèí è
äðóãèå öåëëþëîçîñîäåðæàùèå âèäû ñûðüÿ.
 ðàáîòå Kirscher [3] ïðîâåäåíà ýêîíîìè-
÷åñêàÿ îöåíêà ïðîèçâîäñòâà áóòàíîëà èç êóêó-
ðóçíîé ìóêè ñ èñïîëüçîâàíèåì øòàììà-ïðîäó-
öåíòà áóòàíîëà Ñ. beijerinckii BA101. Óñòàíîâëå-
íî, ÷òî óâåëè÷åíèå âûõîäà áóòàíîëà íà 19,0 %
(0,42–0,50 ã áóòàíîëà íà 1 ã ãëþêîçû) ïðèâî-
äèò ê ñíèæåíèþ ñòîèìîñòè áóòàíîëà íà 14,7 %.
Ïðè èñïîëüçîâàíèè «ñóõîãî ñïðåÿ» èç ñîåâîé
ìåëàññû (SDSM) â êà÷åñòâå ñóáñòðàòà äëÿ ôåð-
ìåíòàöèè ÀÁÝ ñ ïîìîùüþ Ñ. beijerinckii BA
101 âûñîêèå êîíöåíòðàöèè SDSM ïðèâîäèëè
ê íèçêîìó âûõîäó ðàñòâîðèòåëåé. Äîáàâëåíèå
ê ñóáñòðàòó ãëþêîçû â êîíöåíòðàöèè 25,3 ã/ë
ïðèâåëî ê óâåëè÷åíèþ ïðîèçâîäñòâà ABÝ íà
113 % (10,7–22,8 ã/ë). Íà îñíîâàíèè ðåçóëü-
òàòîâ èññëåäîâàíèé ñäåëàí âûâîä, ÷òî èñ-
ïîëüçîâàíèå SDSM â êà÷åñòâå ñóáñòðàòà ñ äî-
áàâëåíèåì ãëþêîçû èëè ñàõàðîçû ìîæåò áûòü
óñïåøíûì äëÿ ïðîèçâîäñòâà ðàñòâîðèòåëåé. Â
òàáë. 4 ïðèâåäåíû âèäû êëîñòðèäèé è íåêîòî-
ðûå àëüòåðíàòèâíûå ñóáñòðàòû.
Kirscher [3] óñòàíîâèë, ÷òî áóòàíîë ìîæíî
ïîëó÷àòü èç ñûðüÿ ñ íèçêîé ñòîèìîñòüþ, òàêî-
ãî êàê êîæóðà àðàõèñà è äðóãèå ñåëüñêîõîçÿé-
ñòâåííûå îòõîäû, è ñäåëàë âûâîä îá óíèâåð-
ñàëüíîñòè øòàììà Ñ. beijerinckii BA 101.
Êðîìå òîãî, áûë èçó÷åí åùå îäèí ñóáñòðàò –
êñèëàí, ïîëó÷àåìûé èç âîëîêîí êóêóðóçû. Ãè-
Òàáëèöà 2. Íåêîòîðûå âèäû ïðîäóöåíòîâ áóòàíîëà
è áóòèðàòà
Ïðîäóöåíò Èñòî÷íèê óãëåðîäà
Clostridium butyricum
tyrobutyricum
beijerinckii
pasterianum
barkeri
acetobutylicum
thermobutyricum
thermopalmarium
Butyribacterium methylotro-
phicum
Pseudobutyrivibrio ruminis
Êðàõìàë, êñèëîçà, ëàê-
òîçà, ãëþêîçà, ñàõàðî-
çà, ãëèöåðèí, öåëëþ-
ëîçà
Ëàêòàò, ãåêñîçû, ïè-
ðóâàò, ÑÎ, ÑÎ2/Í2,
ìåòàíîë
Ãëþêîçà
Òàáëèöà 3. Ïðîäóêòû ñèíòåçà è ïðîäóêòèâíîñòü
øòàììîâ
Âèä è øòàìì
Ïðîäóêòû, ã/ë
Áóòàíîë Ýòàíîë Àöåòîí
C. beijerinckii
NCIMB 8052*
BA 101*
ÀÒÑÑ 25752**
C. butylicum
NRRL B-592**
NRRL B-593**
C. aurantibutyricum
ATCC 17777**
NCIB 10659**
C. acetobutylicum
ATCC 4259**
ATCC 824**
ATCC 8529**
9,2
18,6
5,0
4,5
4,6
3,4
3,1
1,5
1,3
0,3
0,9
0,3
–
–
–
–
–
–
–
–
4,4
8,6
0,4
1,1
0
1,2
0,8
0,3
0,4
0,09
Ïðèìå÷àíèå.  êà÷åñòâå èñòî÷íèêà óãëåðîäà èñïîëü-
çîâàëè ãëþêîçó. *[34]; ** [35].
58 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Å.À. Òèãóíîâà, Ñ.Ì. Øóëüãà, ß.Á. Áëþì
äðîëèç êñèëàíîâûõ âîëîêîí êóêóðóçû è ôåð-
ìåíòàíèöèÿ AÁÝ âûïîëíåíû â åäèíîé èíòåãðè-
ðîâàííîé ñèñòåìå îáîðóäîâàíèÿ, ÷òî ïðèâåëî
ê âûñîêèì âûõîäàì ðàñòâîðèòåëåé – 0,44 ã íà
1 ã ñóáñòðàòà è ïðîèçâîäèòåëüíîñòè 0,47 ã/ë·÷–1.
 ïðîöåññå íåïðåðûâíîãî êóëüòèâèðîâàíèÿ
øòàììà Ñ. beijerinckii P260 ñ èñïîëüçîâàíèåì
â êà÷åñòâå èñòî÷íèêà óãëåðîäà ãèäðîëèçàòà
ïøåíè÷íîé ñîëîìû áûëî äîñòèãíóòî óâåëè÷å-
íèå ïðîèçâîäèòåëüíîñòè áóòàíîëà íà 214 % [3].
Óñòàíîâëåíî ñòèìóëèðóþùåå äåéñòâèå íà ðîñò
Ñ. beijerinckii BA101, à òàêæå íà ñèíòåç áóòàíî-
ëà, ôóðôóðîëà è ôóðôóðîë-ãèäðîêñèìåòèëà.
5. Сырьевая база
Îäíîé èç îñíîâíûõ çàäà÷ áèîòåõíîëîãè÷å-
ñêèõ èññëåäîâàíèé â îáëàñòè ýíåðãåòèêè ÿâ-
ëÿåòñÿ ñîçäàíèå ýêîíîìè÷åñêè ýôôåêòèâíîé
ñõåìû ïðîèçâîäñòâà áèîòîïëèâà [50]. Âïîëíå
ïîíÿòíî, ÷òî ýêîíîìè÷åñêè öåëåñîîáðàçíî èñ-
ïîëüçîâàòü äëÿ ïîëó÷åíèÿ áèîòîïëèâà äåøåâîå
è âîçîáíîâëÿåìîå ñûðüå, íàïðèìåð ðàñòèòåëü-
íóþ áèîìàññó (äðåâåñíûå îòõîäû, ñîëîìó è
äðóãèå âèäû íåïèùåâîãî ðàñòèòåëüíîãî ñûðüÿ).
Èíôîðìàöèÿ î ðåãóëÿòîðíûõ ïóòÿõ, îïðå-
äåëÿþùèõ ôîðìó è ðàçìåð êëåòî÷íûõ ñòåíîê,
äàåò âîçìîæíîñòü ïîëó÷èòü ðàñòåíèÿ ñ óëó÷-
øåííûìè õàðàêòåðèñòèêàìè äëÿ ïðîèçâîäñòâà
áèîòîïëèâà, ñ ëåãêî ôåðìåíòèðóåìûìè èëè
áîëåå òîëñòûìè êëåòî÷íûìè ñòåíêàìè. Ïî-
íèìàíèå ïðîöåññîâ ðîñòà è ðàçâèòèÿ êëåòîê
ïîçâîëÿåò ñîçäàâàòü ðàñòåíèÿ ñ óëó÷øåííûìè
ñâîéñòâàìè äëÿ ïðîèçâîäñòâà áèîòîïëèâà. Ñî-
êðàùåíèå ïðåäâàðèòåëüíîé îáðàáîòêè ñûðüÿ
ìîæåò ñóùåñòâåííî ñíèçèòü öåíó íà áèîòîïëè-
âî. Íîâûå ïðåäñòàâëåíèÿ î ñòðóêòóðå ðàñòåíèé
óæå äàëè âîçìîæíîñòü äëÿ ðàçðàáîòêè âûñîêî-
ýôôåêòèâíîãî ñûðüÿ äëÿ áèîòîïëèâà [51].
Ïðîâåäåí ñêðèíèíã ðàñòåíèé, èñïîëüçóåìûõ
â êà÷åñòâå ñûðüÿ äëÿ áèîòîïëèâà. Îäíèì èç
ïåðñïåêòèâíûõ ðàñòåíèé ïðèçíàí òîïîëü [52].
Îïðåäåëåíû îáðàçöû òîïîëÿ, êîòîðûå äàâàëè
âûñîêèé âûõîä ñàõàðîâ áåç êàêîé-ëèáî ïðåä-
âàðèòåëüíîé îáðàáîòêè. Ê ñîæàëåíèþ, ïîêà íå
ïîëó÷åí îòâåò, êàêèå èìåííî ïðèçíàêè ôåíî-
òèïà òîïîëÿ ïîâûøàþò ýôôåêòèâíîñòü âûäå-
ëåíèÿ ñàõàðà.
Âîçìîæíî òàêæå ïîëó÷åíèå óæå ïîäãîòîâ-
ëåííîãî ñûðüÿ äëÿ ñáðàæèâàíèÿ, íàïðèìåð
öåëëþëîçû, êîòîðóþ ìîæíî íàïðÿìóþ ïåðå-
ðàáàòûâàòü â áèîòîïëèâî. Â ýòîì íàïðàâëåíèè
ðàáîòàåò ðÿä àâòîðîâ [53], çàíèìàþùèõñÿ ñîç-
äàíèåì ìèêðîîðãàíèçìîâ, êîòîðûå ñèíòåçèðó-
þò ëåãêîêîíâåðòèðóåìóþ öåëëþëîçó. Íàéäå-
íû íîâûå ìèêðîîðãàíèçìû (öèàíîáàêòåðèè),
èñïîëüçóþùèå ñîëíå÷íûé ñâåò êàê èñòî÷íèê
ýíåðãèè äëÿ ïðîèçâîäñòâà ïîëèñàõàðèäîâ è
öåëëþëîçû. Òàêèå öèàíîáàêòåðèè ñèíòåçèðî-
âàëè îòíîñèòåëüíî ÷èñòóþ öåëëþëîçó â ôîðìå
ãåëÿ, êîòîðàÿ ëåãêî ðàñùåïëÿëàñü.
Òàáëèöà 4. Íåêîòîðûå àëüòåðíàòèâíûå âèäû ñóáñòðàòîâ [17]
Ñóáñòðàò Èñòî÷íèêè óãëåâîäîâ Âèäû êëîñòðèäèé Ññûëêà
ßáëî÷íûé æìûõ Ôðóêòîçà, ãëþêîçà, ñàõàðîçà C. beijerinckii [40]
Òîïèíàìáóð Ïîëèôðóêòîçà C. beijerinckii [41]
Ñûâîðîòêà Ëàêòîçà C. acetobutylicum [42]
C. beijerinckii [43]
Íå êîíäèöèîííûé êàðòîôåëü Ãëþêîçà, êðàõìàë C. beijerinckii [44]
Ñîåâàÿ ìåëëàñà Äåêñòðîçà, ñàõàðîçà C. beijerinckii [45]
Ãèäðîëèçàò äðåâåñèíû Ãëþêîçà, ìàííîçà, ëèãíîöåëëþëîçà C. acetobutylicum [46]
Òîðô Ãëþêîçà, êñèëîçà, ëèãíîöåëëþëîçà C. beijerinckii [47]
Ñòîêè ïðîèçâîäñòâà ïàëüìî-
âîãî ìàñëà
Ëèãíîöåëëþëîçà, ìàñëî, ãëþêîçà, êñèëîçà C. aurantibutyri-
cum
[48]
Îðãàíè÷åñêèå áûòîâûå
îòõîäû
Ãëþêîçà, êñèëîçà, ëèãíîöåëëþëîçà C. acetobutylicum [49]
59ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Àëüòåðíàòèâíûé âèä òîïëèâà – áèîáóòàíîë
Âàæíûì øàãîì â ïîäãîòîâêå ñûðüÿ áûëî
ñîçäàíèå ýíçèìîâ, ñïîñîáíûõ âëèÿòü íà ïðî-
öåññ âûðàáîòêè ëèãíèíà – êëþ÷åâîãî êîìïî-
íåíòà êëåòî÷íîé ñòåíêè ðàñòåíèé. Ïðåäëîæåíà
ìîäåëü, ïðè êîòîðîé ìåòèëèðîâàíèå ïðåäøå-
ñòâåííèêà ëèãíèíà ìîæåò áûòü îäíèì èç ñïî-
ñîáîâ èíãèáèðîâàíèÿ ñèíòåçà ëèãíèíà [54], òàê
êàê ÷åì ìåíüøå ëèãíèíà, òåì ëåã÷å ðàáîòàòü ñ
êëåòî÷íûìè ñòåíêàìè.
Íåîáõîäèìî îòìåòèòü èññëåäîâàíèÿ ïî èí-
òåãðèðîâàíèþ ãåíîâ ìèêðîîðãàíèçìîâ â ðàñ-
òåíèÿ [55]. Ýêñïðåññèðîâàíèå ãåíîâ ýíçèìîâ,
äåãðàäèðóþùèõ öåëëþëîçó â ðàñòåíèÿõ, ïðèâî-
äèëî ê íåñòàáèëüíîñòè êëåòî÷íîé îáîëî÷êè âî
âðåìÿ ðîñòà ðàñòåíèÿ.
Èçâåñòåí ñïîñîá ïîâûøåíèÿ âûõîäà ðàñ-
òâîðèòåëåé ïðè êóëüòèâèðîâàíèè áàêòåðèé C.
acetobutylicym íà ìó÷íûõ ñðåäàõ çà ñ÷åò ïðåäâà-
ðèòåëüíîãî ðàçæèæåíèÿ êðàõìàëà ðåöèðêóëè-
ðóåìîé áðàæêîé, ñîäåðæàùåé àêòèâíûå àìîëè-
òè÷åñêèå ôåðìåíòû [56].
Îäíèì èç íàïðàâëåíèé ïîäãîòîâêè ñûðüÿ
ÿâëÿåòñÿ ôåðìåíòèðîâàíèå èëè îñàõàðèâàíèå.
Ïðîöåññ ïðîèñõîäèò ïðè íàãðåâàíèè ñûðüÿ
(ñîëîìû) è äîáàâëåíèè ñïåöèàëüíûõ ôåðìåí-
òîâ [53].
 êà÷åñòâå îòíîñèòåëüíî äåøåâîãî ñûðüÿ
èñïîëüçîâàëè àðàáèíîêñèëàíîâûå âîëîêíà çåð-
íîâûõ êóëüòóð, óñèëèâàÿ ñèíòåç îñàõàðèâàíèåì
èëè ãèäðîëèçàöèåé ñ ïîìîùüþ ýíçèìîâ [57].
Äîáàâëåíèå ðî-êóìóëèíîâîé è ôåðóëèíîâîé
êèñëîò â ýíçèìàòè÷åñêóþ ñðåäó ñ ëèãíîöåëëþ-
ëîçîé îêàçûâàëî ñòèìóëèðóþùèé ýôôåêò íà
ðîñò ìèêðîîðãàíèçìîâ [39].
Ñóùåñòâóåò âîçìîæíîñòü èñïîëüçîâàíèÿ â
êà÷åñòâå ñûðüÿ äëÿ ñèíòåçà áèîòîïëèâà îòõîäîâ
ñòî÷íûõ âîä æèâîòíîâîä÷åñêèõ êîìïëåêñîâ è
áîëüøèõ ãîðîäîâ [10]. Äîáàâëåíèå ãëþêîçû ê
ñðåäå ñ èçáûòî÷íîé êîíöåíòðàöèåé èëà ïðè-
âîäèëî ê óâåëè÷åíèþ ðîñòà áàêòåðèé è çàïóñêó
áèîêîíâåðñèè [58]. Ýêîíîìè÷åñêè âûãîäíîå
ïîëó÷åíèå áóòàíîëà âîçìîæíî è ïðè èñïîëüçî-
âàíèè ðîñòêîâ çåðíîâûõ êóëüòóð [59–64].
Îäíèì èç íàïðàâëåíèé â èññëåäîâàíèÿõ ïî
ïîäãîòîâêå ñûðüÿ ÿâëÿåòñÿ âûäåëåíèå íîâûõ
âèäîâ êëîñòðèäèé, êîòîðûå ìîãëè ñáðàæèâàòü
àëüòåðíàòèâíîå ñûðüå. Îáíàðóæåí íîâûé âèä
êëîñòðèäèé – C. carboxidivorans [65], èñïîëü-
çóþùèé â êà÷åñòâå èñòî÷íèêà óãëåðîäà ñèíòå-
òè÷åñêèé ãàç. C. carboxidivorans ñâÿçûâàåò ÑÎ,
ôèêñèðóåò ÑÎ2 è êîíâåðòèðóåò èõ â àöåòèë-
ÊîÀ. Âñå ãåíû êëàññè÷åñêîé ñõåìû ôåðìåí-
òàöèè ÀÁÝ ïðèñóòñòâóþò â ãåíîìå êëîñòðèäèé
ýòîãî âèäà.
6. Схемы получения биобутанола
Ýíåðãîåìêîñòü áóòàíîëà áëèçêà ê ýíåðãîåì-
êîñòè áåíçèíà. Áóòàíîë êàê æèäêèé âûñîêî-
êàëîðèéíûé ýíåðãîíîñèòåëü ìîæåò ÷àñòè÷íî
èëè ïîëíîñòüþ çàìåíèòü áåíçèí áëàãîäàðÿ õî-
ðîøåé ñìåøèâàåìîñòè ñ óãëåâîäîðîäàìè, âû-
ñîêîìó îêòàíîâîìó ÷èñëó è íèçêîé ëåòó÷åñòè
(òàáë. 1).
6.1. Êëàññè÷åñêàÿ ôåðìåíòàöèÿ
Îäíèì èç ìåòîäîâ ïîëó÷åíèÿ áóòàíîëà ÿâëÿ-
åòñÿ êëàññè÷åñêàÿ ôåðìåíòàöèÿ ÀÁÝ (ðèñ. 2).
Ñ ïîìîùüþ ïîäáîðà ñóáñòðàòà, ðÍ, òåìïåðà-
òóðû, âèäà è øòàììà ìèêðîîðãàíèçìîâ ìîæíî
êîíòðîëèðîâàòü óâåëè÷åíèå èëè óìåíüøåíèå
âûõîäà êîíå÷íûõ ïðîäóêòîâ.
Íåêîòîðûå âèäû êëîñòðèäèé èçíà÷àëüíî èìå-
ëè èçìåíåííûé ýíçèìàòè÷åñêèé ïóòü ñèíòåçà
êîíå÷íûõ ïðîäóêòîâ, âñëåäñòâèå ýòîãî âûõîä
ðàñòâîðèòåëåé çíà÷èòåëüíî îòëè÷àëñÿ îò êëàñ-
ñè÷åñêîãî. Çàòðàòû íà ñûðüå â ñåáåñòîèìîñòè
ïðîäóêòà, ïîëó÷åííîãî êëàññè÷åñêèì ïóòåì ñîñ-
òàâëÿëè 60 % è áûëè ýêîíîìè÷åñêè íåâûãîäíû-
ìè [50]. Ïî êëàññè÷åñêîé ñõåìå ôåðìåíòàöèè
ÀÁÝ ìàêñèìàëüíî èçâåñòíûå óðîâíè áèîñèíòå-
çà áóòèëîâîãî ñïèðòà è àöåòîíà ó ðàçíûõ àâòî-
ðîâ â ëàáîðàòîðíûõ óñëîâèÿõ ñîñòàâëÿëè: 15,0
è 10 ã/ë [66], 14 è 7,6 ã/ë [67], 15,1 è 6,3 ã/ë
[68] ñîîòâåòñòâåííî. Ðåçóëüòàòû ïîëó÷åíû ïðè
èñïîëüçîâàíèè èñêóññòâåííûõ ñðåä, ýêçîòè÷å-
ñêèõ èíóëèíîâûõ ñóáñòðàòîâ (áóãîðêîâ çåìëÿ-
íîé ãðóøè, ãåîðãèíû, êîðíåé öèêîðèÿ è äð.)
è ãèäðîëèçàòîâ îòõîäîâ çåðíîâûõ êóëüòóð [69].
6.2. Ôåðìåíòàöèÿ ìóòàãåííûìè øòàììàìè
Ñëåäóþùèì øàãîì ê óâåëè÷åíèþ âûõîäà êî-
íå÷íûõ ïðîäóêòîâ â êëàññè÷åñêîé ôåðìåíòà-
öèè ÀÁÝ áûëî èñïîëüçîâàíèå íàïðàâëåííîãî
ìóòàãåíåçà èçâåñòíûõ øòàììîâ [70]. Îñíîâíû-
ìè ìóòàãåíàìè ÿâëÿëèñü N-ìåòèë-N-íèòðî-N-
íèòðîçîãóàíèäèí (ÌÍÍÃ) è ýòèëìåòàíñóëüôî-
íàò (ÝÌÑ).
Ïîä âîçäåéñòâèåì àëêèëèðóþùèõ àãåíòîâ
ïîëó÷åí ñòàáèëüíûé øòàìì C. beijerinckii ÂA
101 – «ñóïåðïðîäóöåíò» áóòàíîëà [25], à òàêæå
60 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Å.À. Òèãóíîâà, Ñ.Ì. Øóëüãà, ß.Á. Áëþì
øòàììû-ìóòàíòû C. acetobutylicum, íàêàïëè-
âàþùèå íà ìó÷íûõ ñðåäàõ 9–10 ã/ë áóòàíîëà,
4,3–4,9 ã/ë àöåòîíà è 1,3–1,9 ã/ë ýòàíîëà [71].
Èç øòàììà C. acetobutylicum S ïîëó÷åí íî-
âûé øòàìì C. acetobutylicum S-3716 ïóòåì èí-
äóöèðîâàííîãî ìóòàãåíåçà ñ ïîñëåäóþùèì îò-
áîðîì ìóòàíòíûõ êëîíîâ íà ñåëåêòèâíûõ ñðå-
äàõ. Øòàìì C. acetobutylicum S-3716 ïîçâîëÿë
ïîëó÷èòü äî 15,5 ã/ë áóòàíîëà è äî 6,5 ã/ë àöå-
òîíà íà ñðåäàõ ñ ìåëàññîé è äîáàâëåíèåì ìè-
íåðàëüíûõ ñîëåé. Ïðè ýòîì êîíâåðñèÿ óãëåâî-
äîâ â öåëåâûå ïðîäóêòû äîñòèãàëà 40 % [72].
6.3. Èñïîëüçîâàíèå èììîáèëèçèðîâàííûõ
ïðîäóöåíòîâ
Ðàçëè÷íûå ìåòîäû èììîáèëèçàöèè ðàçðà-
áîòàíû äëÿ ìíîãèõ êóëüòóð, ïðîäóöèðóþùèõ
ðàñòâîðèòåëè [73], è èñïûòàíû ïðè èñïîëü-
çîâàíèè êëîñòðèäèé â ïðîèçâîäñòâå áóòàíîëà.
Èììîáèëèçàöèÿ ïðîäóöåíòîâ äàâàëà ïðåèìó-
ùåñòâî â êóëüòèâèðîâàíèè è óâåëè÷èâàëà âû-
õîä íåîáõîäèìûõ ïðîäóêòîâ. Ïðè ýòîì ïðîäëå-
âàëàñü àêòèâíîñòü è ñòàáèëüíîñòü áèîêàòàëèçà,
óìåíüøàëîñü âðåìÿ ôåðìåíòàöèè, óâåëè÷èâà-
ëàñü òîëåðàíòíîñòü êóëüòóðû ê âûñîêèì êîí-
öåíòðàöèÿì ñóáñòðàòà è êîíå÷íûõ ïðîäóêòîâ,
óëó÷øàëàñü ôèëüòðàöèÿ è ñåïàðàöèÿ. Ýôôåê-
òèâíîñòü èììîáèëèçàöèè çàâèñåëà íå òîëüêî
îò ñïîñîáà èììîáèëèçàöèè, íî è îò ìàòåðè-
àëà, èñïîëüçóåìîãî äëÿ àáñîðáöèè. Äëÿ ïðî-
öåññà íåïðåðûâíîãî êóëüòèâèðîâàíèÿ ýôôåê-
òèâíîñòü èììîáèëèçàöèè áûëà åùå ñâÿçàíà ñ
êîíñòðóêöèåé áèîðåàêòîðà.
Èñïîëüçîâàíèå èììîáèëèçèðîâàííûõ êëå-
òîê äëÿ êóëüòèâèðîâàíèÿ C. beijerinckii ÀÒÑÑ
55025 è C. beijerinckii ÂÀ 101 â áèîðåàêòîðå
óâåëè÷èëî ïðîèçâîäèòåëüíîñòü ïðîöåññà, ïðè
ýòîì êîíöåíòðàöèÿ áóòàíîëà äîñòèãàëà 11,5 è
9,4 ã/ë, ñîîòâåòñòâåííî [16]. Èñïîëüçîâàíèå
èììîáèëèçèðîâàííûõ êóëüòóð C. tyrobutyricum
è C. acetobutylicum ïîçâîëèëî ïîëó÷èòü ñêî-
ðîñòü íàêîïëåíèÿ áóòàíîëà 4,64 ã/ë·÷–1 è âûõîä
42 % îò ìàññû ãëþêîçû [13].
6.4. Ôåðìåíòàöèÿ ÷åðåç ïðîìåæóòî÷íûé
ïðîäóêò (ìàñëÿíóþ êèñëîòó)
Îïòèìèçàöèÿ ïðîöåññà ôåðìåíòàöèè ÀÁÝ
è ïîëó÷åíèå áóòàíîëà ÷åðåç ïðîìåæóòî÷íûé
ïðîäóêò – ìàñëÿíóþ êèñëîòó ïîçâîëèëî ñóùå-
ñòâåííî óâåëè÷èòü âûõîä, ñêîðîñòü íàêîïëå-
íèÿ è êîíöåíòðàöèþ áóòàíîëà [13].
Ïðåäâàðèòåëüíûå ðåçóëüòàòû ïîêàçàëè, ÷òî
áîëüøàÿ êîíöåíòðàöèÿ ìàñëÿíîé êèñëîòû ñîç-
äàâàëà òîðìîçÿùèé ýôôåêò íà ðîñò êëåòîê.
Îòíîñèòåëüíî íèçêèå óðîâíè êîíöåíòðàöèè
ìàñëÿíîé êèñëîòû ïîâûøàëè ýôôåêòèâíîñòü
ôåðìåíòàöèè ÀÁÝ è óâåëè÷èâàëè âûõîä áó-
òàíîëà. Îïòèìàëüíîé êîíöåíòðàöèåé ìàñ-
ëÿíîé êèñëîòû â ñðåäå îïðåäåëåíà âåëè÷èíà
4 ã/ë [16].
6.5. Ñîêóëüòèâèðîâàíèå
Óâåëè÷åíèå âûõîäà áóòàíîëà âîçìîæíî è â
ïðîöåññå ñîâìåñòíîãî êóëüòèâèðîâàíèÿ ñ áàê-
òåðèÿìè äðóãèõ âèäîâ. Ñîâìåñòíîå êóëüòèâè-
ðîâàíèå ïðè óñëîâèè õîðîøî ïîäîáðàííûõ
ïàðòíåðîâ äàâàëî âîçìîæíîñòü óâåëè÷èòü ñêî-
ðîñòü è ãëóáèíó ãèäðîëèçà ñóáñòðàòà, óñêîðèòü
áèîñèíòåç ýíçèìîâ, óâåëè÷èòü èõ àêòèâíîñòü è
óñêîðèòü îáðàçîâàíèå ìåòàáîëèòîâ. Êóëüòèâè-
ðîâàíèå C. acetobutylicum ñ êóëüòóðàìè C. cellu-
lolyticum èëè C. thermocellum ïðèâîäèëî ê êîí-
âåðñèè öåëëþëîçû, îäíàêî ïðè ýòîì óðîâåíü
ñèíòåçà ðàñòâîðèòåëåé áûë íèçêèì, âîçìîæíî
èç-çà íåäîñòàòêà ñàõàðà è ìàñëÿíîé êèñëîòû
â ñðåäå [17]. Äëÿ ïîâûøåíèÿ êîíöåíòðàöèè
ìàñëÿíîé êèñëîòû ïðîâåäåíû èññëåäîâàíèÿ ñî
øòàììàìè – ïðîäóöåíòàìè áóòàíîëà C. ace-
tobutylicum è C. beijerinckii, êîòîðûå ñîêóëüòè-
âèðîâàëè ñî øòàììàìè – ïðîäóöåíòàìè ìàñ-
ëÿíîé êèñëîòû C. butyricum è C. pasteurianum.
Âûõîä ðàñòâîðèòåëåé â òàêîì ïðîöåññå ñîêóëü-
òèâèðîâàíèÿ îêàçàëñÿ íå âûøå, ÷åì äëÿ ìîíî-
êóëüòóðû. Ñîâìåñòíîå êóëüòèâèðîâàíèå C. for-
micoaceticum è Lactococcus lactis [74] ïðèâîäèëî
ê óâåëè÷åíèþ àöåòàòà â ñðåäå âïëîòü äî 75 ã/ë.
Ïðè ýòîì áàêòåðèè àäàïòèðîâàëèñü ê ïîâû-
øåííûì êîíöåíòðàöèÿì àöåòàòà ñ ïîìîùüþ
íàòóðàëüíîãî îòáîðà (òîëåðàíòíûå øòàììû).
Òàêîé ïðîöåññ ñîêóëüòèâèðîâàíèÿ èñïîëüçî-
âàí äëÿ ïîëó÷åíèÿ àöåòàòà èç îòõîäîâ ìîëî÷-
íîãî ïðîèçâîäñòâà [75].
6.6. Èñïîëüçîâàíèå ãåíåòè÷åñêè èçìåíåííûõ
øòàììîâ
Ãåíåòè÷åñêèå ìàíèïóëÿöèè ñ êëîñòðèäèÿìè
íàïðàâëåíû íà èçìåíåíèå ãåíîâ âûáðàííîãî
øòàììà äëÿ ïîâûøåíèÿ âûõîäà áóòàíîëà, óâå-
ëè÷åíèÿ òîëåðàíòíîñòè øòàììà, ðàñøèðåíèÿ
äèàïàçîíà èñïîëüçóåìîãî ñûðüÿ äëÿ ñáðàæèâà-
61ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Àëüòåðíàòèâíûé âèä òîïëèâà – áèîáóòàíîë
íèÿ, èçáèðàòåëüíîãî ïðîèçâîäñòâà áóòàíîëà, íå
ñìåøàííîãî ñ ïîëó÷åíèåì äðóãèõ ðàñòâîðèòå-
ëåé è êèñëîò.
Ðàçðàáîòàíû âåêòîðû pFNK1, êîòîðûå ïîç-
âîëèëè óâåëè÷èòü ýôôåêòèâíîñòü òðàíñôîðìà-
öèè øòàììà C. acetobutylicum ATCC 824 [32].
Îäíîé èç ïåðâûõ áûëà èñïîëüçîâàíà èíòåãðà-
öèîííàÿ ïëàçìèäíàÿ òåõíîëîãèÿ äëÿ èíãèáèðî-
âàíèÿ ìåòàáîëè÷åñêèõ ïóòåé, âåäóùèõ ê ïîëó-
÷åíèþ ñ ïîìîùüþ C. acetobutylicum ATCC 824
àöåòàòà è áóòèðàòà. ×èñëî êîïèé ïëàçìèä, îáû÷-
íî èñïîëüçóåìûõ äëÿ C. acetobutylicum, ñîñòàâ-
ëÿëî ïðèáëèçèòåëüíî 7–20 êîïèé íà êëåòêó [76].
Ïëàçìèäíûå êîíñòðóêöèè ñîäåðæàëè ôðàã-
ìåíòû ãåíà ôîñôîòðàíñàöåòèëàçû (pta) èëè áó-
òèðàòêèíàçû (buk), êîòîðûå áûëè èíòåãðèðî-
âàíû â ãîìîëîãè÷íûå îáëàñòè õðîìîñîìû, ÷òî
ïðèâîäèëî ê ìåõàíèçìó èíàêòèâàöèè îäíîãî
èëè äðóãîãî ãåíà. Èíàêòèâàöèÿ ãåíà pta ñíèæà-
ëà ôîñôîòðàíñàöåòèëàçíóþ è àöåòàòêèíàçíóþ
àêòèâíîñòü, ÷òî ïðèâîäèëî ê çíà÷èòåëüíîìó
ïîíèæåíèþ âûõîäà àöåòàòà. Èíàêòèâàöèÿ ãåíà
buk ñíèæàëà áóòèðàòêèíàçíóþ àêòèâíîñòü, è,
êàê ñëåäñòâèå, ïîíèæàëà ñèíòåç áóòèðàòà è
óâåëè÷èâàëà âûõîä áóòàíîëà [77].
Ìåòèëèðîâàíèå ÷åëíî÷íîãî âåêòîðà ñ ïîìî-
ùüþ ìåòèëòðàíñôåðàçû 3TI ôàãà 3T çíà÷è-
òåëüíî ñíèæàëî èëè ïðåäîòâðàùàëî äåãðàäàöèþ
ïëàçìèäíîé ÄÍÊ, êîòîðàÿ ìîãëà ïðîèçîéòè
èç-çà àòàê ðåñòðèêòàç C. acetobutylicum [78].
Õàðàêòåðèñòèêè ïðîìîóòåðîâ ìíîæåñòâà
êëîñòðèäèé èäåíòèôèöèðîâàíû ñ ïîìîùüþ ðå-
ïîðòåðíîé ñèñòåìû, êîòîðàÿ îñíîâûâàëàñü íà
ãåíå lacZ [79] èç Thermoanaerobacterium ther-
mosulfurigenes (êîäèðóåò -ãàëàêòîçèäàçó) è ãåíå
lucB [80] èç Photinus pyralis (êîäèðóåò ëþöèôå-
ðàçó). Ýôôåêòèâíûå ñèñòåìû íîêäàóíà ðàçðà-
áîòàíû òîëüêî äëÿ ïÿòè ãåíîâ (buk, pta, adhE,
solR, spo0A) C. acetobutylicum [81–84]. Äëÿ êëî-
ñòðèäèé ðàçðàáîòàíà è äðóãàÿ ñèñòåìà íîêäàó-
íà ãåíîâ [85]. Ìîáèëüíûå ãðóïïû ²² èíòðîíîâ
Lactococcus lactis ñîçäàâàëè êîìïëåêñ ìåæäó
ÄÍÊ ìèøåíÿìè è ÐÍÊ èíòðîíà â ÐÍÏ êîì-
ïëåêñå (èíòðîíêîäèðóþùèé áåëîê è âûðåçàí-
íûé èíòðîí ÐÍÊ êîìïëåêñà) [86]. Â ðåçóëüòàòå
ïîëó÷àëèñü óïðàâëÿåìûå ãåíû ìèøåíè, à òàê-
æå âîçìîæíîñòü ïðåäñêàçàíèÿ öåëåâûõ ñàéòîâ
è äèçàéí ïðàéìåðîâ äëÿ èíòðîíà [87]. Ñ ïîìî-
ùüþ òàêîé ñèñòåìû ñîçäàíî íåñêîëüêî ìóòàíò-
íûõ øòàììîâ. Â íàñòîÿùåå âðåìÿ ýòà ñèñòåìà
ÿâëÿåòñÿ ñàìîé ýôôåêòèâíîé äëÿ íîêäàóíà ãå-
íîâ [85].
Ïåðâûì ýôôåêòèâíûì ðåçóëüòàòîì ìåòàáî-
ëè÷åñêîé èíæåíåðèè áûëî óâåëè÷åíèå ïðîèç-
âîäñòâà àöåòîíà. Ýíçèì, ôîðìèðóþùèé àöå-
òîí, ðåêîìáèíàíòíîãî øòàììà C. acetobutylicum
ñ àìïëèôèöèðîâàííûìè adc (êîäèðóåò àöåòî-
àöåòàò äåêàðáîêñèëàçó) è ctfAB (êîäèðóåò ÊîÀ
òðàíñôåðàçó) ãåíàìè â ïðîöåññå ôåðìåíòàöèè,
ñòàíîâèëñÿ àêòèâíûì íàìíîãî ðàíüøå, ÷åì
îáû÷íî, ïîýòîìó ïðîèñõîäèëà ðàííÿÿ èíäóê-
öèÿ ôîðìèðîâàíèÿ àöåòîíà. Ýòî ïðèâåëî ê
óâåëè÷åíèþ êîíöåíòðàöèè àöåòîíà, áóòàíî-
ëà è ýòàíîëà íà 95, 37 è 90 % ñîîòâåòñòâåííî
ïî ñðàâíåíèþ ñ ðîäèòåëüñêèì øòàììîì [88].
Àìïëèôèêàöèÿ ãåíà adhE (êîäèðóåò àëüäåãèä
äåãèäðîãåíàçó) íå ïðèâåëà ê óâåëè÷åíèþ ïðî-
èçâîäèòåëüíîñòè ðàñòâîðèòåëåé. Îäíàêî êîãäà
ðîäèòåëüñêèé øòàìì ïðîèçâîäèë ìåíåå 10 ã/ë
áóòàíîëà, òî ãèïåðýêñïðåññèÿ adhE óâåëè÷èâà-
ëà âûõîä ýòàíîëà [89, 84].
Äëÿ ïîíèìàíèÿ ðåãóëÿöèîííîé ñèñòåìû
ôîðìèðîâàíèÿ ðàñòâîðèòåëåé äàëüíåéøèå èñ-
ñëåäîâàíèÿ áûëè íàïðàâëåíû íà âûÿñíåíèå
õàðàêòåðèñòèêè ýêñïðåññèè ãåíà solR (êîäè-
ðóåò ðåãóëÿòîð ïðîäóöèðóþùèõ ãåíîâ ðàñòâî-
ðèòåëåé) ìóòàíòíîãî øòàììà. Ãåí solR ÿâëÿëñÿ
òðàíñêðèïöèîííûì ðåïðåññîðîì è íåãàòèâíûì
ðåãóëÿòîðîì ìåòàáîëèçìà ïðîäóêöèè ðàñòâîðè-
òåëåé. Óäàëåíèå ðåãèîíà ãåíà solR óâåëè÷èâàëî
âûõîä áóòàíîëà [90–92].
Èçó÷åíî òàêæå âëèÿíèå siÐÍÊ íà ôîðìè-
ðîâàíèå ôåðìåíòîâ C. acetobutylicum [93]. Äëÿ
ãåíîâ pta è buk ðàçðàáîòàíû siÐÍÊ, èñïîëüçî-
âàíèå êîòîðûõ ïðèâåëî ê ñîêðàùåíèþ àêòèâ-
íîñòè áóòèðàòêèíàçû è ôîñôîòðàíñáóòèðèëàçû
íà 85 è 70 % ñîîòâåòñòâåííî.
Òîëåðàíòíîñòü ê ðàñòâîðèòåëÿì ÿâëÿåòñÿ
îòâåòîì íà ñòðåññ áàêòåðèé C. acetobutylicum è
âêëþ÷àåò â ñåáÿ ãèïåðýêñïðåññèþ ìíîãèõ ãå-
íîâ. Èçáûòî÷íàÿ ýêñïðåññèÿ áåëêîâ òåïëîâî-
ãî øîêà GroES è GroEL ïîçâîëèëà óâåëè÷èòü
âûõîä ðàñòâîðèòåëåé. Ñêðèíèíã ãåíîìíîé áè-
áëèîòåêè äëÿ èäåíòèôèêàöèè îòäåëüíûõ ãåíîâ
ìîæíî èñïîëüçîâàòü äëÿ ïîâûøåíèÿ òîëåðàíò-
íîñòè øòàììîâ ê ðàñòâîðèòåëÿì [94–97].
Ðàñøèôðîâêà ãåíîìà C. acetobutylicum ATCC
824 ïîçâîëèëà íà÷àòü ãåíåòè÷åñêèå ìàíèïóëÿ-
öèè è ïîñëóæèëà òîë÷êîì ê èçó÷åíèþ ìåõàíèç-
ìîâ äåéñòâèÿ ðàçëè÷íûõ ãåíîâ [98]. Ðåçóëüòàòû
62 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Å.À. Òèãóíîâà, Ñ.Ì. Øóëüãà, ß.Á. Áëþì
ôåðìåíòàöèè ïðè ãåíåòè÷åñêèõ ìàíèïóëÿöèÿõ
ñ áàêòåðèÿìè C. acetobutylicum ïðåäñòàâëåíû â
òàáë. 5. Ïðîöåññ ñèíòåçà ðàñòâîðèòåëåé òåñíî
ñâÿçàí ñ ïðîöåññîì ñïîðóëÿöèè. Òðàíñêðèï-
öèîííûé ôàêòîð, îòâåòñòâåííûé çà àêòèâàöèþ
ñïîðóëÿöèè (Spo0A), òàêæå èíèöèèðóåò ñèíòåç
ðàñòâîðèòåëåé â Ñ. àcetobutylicum ñ ïîìîùüþ
àêòèâàöèè ãåíîâ òðàíñêðèïöèè àöåòîàöåòàòà
äåêàðáîêñèëàçû (adc), àëêîãîëüäåãèäðîãåíàçû
(adhE) è ÊîÀ òðàíñôåðàçû (ctfAB) [3].
Òàáëèöà 5. Ôåðìåíòàöèÿ ðàçíûõ ãåíåòè÷åñêè èçìåíåííûõ øòàììîâ C. acetobutylicum [3]
Íàäñèíòåç
ãåíà
Óãíå-
òåíèå
ãåíà
Àöå-
òàò
Áóòè-
ðàò
Àöå-
òîí
Áóòà-
íîë
Ýòà-
íîë
Ñóììàð-
íîå êî-
ëè÷åñòâî
ðàñòâî-
ðèòåëåé
ÁÝ/À Êîììåíòàðèè
Ññûë-
êà
Êîíòðîëü 4,2 6,7 4,5 9,5 0,74 14,7 2,28 Âûõîä ðàñòâîðèòåëåé íà
50 % âûøå (ã/ë) íà ãëþ-
êîçå
[88]
adc, ctfAB – 0,6 0 8,7 13 1,4 23,1 1,66
Êîíòðîëü 3,2 3,2 3,8 5,5 0,7 16,4 1,63 ÁÝ/À óâåëè÷åí [98]
adhE – 1,9 0,4 4,2 8,7 1,9 14,8 2,53
pSOL1 6,4 14,9 0 0 0 0 – Àöåòîí íå ïðîèçâîäèëñÿ/
adhE îòâå÷àåò çà ÀÁÝ ïðî-
äîêöèþ
[89]
adhE pSOL1 6,1 8,7 0 6,2 0,4 6,6 –
Êîíòðîëü 6,7 9,5 4,6 9,7 0,5 14,8 2,2 Óðîâåíü áóòèðàòà èìååò ðå-
øàþùåå çíà÷åíèå äëÿ ñèí-
òåçà áóòàíîëà
[82]
– pta 5,2 14 4,2 9,9 0,6 14,7 2,5
– buk 9 3,3 2,3 10,8 0,7 13,8 5
Êîíòðîëü 3,1 3,1 3,8 5,5 0,7 10,0 1,63 Ïîâûøåííûé âûõîä ðàñ-
òâîðèòåëåé íà ãëþêîçå
[84]
solR 1,7 1,9 8,1 17,8 1,0 26,9 2,32
buk 6,7 1,6 4,4 16,7 2,6 23,7 4,38 Äîñòèãíóò âûñîêèé óðî-
âåíü áóòàíîëà
[99]
adhE buk 6,8 1,6 3,8 16,7 4,5 25 5,58
Êîíòðîëü 3,6 3,6 5 11,7 0,73 17,4 2,5 Óïðàâëåíèå ðåãóëÿòîðíû-
ìè ãåíàìè ïðîèçâîäñòâà
ðàñòâîðèòåëåé. Äîñòèãíóò
âûñîêèé óðîâåíü êîíöåí-
òðàöèè áóòàíîëà
[90]
– solR 4,1 1,4 5,6 14,6 4,4 24,6 3,4
adhE solR 5,1 1,1 8,2 17,6 2,1 27,9 2,4
Êîíòðîëü 4 4 4,4 10,4 0,2 15 2,4 asRNA ðåãóëèðîâàíèå, äî-
ñòèãíóò âûñîêèé óðîâåíü
âûõîäà ýòàíîëà
[100]
adhE – 7 0 1,4 10 8,8 20,2 13,4
– atB 6,8 10 0,3 1,2 0,2 1,7 4,7
Äèêèé òèï 4,5 2,2 3 11 1,1 15,1 4 Øòàìì, òîëåðàíòíûé ê
ðàñòâîðèòåëþ
[95]
Êîíòðîëü 5,4 3,5 6 13 1,1 20,1 2,4
groESL – 4,5 3,5 8 17 1,1 26,1 2,3
Ïðèìå÷àíèå. ÁÝ/À – (áóòàíîë+ýòàíîë)/àöåòîí (ã/ã).
Íà îñíîâå èíôîðìàöèè î ãåíîìå ìîæåò
áûòü ïîñòðîåíà ìåòàáîëè÷åñêàÿ ãåíîìíàÿ ìî-
äåëü. Ýòà ìîäåëü äîëæíà ïðîéòè ïðîöåññ ïðî-
âåðêè ïóòåì ñðàâíåíèÿ òåîðåòè÷åñêèõ äàííûõ
è ïîëó÷åííûõ îò ðåàëüíîé ôåðìåíòàöèè. Ñòðà-
òåãèÿ ðàçâèòèÿ èíòåãðèðîâàííîãî ïðîöåññà ïî-
ëó÷åíèÿ áèîòîïëèâà ïðåäñòàâëåíà íà ðèñ. 3.
Äëÿ âûÿâëåíèÿ ãåíîâ-ìèøåíåé è ïóòåé äëÿ
ìàíèïóëÿöèè ñ íèìè, êðîìå ãåíåòè÷åñêîãî
àíàëèçà, íåîáõîäèìî èçó÷èòü òðàíñêðèïòîìíîå
63ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Àëüòåðíàòèâíûé âèä òîïëèâà – áèîáóòàíîë
è ïðîòåîìíîå ïðîôèëèðîâàíèå øòàììîâ â ðàç-
ëè÷íûõ ãåíîòèïè÷åñêèõ è ýêîëîãè÷åñêèõ óñëî-
âèÿõ. Èíòåãðàöèÿ âñåõ ýòèõ ðåçóëüòàòîâ ìîæåò
ïðèâåñòè ê ñîçäàíèþ øòàììà ñ ïîâûøåííûì
âûõîäîì áóòàíîëà.
6.7. Ñáðàæèâàíèå öåëëþëîçû
Íåêîòîðûå êëîñòðèäèè ñèíòåçèðóþò êñèëàíà-
çó è ïîýòîìó ñïîñîáíû èñïîëüçîâàòü êñèëàí –
ãëàâíûé êîìïîíåíò ãåìèöåëëþëîçû – â êà÷åñòâå
îñíîâíîãî èñòî÷íèêà óãëåðîäà. Ñóõàÿ ðàñòèòåëü-
íàÿ áèîìàññà ñîäåðæèò 15–40 % ãåìèöåëëþëîç.
Êðîìå ñáðàæèâàíèÿ, êëîñòðèäèè ñïîñîáíû îñó-
ùåñòâëÿòü ãèäðîëèç öåëëþëîçû ñ ïîìîùüþ ôåð-
ìåíòàòèâíîãî êîìïëåêñà, êîòîðûé íàõîäèòñÿ â
öåëëþëîñîìå êëîñòðèäèé [50, 101].
Áîëüøèíñòâî öåëëîëèòè÷åñêèõ êëîñòðèäèé
â êà÷åñòâå ïðîäóêòîâ ôåðìåíòàöèè ïðîèçâîäÿò
óêñóñíóþ è ìàñëÿíóþ êèñëîòû, äâóîêèñü óãëå-
ðîäà, ãèäðîãåí, ýòàíîë, àöåòàò, ëàêòàò è ôîð-
ìèàò. Ýòè ïðîäóêòû ìîãóò áûòü èñïîëüçîâàíû
â äàëüíåéøåé ïåðåðàáîòêå [102]. Äëÿ ýêîíî-
ìè÷åñêè âûãîäíîãî àöåòîíîáóòèëîâîãî áðîæå-
íèÿ íåîáõîäèìû ñîëüâåíòîãåííûå øòàììû, êî-
òîðûå èñïîëüçóþò âîçîáíîâëÿåìîå è äåøåâîå
ñûðüå. Òàêèì ñûðüåì ìîæåò âûñòóïàòü áèî-
ìàññà íåïèùåâûõ ðàñòåíèé èëè îòõîäû ñåëüñ-
êîãî õîçÿéñòâà.
 ðàáîòå Áåðåçèíîé [103] ïîêàçàíî, ÷òî øòàì-
ìû C. acetobutylicum 6, C. acetobutylicum 7 è C.
acetobutylicum VKPM B-4786 îáëàäàþò âûñîêîé
ãåìèöåëëþëîçíîé àêòèâíîñòüþ, ÷òî îòêðûâàåò
Ðèñ. 3. Ñòðàòåãèÿ ðàçâèòèÿ èíòåãðèðîâàííîãî áèîïðîöåññà äëÿ ïîëó÷åíèÿ áèîòîïëèâà [3]
64 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Å.À. Òèãóíîâà, Ñ.Ì. Øóëüãà, ß.Á. Áëþì
âîçìîæíîñòü èñïîëüçîâàíèÿ äëÿ ïðîèçâîäñòâà
áóòàíîëà äåøåâîé ðàñòèòåëüíîé áèîìàññû, ÷àñ-
òè÷íîé çàìåíû ïøåíè÷íîé ìóêè íà áèîìàññó
â òðàäèöèîííûõ ñðåäàõ.
ßñòðåìñêîé è äð. [104] ïðè ìíîãîêðàòíûõ
«ïåðåñåâàõ» ñåëåêòèðîâàí øòàìì C. thermocel-
lum 5CT ñ âûñîêîé öåëëþëîëèòè÷åñêîé àêòèâ-
íîñòüþ. Òàêîé øòàìì ïîçâîëÿåò ïîëó÷àòü òåð-
ìîñòàáèëüíûå ýíçèìû, ïîýòîìó ïåðñïåêòèâåí
äëÿ èñïîëüçîâàíèÿ â áèîòåõíîëîãè÷åñêèõ ïðî-
öåññàõ äëÿ ïðîèçâîäñòâà ðàñòâîðèòåëåé.
6.8. Àýðîáíûå ïðîäóöåíòû
Êëàññè÷åñêàÿ ôåðìåíòàöèÿ ÀÁÝ øòàììà-
ìè ðîäà Clostridium ïðîõîäèò â àíàýðîáíûõ
óñëîâèÿõ. Îäíèì èç ïóòåé ïîâûøåíèÿ ìàêñè-
ìàëüíîãî âûõîäà áóòàíîëà è ïðîèçâîäèòåëüíî-
ñòè ïðîöåññà áûëî ñîçäàíèå ïðîöåññà ñèíòåçà
áóòàíîëà â àýðîáíûõ óñëîâèÿõ ñ ïîìîùüþ E.
coli. Ñîîòâåòñòâóþùèå ãåíû êëîñòðèäèé, îòâå-
÷àþùèå çà ñèíòåç áóòàíîëà, ïåðåñàæåíû â E.
coli. Êàê ðåçóëüòàò, ïîëó÷åí øòàìì, ñèíòåçèðó-
þùèé áóòàíîë â àýðîáíûõ óñëîâèÿõ, õîòÿ è â
íåáîëüøèõ êîëè÷åñòâàõ [105].
Ïîëó÷åíû è äðóãèå øòàììû-ïðîäóöåíòû,
êîòîðûå ñèíòåçèðîâàëè áóòàíîë â àýðîáíûõ
óñëîâèÿõ [106]. Òàê, íà îñíîâå äðîææåé Sac-
charomyces ñîçäàíû íîâûå øòàììû ñ ïîìîùüþ
ïåðåíîñà ñîîòâåòñòâóþùèõ ãåíîâ èç áèîñèíòåòè-
÷åñêîé ñõåìû ïîëó÷åíèÿ áóòàíîëà â êëîñòðèäè-
ÿõ è äîáàâëåíèÿ íåñêîëüêèõ ýíçèìîâ èç äðóãèõ
ìèêðîîðãàíèçìîâ (S. cerevisiae, E. coli, C. beijerinc-
kii è Ralstonia eutropha). Ïðîàíàëèçèðîâàíû ðàç-
ëè÷íûå ñî÷åòàíèÿ ýíçèìîâ ìèêðîîðãàíèçìîâ è
èõ âëèÿíèå íà ïðîöåññ ñèíòåçà áóòàíîëà. Íàèáî-
ëåå ïðîäóêòèâíûå øòàììû ñîäåðæàëè 3-ãèäðîê-
ñèáóòèðèë-ÊîÀ äåãèäðîãåíàçû, ïîëó÷åííûå èç Ñ.
beijerinckii. Ïîäáîð ñîîòâåòñòâóþùèõ ýíçèìîâ äàë
âîçìîæíîñòü ïîâûñèòü êîíöåíòðàöèþ áóòàíîëà â
êóëüòóðàëüíîé ñðåäå â 10 ðàç.
Ðåçóëüòàòîì èññëåäîâàíèé Lin et al. [107]
áûëî ñîçäàíèå øòàììîâ Lactococcus lactis è
Lactobacillus buchneri ñ âêëþ÷åíèåì ãåíà thl, ïî-
ëó÷åííîãî èç Clostridium beijerinckii P260. Òàêèå
ðåêîìáèíàíòíûå øòàììû ñèíòåçèðîâàëè 28 è
66 ìã/ë áóòàíîëà ñîîòâåòñòâåííî.
Àâòîðû ðàáîòû [108] ñîçäàëè íîâûé øòàìì-
ïðîäóöåíò íà îñíîâå E. coli, êîòîðûé ñèíòåçè-
ðîâàë 30 ã/ë áóòàíîëà. Êðîìå îñíîâíûõ ãåíîâ
Clostridium spp., ó÷àñòâóþùèõ â ñèíòåçå áóòà-
íîëà, â E. coli áûë âñòðîåí ãåí òðàíñ-ýíîóë-
ÊîÀ-ðåäóêòàçû (Ter) èç Treponema denticol. Ýòîò
ýíçèì èìåë ñïåöèôè÷åñêóþ àêòèâíîñòü ïî îò-
íîøåíèþ ê êðîòîíèë-ÊîÀ ðåäóêòàçå è èñïîëü-
çîâàë ÍÀÄÍ. Òàêîé ïóòü ñèíòåçà áóòàíîëà ñ
èñïîëüçîâàíèåì òðàíñ-ýíîóë-ÊîÀ-ðåäóêòàçû
ïîçâîëÿë îñóùåñòâèòü ñèíòåç ïî àëüòåðíàòèâ-
íîé ñõåìå, áåç âîâëå÷åíèÿ ôåððåäîêñèíà èëè
ôëàâîïðîòåèíà.
 ðåçóëüòàòå ýòèõ èññëåäîâàíèé óñòàíîâëå-
íî, ÷òî ÍÀÄÍ è àöåòèë-ÊîÀ ÿâëÿþòñÿ îñíîâ-
íûìè ðåãóëÿòîðàìè, êîòîðûå ìîãóò óâåëè÷è-
âàòü âûõîä ðàñòâîðèòåëåé. Êðîìå ïîëó÷åíèÿ â
ïðîöåññå ñèíòåçà âûñîêîé êîíöåíòðàöèè áóòà-
íîëà, áûë äîñòèãíóò åùå è âûñîêèé óðîâåíü
ýêîíîìè÷íîñòè (70–88 % îò òåîðåòè÷åñêîãî).
7. Очистка и выделение
Êóëüòóðàëüíàÿ æèäêîñòü ïîñëå ïðîöåññà
êóëüòèâèðîâàíèÿ ñîäåðæèò ñìåñü áóòàíîëà è
äðóãèõ ïðîäóêòîâ. Äëÿ ïîëó÷åíèÿ ÷èñòîãî áóòà-
íîëà íåîáõîäèìî êàêèì-òî îáðàçîì ðàçäåëèòü
ýòó ñìåñü. Îäíèì èç âîçìîæíûõ ïðîöåññîâ
âûäåëåíèÿ áóòàíîëà ÿâëÿåòñÿ èíòåãðèðîâà-
íèå ñòàäèè âûäåëåíèÿ áóòàíîëà ñ ïðîöåññîì
íåïðåðûâíîé ôåðìåíòàöèè – òàê íàçûâàåìàÿ
ýêñòðàêòèâíàÿ ôåðìåíòàöèÿ. Ïðîâåäåíèå ïðî-
öåññà ïî òàêîé òåõíîëîãèè ïîçâîëèëî ìèíèìè-
çèðîâàòü èíãèáèðóþùåå äåéñòâèå áóòàíîëà è
óìåíüøèòü ýíåðãîçàòðàòû íà êîíöåíòðèðîâà-
íèå öåëåâîãî ïðîäóêòà.
Ñóùåñòâóþò òðè îñíîâíûõ ñïîñîáà âûäåëå-
íèÿ îòäåëüíûõ êîìïîíåíòîâ èç ñìåñè êîíå÷-
íûõ ïðîäóêòîâ ôåðìåíòàöèè: ðåêòèôèêàöèÿ,
ïåðâàïîðàöèÿ è ïðîòèâîòî÷íàÿ æèäêîñòíàÿ
ýêñòðàêöèÿ (ðèñ. 4).
Ïðîöåññ ïåðâàïîðàöèè – âûäåëåíèå îðãà-
íè÷åñêèõ ïðîäóêòîâ èç ýíçèìàòè÷åñêîé ñðåäû.
Îí îñíîâàí íà ìåáðàííîì ñïîñîáå ðàçäåëåíèÿ
æèäêîñòè (èñïàðåíèå ÷åðåç ìåìáðàíó).  ýòîì
ïðîöåññå ñìåñü æèäêèõ êîìïîíåíòîâ (ïèòà-
òåëüíûé ïîòîê) ïðèâîäèëàñü â êîíòàêò ñ îäíîé
ñòîðîíû ìåìáðàíû, à ïåðìèàò – ïðîäóêò, îáîãà-
ùåííûé öåëåâûìè êîìïîíåíòàìè ñìåñè, êîòî-
ðûå ïðîíèêàþò ÷åðåç ìåìáðàíó, óäàëÿëñÿ â
âèäå ïàðà ñ îáðàòíîé ñòîðîíû ìåìáðàíû. Ïåð-
âàïîðàöèÿ îòëè÷àåòñÿ îòíîñèòåëüíî íèçêèì
óäåëüíûì ýíåðãîïîòðåáëåíèåì ïî ñðàâíåíèþ
ñ ìåìáðàííûìè òåõíîëîãèÿìè, èñïîëüçóþùè-
ìè ïîðèñòûå ìåìáðàíû. Ðàçäåëåíèå ïðîäóêòîâ
65ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Àëüòåðíàòèâíûé âèä òîïëèâà – áèîáóòàíîë
ôåðìåíòàöèè èäåò íà ìîëåêóëÿðíîì óðîâíå, ÷òî
ïîâûøàåò èçáèðàòåëüíîñòü âûõîäà ïðîäóêòà, à
òàêæå åãî ÷èñòîòó.
Äëÿ ïîääåðæàíèÿ è îáåñïå÷åíèÿ ñòàöèî-
íàðíîãî ðàçäåëåíèÿ ïåðâàïîðàöèþ ïðîâîäèëè
ðàçëè÷íûìè ìåòîäàìè: ñ ïîìîùüþ âàêóìà èëè
â ïîòîêå ãàçà-íîñèòåëÿ. Â ïðîìûøëåííîñòè
áëàãîäàðÿ ïðîñòîòå ðåàëèçàöèè è ìèíèìàëüíîé
ïîòðåáíîñòè â îáîðóäîâàíèè, îáû÷íî èñïîëü-
çóåòñÿ èìåííî âàêóóìíàÿ ïåðâàïîðàöèÿ. Ïðî-
öåññ ïåðâàïîðàöèè âêëþ÷àåò â ñåáÿ òðè ïîñ-
ëåäîâàòåëüíûå ñòàäèè: ñåëåêòèâíàÿ ñîðáöèÿ íà
âõîäíîé ïîâåðõíîñòè ìåìáðàíû; ñåëåêòèâíàÿ
äèôôóçèÿ ÷åðåç ìåìáðàíó; äåñîðáöèÿ â ïàðî-
îáðàçíóþ ôàçó íà âûõîäíîé ïîâåðõíîñòè. Äëÿ
î÷èñòêè áóòàíîëà èñïîëüçîâàëè ðàçëè÷íûå âèäû
ìåìáðàí. Ðàçðàáîòàíûå ñïåöèàëüíî äëÿ î÷èñòêè
áóòàíîëà ìåìáðàíû äàëè âîçìîæíîñòü â äâà
ðàçà ïîâûñèòü âûõîä è ñêîðîñòü íàêîïëåíèÿ
ïðîäóêòà [109–111].
Âòîðûì ñïîñîáîì ÿâëÿåòñÿ ïîëó÷åíèå î÷è-
ùåííîãî áóòàíîëà ñ ïîìîùüþ ðåêòèôèêàöèè.
 ýòîì ïðîöåññå ðàçäåë ñìåñè ïðîäóêòîâ ôåð-
ìåíòàöèè ïðîõîäèë çà ñ÷åò ïðîòèâîòî÷íîãî
ìàññî- è òåïëîîáìåíà ìåæäó ïàðîì è æèäêî-
ñòüþ. Ðåêòèôèêàöèÿ ïðîâîäèëàñü êàê ïðè íå-
ïðåðûâíîé, òàê è ïðè ïåðèîäè÷åñêîé ôåðìåí-
òàöèè. Ðàçäåëåíèå ýíçèìàòè÷åñêîé ñìåñè íà
ïðàêòè÷åñêè ÷èñòûå êîìïîíåíòû ïðîèñõîäèëî
çà ñ÷åò ðàçëè÷íûõ òåìïåðàòóð êèïåíèÿ ïðîäóê-
òîâ, ïóòåì ìíîãîêðàòíûõ èñïàðåíèé æèäêîñòè
è êîíäåíñàöèè ïàðîâ. Èíòåãðèðîâàíèå ðåêòè-
ôèêàöèè â ôåðìåíòàöèîííûé ïðîöåññ óâåëè-
÷èëî áèîêîíâåðñèþ ñàõàðîâ â äâà ðàçà è ïîçâî-
ëèëî èñïîëüçîâàòü ïîâûøåííûå êîíöåíòðàöèè
ñàõàðà äëÿ ñáðàæèâàíèÿ [112–115].
Ïðè ðàçäåëåíèè áóòàíîëà íà óñòàíîâêå
ïðîòèâîòî÷íîãî äåéñòâèÿ èñïîëüçóåòñÿ ðàçëè-
÷èå â êîýôôèöèåíòàõ ðàñïðåäåëåíèÿ âåùåñòâ.
Áóòàíîë õîðîøî ðàñòâîðÿåòñÿ â îðãàíè÷åñêèõ
ðàñòâîðèòåëÿõ (ýêñòðàãåíò) è ãîðàçäî õóæå â
âîäíîé ôàçå (ýíçèìàòè÷åñêàÿ ñìåñü), ïîýòîìó
áóòàíîë èçáèðàòåëüíî êîíäåíñèðîâàëñÿ â ýêñ-
òðàãåíòå. Äëÿ âûäåëåíèÿ è î÷èñòêè áóòàíîëà
÷àùå âñåãî èñïîëüçîâàëè èçâåñòíûå ýêñòðàãåí-
òû, âêëþ÷àÿ äåêàíîë è îëåèëîâûé ñïèðò. Äëÿ
îãðàíè÷åíèÿ òîêñè÷åñêîãî âîçäåéñòâèÿ ýêñ-
òðàãåíòà íà êóëüòóðû êóëüòèâàöèîííàÿ ñðåäà
áûëà îòäåëåíà ìåìáðàíîé (ïðîöåññ «ïåðñòðàê-
øîí»). Âûñîêèå ïîêàçàòåëè ñêîðîñòè íàêîïëå-
íèÿ áóòàíîëà ïîëó÷åíû ïðè èíòåãðèðîâàíèè
æèäêîñòíîé ýêñòðàêöèè c èñïîëüçîâàíèåì
äåêàíîëà è îëåèëîâîãî ñïèðòà â ïðîöåññå íå-
ïðåðûâíîãî êóëüòèâèðîâàíèÿ C. acetobutylicum
[116–118].
Заключение
Âñå áèîòåõíîëîãè÷åñêèå ïðîöåññû ñóùå-
ñòâåííî çàâèñÿò îò ïðîäóêòèâíîñòè øòàììà è
óñëîâèé ôåðìåíòàöèè. Â ïðîèçâîäñòâå áóòàíî-
Ðèñ. 4. Ñõåìà íåïðåðûâíîãî òåõíîëîãè÷åñêîãî ïðîöåññà ôåðìåíòàöèè è ýêñòðàêöèè áóòàíîëà: a – ðåêòèôè-
êàöèÿ, á – æèäêîñòíàÿ ýêñòðàêöèÿ, â – ïåðâàïîðàöèÿ [3]
66 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 6
Å.À. Òèãóíîâà, Ñ.Ì. Øóëüãà, ß.Á. Áëþì
ëà ýòó ïðîáëåìó ðåøàþò ðàçëè÷íûìè ñïîñîáà-
ìè: ñåëåêöèåé è ãåííîé èíæåíåðèåé øòàììîâ-
ïðîäóöåíòîâ, îïòèìèçàöèåé ýíçèìàòè÷åñêîé
ñðåäû è ñàìîãî ïðîöåññà ôåðìåíòàöèè. Îáúå-
ìû âûõîäà áóòàíîëà ïðè èñïîëüçîâàíèè ðàç-
ëè÷íûõ èñòî÷íèêîâ óãëåðîäà ìîæíî óâåëè÷èòü
çà ñ÷åò îïòèìèçàöèè ìåòàáîëè÷åñêèõ ïóòåé
ñèíòåçà.
 äîïîëíåíèå ê èíæåíåðèè ìåòàáîëè÷åñêèõ
ïóòåé ìîæíî îïòèìèçèðîâàòü ãëîáàëüíûå ðåãó-
ëÿòîðû ñèíòåçà è ôàêòîðû òðàíñêðèïöèè, êî-
òîðûå ñîïðîâîæäàþò êëåòî÷íûå áåëêè, óâåëè-
÷èâàþùèå âûõîä áóòàíîëà è ñïîñîáñòâóþùèå
òîëåðàíòíîñòè øòàììîâ.
Ðàçðàáîòêà íîâûõ ïîäõîäîâ ê ïðîöåññó ôåð-
ìåíòàöèè íà îñíîâå Ì-øòàììîâ ìîæåò îêàçàòü
ñóùåñòâåííîå âëèÿíèå íà ýêîíîìèêó ïðîöåññà
ïîëó÷åíèÿ áèîáóòàíîëà.
Îäíèì èç íàèáîëåå âàæíûõ ôàêòîðîâ íå
òîëüêî äëÿ ïîëó÷åíèÿ áèîáóòàíîëà, íî è äëÿ
âñåé èíäóñòðèè áèîòîïëèâ ÿâëÿåòñÿ èñïîëü-
çîâàíèå äåøåâûõ ñóáñòðàòîâ. Âàæíîå çíà÷å-
íèå áóäåò èìåòü òàêæå ñòîèìîñòü ïðîèçâîäñòâà
áèîìàññû (ñûðüÿ), åå òðàíñïîðòèðîâêè è õðà-
íåíèÿ. Îñîáîå ìåñòî â ïîëó÷åíèè áèîòîïëèâ
îòâîäèòñÿ èñïîëüçîâàíèþ öåëëþëîçíîé è ëèã-
íîöåëëþëîçíîé áèîìàññû. Ïðîöåññ ïðîèçâîä-
ñòâà áóòàíîëà ìîæåò ñòàòü êîíêóðåíòíûì íà
îñíîâå ñèñòåìíîãî ïîäõîäà ê èçó÷åíèþ âñåõ
àñïåêòîâ ôåðìåíòàöèè, ìåòàáîëè÷åñêîé èíæå-
íåðèè øòàììîâ ñ ó÷åòîì îïðåäåëåíèÿ ïîëíûõ
ïîñëåäîâàòåëüíîñòåé ãåíîìîâ è âûáîðà àëüòåð-
íàòèâíîãî äåøåâîãî ñûðüÿ.
O. Tigunova, S. Shulga, Y. Blume
Institute of Food Biotechnology and Genomics
of National Academy of Sciences of Ukraine, Kyiv
E-mail: Shulga5@i.ua
ALTERNATIVE TYPE OF FUEL – BIOBUTANOL
Butanol – an alternative fuel that on amid dwindling
global (accessible) oil reserves can serve as a source of
energy. In the industrial-scale butanol is produced by
chemical synthesis, although initially butanol production
was due to microbiological synthesis. For cost-efficient
production, a strain of microorganisms must have over
production of butanol. In the review of butanol synthesis
pathway with the help of microorganisms, their regu-
lation, the principles and techniques of increasing the
productivity of the most promising strains and producer
strains for industrial production.
Î.Î. Ò³ãóíîâà, Ñ.Ì. Øóëüãà, ß.Á. Áëþì
ÀËÜÒÅÐÍÀÒÈÂÍÈÉ ÂÈÄ ÏÀËÈÂÀ –
Á²ÎÁÓÒÀÍÎË
Áóòàíîë – àëüòåðíàòèâíå ïàëèâî, íà òë³ ñâ³òîâèõ
(äîñòóïíèõ) çàïàñ³â íàôòè, ÿê³ âèñíàæóþòüñÿ, º ïî-
òåíö³éíèì äæåðåëîì åíåð㳿. Ó ïðîìèñëîâèõ ìàñøòà-
áàõ áóòàíîë îäåðæóþòü õ³ì³÷íèì ñèíòåçîì, õî÷à
ñïî÷àòêó âèðîáíèöòâî áóòàíîëó áóëî ïîâ’ÿçàíî ç
ì³êðîá³îëîã³÷íèì ñèíòåçîì. Äëÿ åêîíîì³÷íî âèã³ä-
íîãî âèðîáíèöòâà øòàìè ì³êðîîðãàí³çì³â ïîâèíí³
ìàòè çäàòí³ñòü äî íàäñèíòåçó áóòàíîëó.  îãëÿä³
ðîçãëÿíóòî øëÿõè ñèíòåçó áóòàíîëó ì³êðîîðãàí³ç-
ìàìè ³ ¿õ ðåãóëÿö³ÿ, íàéá³ëüø ïåðñïåêòèâí³ øòàìè-
ïðîäóöåíòè äëÿ ïðîìèñëîâîãî âèðîáíèöòâà òà ìå-
òîäè ï³äâèùåííÿ ïðîäóêòèâíîñò³ øòàì³â.
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]
/OmitPlacedBitmaps false
/OmitPlacedEPS false
/OmitPlacedPDF false
/SimulateOverprint /Legacy
>>
<<
/AddBleedMarks false
/AddColorBars false
/AddCropMarks false
/AddPageInfo false
/AddRegMarks false
/ConvertColors /ConvertToCMYK
/DestinationProfileName ()
/DestinationProfileSelector /DocumentCMYK
/Downsample16BitImages true
/FlattenerPreset <<
/PresetSelector /MediumResolution
>>
/FormElements false
/GenerateStructure false
/IncludeBookmarks false
/IncludeHyperlinks false
/IncludeInteractive false
/IncludeLayers false
/IncludeProfiles false
/MultimediaHandling /UseObjectSettings
/Namespace [
(Adobe)
(CreativeSuite)
(2.0)
]
/PDFXOutputIntentProfileSelector /DocumentCMYK
/PreserveEditing true
/UntaggedCMYKHandling /LeaveUntagged
/UntaggedRGBHandling /UseDocumentProfile
/UseDocumentBleed false
>>
]
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice
|