Transcript 발효화학-8.

발효화학 (Fermentation Chemistry), Bacterial Physiology and Metabolism
Chapter 8. Anaerobic Fermentation
Yong-Cheol Park
(http://park.openwetware.org)
Department of Advanced Fermentation
Fusion Science & Technology
Introduction
 Anaerobic conditions are maintained in some ecosystems where the rate of
oxygen supply is lower than that of consumption.
 Organic compound are removed from anaerobic ecosystems through the
concerted action of fermentative and anaerobic respiratory microorganisms
 In microbiology, the term ‘fermentation’ can be used to describe either
(1) microbial processes that produce useful products or
(2) a form of anaerobic microbial growth using internally supplied electron
acceptors and generating ATP mainly through substrate-level phosphorylation
(SLP).
 This chapter describes the fermentation processes carried out by various
anaerobic prokaryotes.
 In fermentation, ATP is generated not only through SLP but also by other
mechanisms such as the reactions catalyzed by fumarate reductase and Na+dependent decarboxylase, and lactate/H+ symport as described earlier (Sec.5.8.6).
8.1 Electron acceptors used in anaerobic metabolism
8.1.1 Fermentation and anaerobic respiration
 Respiration : the reduction of oxygen by electrons from the electron transport
chains coupled to the generation of a proton motive force through electron
transport phosphorylation (ETP, Sec.5.*)
 Anaerobic respiration : ETP process with externally supplied oxidized compounds
other than oxygen as the terminal electron acceptor under anaerobic conditions
 Fermentative process : ATP generation through SLP with the oxidation of
electron donors coupled to the reduction of electron carriers such as NAD(P)+
or flavin adenine dinucleotide (FAD). The reduced electron carriers (NAD(P)H or
FADH2) are reoxidized reducing the metabolic intermediate.
8.1.2 Hydrogen in fermentation
 The ratio of product formed/substrate consumed is constant in some
fermentations such as the ethanol (Sec.8.3) and homolactate (Sec.8.4)
fermentations.
 While the ratio is variable in others such as the clostridal fermentation (Sec.8.5).
8.1 Electron acceptors used in anaerobic metabolism
8.1.2 Hydrogen in fermentation (continued)
 Fermentation process with a constant ratio are referred to as linear pathways,
while the others are referred to as branched fermentative pathways (Fig.8.1).
 A branched pathway yields more ATP and more oxidized products than a linear
pathway.
 To produce more oxidized products, a proportion of the reduced electron
carriers such as NAD(P)H should be oxidized, coupled to the reduction of H+ to
H2.
 The formation of products in a branched pathway is dictated by the
environmental growth
conditions, especially
the hydrogen partial
pressure.
8.2 Molecular oxygen and anaerobes
 Classification of microbes according to their response to molecular oxygen (O2),
(1) aerobes, (2) facultative anaerobes,
(3) obligate anaerobes (aerotolerant obligate anaerobes and strict anaerobes)
 O2 comprises about 20% of air, and air-saturated liquid media contains about
7~8 mg/L O2 at ambient temperature.
 Strict anaerobes and microaerophiles are inhibited by molecular oxygen or it
metabolites.
[Hypotheses of the inhibitory mechanism]
 Molecular oxygen reacts with reduced flavoproteins, Fe-S proteins and
cytochromes to be reduced to hydrogen peroxide (H2O2) or superoxide (O2.-),
 Which are very strong oxidants with high redox pontentials and destroy cellular
polymers such as DNA, RNA, proteins and other essential components.
8.2 Molecular oxygen and anaerobes
 Superoxide dismutase (SOD), catalase, peroxidase : enzymes detoxifying
hydrogen or superoxide present in aerobes and facultative anaerobes.
 Strict anaerobes have been thought to be
sensitive against O2 because they may not
possess SOD and catalase.
 Though some strict anaerobes have SOD
and catalase activities (Table8.1), they do not
grow under aerobic conditions in the Lab.
 because dissolved oxygen increases the
redox potential and a high redox potential
inhibits the growth of some strict anaerobes.
8.3 Ethanol fermentation
 An alcohol yeast, Saccharomyces cerevisiae ferments carbohydrates through the
EMP pathway to ethanol.
 A bacterium, Zymomonas mobilis produce ethanol via the ED pathway.
 Pyruvate is decarboxylated to
acetaldehyde, which is used as the
electron acceptor.
 Acetaldehyde is reduced to ethanol,
which consumes the electrons
generated during the glycolytic
process where ATP is generated
through SLP (Fig.8.2).
8.3 Ethanol fermentation
 S. cerevisiae generate 2 ATP from 1 hexose molecule but Z. mobilis does a single
ATP from 1 hexose molecule.
 Pyruvate decarboxylase has thiamine pyrophosphate as a prosthetic group as in
pyruvate dehydrogenase and is key enzyme of ethanol fermentation.
 Pyruvate decarboxylase is known mainly in eukaryotes.
 In addition to Z. mobilis, pyruvate decarboxylase is found in a facultative anaerobe,
Ervinia amylovora, and in a strictly anaerobic acidophile, Sarcina ventriculi.
 Saccharolytic clostrida, heterofermentative lactic acid bacteria and enteric bacteria
oxidize pyruvate to acetyl-CoA before reducing it to ethanol.
 Because these bacteria do not
possess pyruvate
decarboxylase.
8.3 Ethanol fermentation
 Ethanol fermentation through pyruvate decarboxylase in a linear fermentative
pathway does not produce any by-products except CO2 and water,
 While ethanol fermentation through acetyl-CoA is a branched fermentative
pathway and produces various fermentation products such as lactate, acetate and
H2.
포도주 양조기술
포도주 개요
• 포도 및 그 즙액을 발효한 알코올 함유 음료
Wine의 정의 : 과실, 딸기 등의 속 혹은 그 즙액 또는 꿀과 같
은 적당한 농산물의 발효로 만들어진 알코올함유 음료
• 과실주란?
과실즙을 주된 원료로 하여 당질 또는 효모, 물을 첨가하
여 발효시킨 주요를 여과, 제성한 것
(법제 3조 제 5호)
• 당분첨가량(원료중량 대비)
AL. 10%이하 : 100 분의 30
AL. 10%이상 : 100 분의 50
와인의 분류
1) 색에 의한 분류
• 화이트 와인(White wine)
- 당분 함량 小, 숙성기간 짧다
- 연한녹색에서 황색까지의 와인을 총칭
• 레드와인(Red wine)
- 색 : 암적색 – 담적색 와인
- 수확된 포도의 껍질까지 즙을 내어 발효한 와인
• 로제와인(Rose wine)
- 홍색(pink)색 와인
- 백포도주+적포도주 혼합방법
와인의 분류
2) 성질에 의한 분류
• 비발포성 와인(Natural still wine)
- 보편적 와인, 순수한 자연상태 포도
• 발포성 와인(Sparking wine)
- 프랑스 상파뉴 지방의 삼패인
- 2차 발효에서 생긴 탄산가스를 함유한 발포성 와인
• 주정강화 와인(Fortified wine)
- 발효과정에 브랜디를 첨가 발효를 정지함
- 알코올 20%내외 드라이(dry)  스위트(sweet)
• 혼성와인(Aromatized wine)
- 약초, 향료, 색소, 감미재료를 첨가한 맛과 항을 갖는 와인
- 벌무스(Vermouth), 두보넷(Dubonnet), 아메르피콘(Amerpicon) 등
와인의 분류
3) 맛에 의한 분류
• 스위트 와인(Sweet wine)
- 당분이 남아있는 상태에서 발효를 중지, 또는 당분 가미
- 식후주와인(Dessert wine) : 소화촉진효과
- 포트와인, 크림셔리
• 드라이 와인(Dry wine)
- 당분이 거의 없는 와인
- 식욕촉진 주 와인
• 미디엄 드라이 와인(Midium dry wine)
- 단맛이 약간 있는 와인
- Semi Dry
와인의 분류
4) 식사에 의한 분류
• 식전주 와인(Appetizer wine)
- 식욕촉진을 위해 식전에 제공, 전체요리와 함께 마심
• 테이블 와인(Table wine)
- 식사 중 사용, 주요리(main dish)와 함께 마심
- 백포도주 : 흰살의 육류, 생신
- 적포도주 : 붉은 살의 육류
• 식후주와인(Dessert wine)
- 식사 후 소화촉지
- 포트와인(port wine), 크림셰리(Cream sherry)
• 기타
- Young wine(1-2년), Age wine(5-15년), Great wine(15년 이상)
포도주 제품 특성
• 빛깔- 화이트와인: 순백색으로부터 호박색
• 향기- 고유향 Aroma, 숙성향 부케(bouquet)
• 맛
- 느끼하지 않고 은은한 감미
• 투명도- 크리스탈과 같이 광채, 맑은 상태
• 수확년도- 와인의 품질에 큰 영향
포도주의 보관 및 마시는 법
• 보관방법
- 보관시 직사광선을 피하고 온도변화가 거의 없는 곳
- Cork 마개로 밀봉된 와인 눕혀서 보관
• 마시는법
- 빛깔, 향기, 맛을 음미
포도주의 기능성 (프랜치 패러독스)
• CHD(coronary heart disease)로 인한 사망률을 낮춘다.
• 동맥경화감소 – 포도주의 폴리페놀(polyphenol)
• 적포도주의 페놀계화합물의 플라보노이드(flavonoids)
- 플라보놀 (flavonols)
- 안토시안 (anthocyanins)
- 카테킨 (catechins),
- 프로시아니딘 (카테킨의 oligomer), 탄닌 (tannins)
• 항산화작용, 종양 발생 및 진행의 억제 효과
• 혈소판 응집의 억제, 항염증제, 항균제, 중금속에 의한 독성의 억제
 resveratrol (레스베라트롤)
* 포도주의 해로운 성분 - 펙틴성분에서 유래되는 메탄올, 발효 전 원료에
아황산 처리 후 남아있는 SO2, ethyl carbamate(urethane), diethylene
glycol(DEG), histamine, tyramine
포도주의 제조 공정도
포도
파쇄
제경
아황산 참가
가당
과즙
발효
압착여과
백포도주의 경우
효모
종자, 과피 제거
후발효
앙금분리
저장
여과
병입
제품
적포도주의 제조
1) 원료포도 품종의 선택
• Red - Cabernet Sauvignon, Merlot, Pinot Noir, Shiraz,
• White – Chardonnay, Riesling, Sauvignon blanc
2) 포도의 주요성분
•
•
•
•
•
•
•
포도과 중 과경 2 - 5%
과즙량 80 – 90 %
종자 0 – 5%, 과피량 5 – 12%
보통 1,000kg의 포도로 부터 신선 과즙 650 – 750L
당분 : 포도당(glucose)와 과당(fructose) 이 1:1 함유
유기산 (1.0%) : 주석산 0.5%, 사과산 0.2%, 구연산 0.05%
색소 : 안토시아닌(anthocyanin)
적포도주의 제조
3) 원료포도의 수확과 운반
• 포도밭에서 파쇄한 것에 메타카리(술덧 1톤당 메타카리
200g(100ppm)사용) 등 아황산을 가해서 발효를 억제시켜, 제
조장으로 운반
4) 파쇄 및 제경
•
•
•
•
제경은 파쇄 후 실시
파쇄와 제경을 연속적으로 행할 수 있는 파쇄 제경기를 사용
Stainless steel이나 목재 재질 사용함
철분, 구리, 이온 혼입방지
5) 아황산의 첨가
• 건조한 적포도주 술덧에는 100ppm(메타카리를 100L에 대해
20g사용)
• 부패과에는 100 – 200ppm
아황산 (SO2)
아황산의 효과
•
•
•
•
•
중세 때부터 사용되어온 포도주의 산화방지제
과즙의 유해한 미생물의 번식 억제와 살균 효과
와인효모는 아황산에 대한 내성이 강하여 정상 알코올 발효
백포도주의 착색 방지, 적포도주의 색소 추출 상승효과
다양한 향미성분과 결합하여 와인의 향미를 높임
아황산의 첨가방법
•
•
•
•
•
•
방법 : 메타카리(K2S2O5), 무수아황산
포도주 1kg 중 350mg(350ppm)
1L에 메타카리를 약 606mg 첨가한 양에 상당
적포도주 술덧에는 75-100ppm(메타카리로는 100L 당 20g)
메타카리 : SO2함량 57.6%함유
무수아황산 : 가격이 싸다. 많이 사용해도 칼륨의 이미가 없다
적포도주의 제조
6) 밑술(주모)의 첨가
• 제조: 포도과즙 1L  증기살균 30분  효모접종  배양(25oC, 2일)
 10L과즙+메타카리 2g에 접종  12일 배양  밑술
• 소량인 경우(5kg 내외)
0.5% 설탕용액  건조효모 1g c첨가  30분 실온방치  밑술
• 주모첨가량
포도과즙에 아황산 첨가 후 5 – 24시간 경과 후 술덧의 용량에 대하여 3 –
5% 첨가한다
적포도주의 제조
7) 주발효의 관리
• 적포도주에서는 품온 25-30oC로 발효시키는 것이 바람직
• 담금 후 5-7일이 적당
• 30도 이상의 고온에서 발효시킨 것은 생성된 알코올 증발에
의한 결감과 향기의 손실이 있다
• 술덧 중의 과육과 과피가 직접 공기에 접촉되지 않도록 한다
8) 압착
• 예정된 색소와 Tannin이 즙액 중에 용출되고 나면 압착
여과를 하여 과피와 씨를 제거한다
적포도주의 제조
9) 후발효 (2차 발효) – 감산발효 또는 유산발효라고 함
• 주발효 /압착 후 오크통 또는 탱크로 옮겨 잔류당의 발효진행
• 주발효의 탄산가스가 줄어듬
• 적포도주의 사과산(malic acid)가 유산균에 의해 젖산(lactic acid)
로 전환되어 신맛의 감소
• 와인 내의 탁한 색의 효모가 대부분 밑으로 침전
• 후발효를 통해 최상품과 생물학적 안정을 기대함
10) 침전물 제거와 저장
• 효모, 주석 또는 단백질 등이 침전  포도주 맑아짐
• 암금분리 - 효모의 자기분해와 생물적인 감산작용의 방지
- 산화에 의한 숙성을 촉진
• 앙금분리 후 같은 type의 술로 용기에 가득 채운 후 아황산
보충(50ppm)
적포도주의 제조
11) 저장과 숙성
•
•
•
•
•
목통저장 : 나무통 재질의 세공을 통하여 산화되고 변화
병저장 : 공기차단으로 묵힘(aging)이 일어남
저장에 의해 알코올, 산류, 아미노산 등이 산화
특유의 향미가 없어지고, 향기 좋고 맛이 좋은 향미로 발전
저장, 숙성기간
가베르네 쇼비농 품종(떫은 맛이 많은)
- 소형 오크통(200 - 400L) : 2 – 3년 저장
마스카트, 베리 A 품종(맛이 담백, 가벼운 type술)
- 소형 오크통 : 1 – 2년 저장
• 저장온도 : 12 – 15 oC
적포도주의 제조
12) 청 징
• Pectin, 단백질, 금속 등을 제거를 목적으로 함
• 청징제 자체의 성질, 처리량, 처리방법을 참고로 소량 시험 후 적정량
결정
• 청징제는 각각 양전하/음전하를 띄어 상대 물질과 결합, 침전시킴
• 청징제 및 방법
 효소청징
- pectin 분해효소제를 0.01 – 0.1% 첨가
 Gelatin과 Tanin 청징
- Tanin과 단백질이 결합하여 응집, 침전 시 혼탁물질도 침전, 청징됨
- 소량시험 후 필요 최소량 결정
Ex) 포도주 1L  1% 탄닌액 15ml 첨가  1일 방치  1% 젤라틴액 15ml 첨가 후 3 – 4일 경과  청
징
*기타 청징제 : 카제인, 벤토나이트(산성백토)
적포도주의 제조
13) 출하 시 열처리법
 병, Cork마개 살균
- 병 : 약알카리성 세제로 세척 후 수세
- 코르크 : 1.5 – 2.0%의 아황산수에 1 – 2 시간 침적 후 사용
 열처리
- 플레이트 히터 등의 열교환기 사용
- 70 – 80 oC에서 1 – 2분 정도
14) 제품 출하
일반적인 포도주의 성분 함량
일반포도주
(Table wine)
후식용 포도주
(Dessert wine)
White
Red
White
Red
수분
에탄올
휘발성 성분
추출물질 합계
당(sugar)
펙틴 관련물질
글리세린 물질
유기산
회분
페놀성 화합물
아미노산 및 관련성분
지방성분 및 터핀계 물질
기타 비타민 등 미량성분
87
10
0.04
2.96
0.38
1.1
0.7
0.7
0.2
0.01
0.25
0.01
0.01
87
10
0.04
2.96
0.05
1.1
0.83
0.83
0.2
0.02
0.25
0.02
0.01
76
14
0.05
9.95
0.25
0.9
0.5
0.5
0.2
0.2
0.2
0.01
0.01
74
14
0.05
11.95
0.25
0.9
0.5
0.5
0.2
0.2
0.2
0.02
0.01
합 계(%)
100
100
100
100
8.4 Lactate fermentation
 Lactate is a common fermentation
product in many facultative and
obligate anaerobes.
 Lactic acid bacteria (LAB) produce
lactate as a major fermentation
product, and most of them have a
limited ability to synthesize
monomers for biosynthesis and
vitamins.
 Homofermentative Lab produce only
lactate from sugars through the EMP
pathway,
 while heterofermentative LAB
produce acetate and ethanol in
addition to lactate via the
phosphoketolase pathway (Table8.2
and Sec.4.5).
8.4 Lactate fermentation
8.4.1 Homolactate fermentation
 Homofermentative LAB include most species of Lactobacillus, Sporolactobacillus,
Pediococcus, Enterococcus and Lactococcus.
 They use hexoses though the EMP pathway to generate ATP.
 Lactate dehydrogenase reoxidizes the NADH reduced during the EMP pathway
using pyruvate as the electron acceptor (Fig.8.3).
 Lactate is accumulated lowering the intracellular pH.
 Lactate dehydrogenase is active in acidic
conditions producing lactate as the major
product.
 Under alkaline conditions,
homofermentative LAB produce large
quantities of acetate and ethanol.
8.4 Lactate fermentation
8.4.2 Heterolactate fermentation
 Species of Leuconostoc and Bifidobacterium
produce ethanol and acetate in addition to
lactate via the phosphoketolase pathway.
 In this pathway, glucose  glucose-6phosphate  ribulose-5-phosphate
 xylulose-5-phosphate  glyceraldehyde-3phosphate + acetyl-phosphate
 Glyceraldehyde-3-phosphate is metabolized
to lactate as in the homolactate fermentation
producing ATP.
 Acetyl-phosphate is reduced to ethanol
acting as the electron acceptor to oxidize the
NADH.
 One ATP per hexose is available from the
heterolactate fermentation.
8.4 Lactate fermentation
8.4.2 Heterolactate fermentation (continued)
 Pentoses are converted to xylulose-5-phosphate without reducing NAD+.
 In this case, acetyl-phosphate is not used as the electron acceptor but is used
to synthesize ATP through acetate formation.
 L. mesenteroides synthesize 1 ATP from a molecule of hexose and 2 ATP from a
molecule of pentose.
 Bifidobacterium bifidum ferments 2 molecules of hexose to 2 molecules of
lactate, 3 molecules of acetate and 5 ATP employing two separate
phosphoketolase active on fructose-6-phosphate and xylulose-5-phosphate,
respectively (Fig.4.10, Sec.4.5).
8.4 Lactate fermentation
8.4.3 Biosynthesis in lactic acid bacteria
 LAB require many amino acids, vitamins, nucleic acid bases and other
substances as growth factors because LAB cannot synthesize them.
 However, LAB can synthesize a few monomers and polymers for biosynthesis
from acetyl-CoA, which is not an intermediate in homolactate fermentation.
 Pyruvate is oxidized to acetyl-CoA through different routes depending on the
strain.
(1) Pyruvate dehydrogenase complex : in most species of Lactococcus and
Enterococcus as in aerobic bacteria
(2) Pyruvate:formate lyase : in Enterococcus faecalis, Bifidobacterium bifidum
and Lactobacillus casei. This enzyme is found in anaerobic metabolism in
facultative anaerobic enteric bacteria (Sec.8.6).
8.4 Lactate fermentation
8.4.3 Biosynthesis in lactic acid bacteria (continued)
(3) Pyruvate oxidase and phosphotransacetylase : converting pyruvate to acetylCoA in other LAB including Lactobacillus delbruechii and Lactobacillus
plantarum.
8.4.5 Lactate/H+ symport
 When the intracellular lactate concentration is higher than that of the medium,
lactate is exported with 2H+ generating a proton motive force in Lactococcus
cremoris (Fig.5.28, Sec.5.8.6.3).
8.4 Lactate fermentation
8.4.6 LAB in fermented food
 Dairy products and fermented vegetables are typical LAB fermented foods and
LAB produce flavours specific for each food as well as lactate.
<Citrate metabolism in LAB>
 Citrate is produced in the initial phase of soybean
sauce fermentation and converted to acetate in
the later stages by LAB.
 Pediococcus halophilus, isolated from maturing
soybean sauce, ferments citrate to acetate and
formate (Fig.8.4).
 Decarboxylation reaction of OAA is not coupled to
proton motive force generation as in other LAB.
 Since pyruvate is not needed to dispose of
electrons, it is converted to acetyl-CoA through a
reaction catalyzed by pyruvate:formate lyase.
 Salmonella typhimurium and Klebsiella
pneumoniae ferment citrate through a similar
metabolism (Fig.8.14).
8.4 Lactate fermentation
8.4.6 LAB in fermented food (continued)
 Milk contains about 1.5 g/L citrate, which is
converted to diacetyl, acetoin, acetate and lactate
during the butter fermentation by LAB such as
Lactococcus lactis subsp. diacetyllactis,
Leuconostoc cremoris, Leuconostoc oenos and
Leuconostoc mesenteroides (Fig.8.5).
 When NADH/NAD+ ratio is high, pyruvate is
reduced to lactate. When the ratio is low, acetoin
is produced through 2-acetolactate.
 A similar reaction is found in some Bacillus species
(Fig.7.40) and in enteric bacteria (Fig.8.12).
 ATP is formed by acetate, but energy is not
conserved in the decarboxylation of OAA.
 A proton motive force is generated through
citrate/lactate exchange, similar to the
malate/lactate exchange (Sec.5.8.6).
8.4 Lactate fermentation
8.4.6 LAB in fermented food (continued)
<Malolactic fermentation in LAB>
 Malolactic fermentation in wine fermentation : conversion of malate to lactate
by Lactobacillus plantarum, Lb. casei, Leuconostoc mesenteroides, Leu. oenos
and Lactococcus lactis.
 Since these bacteria cannot use malate as their carbon and energy source, the
malolactic fermentation is possible in the presence of fermentable substrates
such as glucose.
 A proton motive force is generated in this fermentation through H+
consumption in the carboxylation of malate to lactate and through malate2/lactate- exchange (Sec.5.8.6).
8.4 Lactate fermentation
8.4.6 LAB in fermented food (continued)
<Glycerol fermentation in LAB>
 Glycerol is dehydrated to 3-hydroxypropionaldehyde that serves as an electron
acceptor in heterofermentative Lactobacillus species.
 Since these bacteria preferentially use this electron acceptor, pyruvate is
oxidized to acetate, synthesizing ATP, through the reaction catalyzed by acetate
kinase.
 Consequently, the growth yield of heterofermentative LAB is higher on glucose
with glycerol than on glucose alone.
 A similar fermentation is found in the metabolism of glycerol by enteric bacteria
(Fig.8.13).
8.6 Mixed acid and butanediol fermentation
8.6.1 Mixed acid fermentation
 Some G(-) facultative anaerobic bacteria including species of Escherichia,
Salmonella, Shigella and Enterobacter ferment glucose, producing various
products including lactate, acetate, succinate, formate, CO2 and H2 (Fig.8.11).
 Phosphoenolpyruvate (PEP)
carboxylase synthesizes OAA from
PEP before being reduced to
succinate using 2 NADH.
 Pyruvate is either reduced to lactate,
or cleaved to acetyl-CoA and formate
by pyruvate:formate lyase.
 According to the availability of
electrons, acetyl-CoA is either
reduced to ethanol or used to
synthesize ATP.
 Strictly anaerobes such as Anaerobiospirillum
succiniciproducens and Actinobacillus
succinogenes ferment carbohydrate mainly
to succinate.
8.6 Mixed acid and butanediol fermentation
8.6.2 Butanediol fermentation <Glucose to butanediol>
 Some Erwinia, Klebsiella, Serratia, Bacillus and LAB species produce 2,3butanediol (2,3-BDO) in addition to lactate and ethanol from pyruvate (Fig.8.12).
 In these bacteria, pyruvate is the substrate for three enzymes such as lactate
dehydrogenase, pyruvate:formate lyase and 2-acetolactate synthase.
 2-Acetolactate synthase condenses two
molecules of pyruvate to 2-acetolactate that
is further decarboxylated and reduced to 2,3butanediol.
 Under anaerobic conditions,
2,3-butanediol-producing
facultative anaerobes produce
acid product, lowering the
external and intracellular pH.
 2-Acetolactate synthase has an optimum at pH
6.0. When the intracellular pH drops, this
enzyme becomes active to divert carbon flux
from acid production to the neutral solvent.
8.6 Mixed acid and butanediol fermentation
8.6.2 Butanediol fermentation <Gycerol to butanediol>
 Klebsiella pneumoniae, Klebsiella
oxytoca and Enterobacter
aerogenes ferment glycerol to
various products including 2,3BDO.
 They oxidize a part of glycerol to
pyruvate, and dispose of the resulting
electrons to reduce the remaining
glycerol to 1,3-propanediol.
 Pyruvate is metabolized as in the 2,3butanediol fermentation.
 Glycerol is reduced to 1,3-propanediol
by LAB while oxidizing carbohydrate
(Sec.8.4.6).
 These diols are important
petrochemical intermediates.
8.6 Mixed acid and butanediol fermentation
8.6.4 Anaerobic enzymes
<Pyruvate to acetyl-CoA and formate>
 Enteric bacteria pyruvate n a reaction catalyzed by the pyruvate dehydrogenase complex
under aerobic conditions, which is not expressed under anaerobic conditions and of which
activity is inhibited by NADH.
 Pyruvate:formate lyase functions only under anaerobic conditions since it is expressed
under fermentative conditions and irreversible inactivated by molecular oxygen.
<Oxidation of formate>
 Under fermentative conditions, formate:hydrogen lyase cleaves the formate
produced by pyruvate:formate lyase to CO2 and H2. This enzyme is a complex of
formate dehydrogenase II (FDHII) and hydrogenase.
 When electron acceptors such as nitrate or fumarate are present, formate dehydrogenase I
(FDHI) oxidizes formate to CO2 transferring the electrons to nitrate reductase or fumarate
reductase via NADH for energy conservation through anaerobic respiration (Fig.8.15).
 These enzymes are present in species of Escherichia and Enterobactor. Their expression is
controlled by the FNR protein (Sec.12.2.4).
8.6 Mixed acid and butanediol fermentation
8.6.4 Anaerobic enzymes (continued)