اجلمهورية العربية السورية اجلمهورية العربية السورية وزارة التعليم العالي وزارة التعليم العالي جامعة دمشق اهليئة العامة للتقانة احليوية كلية السراعة
رسالة مقدمة لنيل درجة الدكتوراه يف اهلندسة الزراعية )علوم األغذية (
بعنوان
أمثلة إىتاج الكحول احليوي مً املوالس باستخداو سالالت مً مخرية .Saccharomyces sp وبكترييا Zymomonas mobilis Optimization of bioethanol production from molasses using strains of Saccharomyces sp. yeast and Zymomonas mobilis bacteria
إعداد المهندسة
بإشراف
المشرف المشرف المشارك األستاذ الدكتور عادل سفر األستاذ الدكتور أمحد مسور اإلبراهيه كلية الزراعة كلية اهليدسة الكينيائية والبرتولية
2014 - أتكدً خبالص الشكس إىل األضتاذ الدكتٕز عاده ضفساألضتاذ يف قطي عمًٕ
األغرٖٛ كمٛٗ الصزاعٛ جاوعٛ دوشل دوشل لتفضمْ باإلشساف عمٜ ِرا البحث ٔوساجعتْ
الٍكدٖٛ لْ ٔجلىٗع املطاعدات اليت قدوّا إلمتاوْ.
- عىٗل الشكس لكمٛٗ الصزاعٛ ممثمٛ بعىٗدِا- ٔٔكالّٟا- ٔاقطاوّا كافٛ اليت
احتضٍت أعىالٍا ٔأظّستّا لمٍٕز ٔاخص بالشكس قطي عمًٕ األغرٖٛ ممثمٛ بالعىادٚ ٔأعضاٞ
اهلٛ٠ٗ التدزٖطٛٗ .
- أتٕجْ بالشكس اجلصٖن إىل األضتاذ الدكتٕز أمحد مسٕز االبساِٗي عضٕ اهلٛ٠ٗ
التدزٖطٛٗ بكمٛٗ اهلٍدضٛ الكٗىاٟٛٗ ٔالبرتٔلٛٗ جاوعٛ البعث, الرٙ مل ٖرتك جّداً ٔوطاعدٚ
ٔخربٚ فٍٛٗ ٔعمىٛٗ إال ٔقدوّا.
- كمىات٘ عاجصٚ عَ شكس اهلٛ٠ٗ العاوٛ لمتكاٌٛ احلٖٕٛٗ لكن وا قدوتْ ٔتكدوْ لدعي
البحث العمى٘ , ٔكاٌت حاضٍٛ دافٛ٠ لبحث٘ ِرا ٔلٕالِا ملا ٔصمت هلرٓ الٍتٗجٛ الساٟعٛ.
ٔاخص بالشكس ٔاالوتٍاُ األضتاذ الدكتٕز عصاً قاضي املدٖس العاً لمٛ٠ّٗ العاوٛ لمتكاٌٛ
احلٖٕٛٗ ملا قدوْ وَ دعي ٔتشجٗع لطالب الدزاضات العمٗا ٔإتاحٛ الفسصٛ جلىٗع الباحثني
لالضتفادٚ وَ إوكاٌٗات اهلٛ٠ٗ
- جصٖن الشكس إىل األضتاذ الدكتٕز حمىد فٕاش العظىٛ, أضتاذ أوساض الٍبات ٔالٕباٟٗات
يف كمٛٗ الصزاعٛ جباوعٛ لكن العُٕ ٔالتٕجْٗ الرٙ أغٍٜ بْ ِرا البحث - الشكس ٔاالوتٍاُ الكبري العىٗل لكن أخٕت٘ ٔأصدقاٟ٘ يف اهلٛ٠ٗ الرَٖ كإٌا عٌٕا
ل٘ يف عىم٘ ِرا ً. ضٕوس العم٘, ً . امياُ عصكٕه , الدكتٕزٚ عسٔب املصسٙ,
الدكتٕزٚ شعمٛ خازٔف,
ً. زضٕاُ بدز الدَٖ ٔ ً. زشا العٗد ٔ اآلٌطٛ وّا حداد هلي وين كن احملبٛ
- االوتٍاُ الكبري لكن لمعاومني يف ٛ٠ِٗ الطاقٛ الرزٖٛ ملطاِىتّي جبصٞ وَ ِرا العىن
أخريا اشكس كن وَ قدً ل٘ ٖد العُٕ ٔاملطاعدٚ ٔضاِي يف دعي ِرا البحث ٔإظّازٓ
لمٍٕز .
نسرين نقشو
اىل ٌٕز عٗين وَ غادزتين قبن أُ تسٝ جناح٘ ِرا
إىل أمي
إىل شسٖكٛ زٔح٘ ٔزفٗكٛ دزب٘ إىل وَ ِ٘ أخيت ٔصدٖكيت إىل الغالية نظام
الرَٖ دعىٌٕ٘ ٔاغدقٕ عم٘ وَ احلب ٔاحلٍاُ وا ال تصفْ الكمىات إىل أخوتي وأخواتي ) رنا , رابعة , امحد , حممد (
اىل وَ قضٗت بٍّٗي أمجن األٔقات ٔأحالِا إىل أصدقائي وزمالئي
اىل وَ وٍحٌٕا وَ ٔقتّي ٔجّدِي ٔعمىّي الكثري إىل أساتذتي
لكي مجٗعا حمبيت نسرين جدول المحتويات
الموضوع رقم الصفحة
1 1 3 1 1 1 1 3 1 2 1 1 3 1 -3-1-1-3-1 4 1 1 3 1 5 1 1 3 1 6 1 1 3 1 7 1 1 3 1 1 3 1 1 8- مالءمة الركيزة األولية إلنتاج اإليتانول -9 - 1-1-3 1 -10- 1-1-3 1
قائمة الجداول
الموضوع رقم الصفحة (1
11
قائمة األشكال
الموضوع رقم الصفحة رباعي األوعية
املكدمة Introduction
1
2
3
الفصل األول الدراسة املرجعية LITERATURE REVIEW
4
1
Davis et al., 2005) Biomass
73
Demirbas et al., 2004; Yu et al., CO2
Piccolo and 98 2003
Bezzo, 2009
(Licht et al., 2007; Buckeridge et al., 2010(،
(Hansen et al., 2005; Patil, 1991)
Altintas et al., 2002) Wyman and Hinman, 1990)
5
2005 2000 95
SourceOECD, 2006; Pilgrim,2009 2010 2005
RFA,2011
Proalcool
1975
2008 30
400
Basso and Rosa, 2010; Amorim et al., 2009
2009 10.600 2008 9
2010 13.230
2022 2015 36 20.5
Ingeldew et al., 2009
Bio fuels Platform
6
2009 2004 1992 44
1250 635
465 750
HGCA, 2011 7700 2010
(Licht,
2007)
70
890 2010 470 2009
132 2011
(RFA, 2011)
Bioethanol 1 1
Altintas et al., 2002)
7
Carvalho et al.,
1993
1 1 1
46.07 C2H5OH
0.789 °78 °117
°12.8 °177 °78.5
(Walker, 2010
2 1 1
CO2
CO2
34 1
Walker, 2010
bio
8
EMP
Embden - Meyerhof - Parnas
CO2
C6H12O6 → 2 C2H5OH + 2 CO2 + 2ATP Olsson and Hahn-Hagerdal 1996; Nigam 1999; Martin et al., 2006
5 95
(Ferrari et al., 1992, Beatriz et al., 2005)
3 1 1
1 3 1 1
Saccharum sp.
(Beta vulgaris
9
Leiper et al., 2006
Lin
and Tanaka, 2006
Tao et
al., 2005; Tanaka et al., 1999
Sreenath and Jeffries, 2000; Mohagheghi et al., 2006
95
1 99.9
Davis et al., 2005; Ruanglek et al., 2006 3
2 3 1 1
10
ÜÇÜNCÜ, 2011
oilseed pulp, sugar beet pulp
Salix viminalis
Miscanthusgigantum
Arundodonax Phalarisarundinaceae
Prescott
and Dunn, 2002
Baptista et al., 2006
5
11
Saccharomyces cerevisiae
S.cerevisiae
Kluyveromyces fragilis
Nigam et al., 1998
2 1
Hinkova and Bubnik, 2001
20 80
62 48 60
14.8 16.7
1.5 0.4 8.5
12
Murtagh, 1999)
0.01 1.6 0.5 2 0.5
Huertaz-Díaz et al., 1991
0.02
formic acid acetic acid
melanoids
Borzani et al., 1993
6 - 4
9
13
Hamdy 4
et al., 1992
6.7 pH
% 1.3
1
0.1% %1.5
(Leiper et al., 2006) 30
90 85
pH
Mariam et al., 2009
Cazetta
et al., 2007
14
55
Jiménez et al., 2004
75
Piggot, 2005
Saccharomyces cerevisiae
Abdel-
Fattah et al., 2000; Eden et al., 2001
Cachot and
Pons, 1991
36-30 85 78 Brix
16 10 15 11 7 4 10 6
4.5 2 4 2 2 1
Carvalho et al., 100 5 2
1993
15
7 pH
Borzani, 2001 5.5 5
Cachot and Pons,
1991
A
B, C
16
27 85
Murtagh, 1999) 2.25 15.5 50
1 2 1
Brix
1.416 80
(Kiss et al., 1999) 80
Refractometer
90 85 80
15 10 90 85
Wang et al., 1985
S. cerevisiae
Doelle and Greenfield 1985; Vasconcelos et al., 1998
17
3 1
S.uvarum
Kluyveromyces sp. S.pombe S.virni
S. cerevisiae
Walker et al., 1990; Moreira
et al., 2005
Tao et al., 2005 S. cerevisiae
Saccharomyces cerevisiae 1 3 1
Fungi
Saccharomycetes Ascomycota
Saccharomyces Saccharomycetaceae Saccharomycetales
CAB International, 2005
Ergun and Freda, 2000
α
Cakar et al., 2005
18
Haq and Ali, 2007
S.cerevisiae
Carrascosa, 2006
Fregonesi et al.,
Schizosaccharomyces 2007
Carrascosa, 2006
C6
CO2 C2
(Converti et al., 2003)
CO2
ATP
ATPs Santos et al., 2008
19
PFK ATP
CO2
Ingledew,1999
Rajoka et
al., 2005
Patrascu et al., 2009
Bai et al.,) 2008
Hirasawa et al., 2007; Pham and Wright, 2008
1 1 3 1
20
1 1 1 3 1
Bx
32-14
% 32
Jones and
Ingledew, 1994
93 90
5 2
(Bai et al., 2008)
40 %24 Bx) 60.83
100/ 23.52 100/ 1.28
Laopaiboon et al., 2009
21 12
15
Mariam et al., 2009 14.92 6.49
30 20 10 5
30 100 40
21
Periyasamy et Vasconcelos et al., 1998; Bai et al., 2004
al., 2009
(30 20 15
(10 8
30
Wilkins et al., 2007 Ingledew,1993 20
2 1 1 3 1
pH
5.5 4.5
4.2 pH 5.7 pH (Nigam,1999)
0.15 56.03 59.1
0.4 0.19 0.08
Periyasamy et al., 2009
12 4 8 1
4 pH
Vasconcelos et al.,1998 4.5 4.2 pH
5.5 4 pH S.cerevisiae
22
6.39 5 4 5.5
Mariam et al., 2009
3 1 1 3 1
30 29
Jamai et °2 32 Saccharomyces cerevisiae
S. cerevisiae al., 2003
°45 °40 °35 30 25
°35
Periyasamy et al.,
2009
Mariam et al., 2009 6.42 °30
Nolan et al.,
1994
: 4 1 1 3 1
Saccharomyces spp.
23
Banat et
al., 1998
S. uvarum 17
S.uvarum ATCC26602
S.cerevisiae Y-1500
4 Comberbach and Bu'Lock, 1984
S.cerevisiae
40
S.cerevisiae MTCC174 S.cerevisiae HAU-11 S.cerevisiae 3B
S.cerevisiae MTCC172
61.5 66.7 67 71
Bajaj et al., 2003 Walker et al., 1990
S.cerevisiae MT15
15
Rajoka et al., 2005 40 72
24
Kluyveromyces marxianus
7.2
Banat and Marchant, 1995) 0.44
S.cerevisiae
Converti et al., 2003; Moreira et al., 2005
18
S.cerevisiae
(Balat et al., 2008)
5 1 1 3 1
glycolysis
Vasconcelos et al., 2004
25
Griffith and Ngo, 1994)
S.cerevisiae .(Thomas et al., 1996)
Arshad, 2005)
26
Rolz and De Leon,
2011
(NH4NO3)
1:5.5 0.275 (KH2PH4)
55.89 100 10.41
60 100 3.89
Alkabbashi et al., 2011
150
8.3
Saf-Instant 59.15
8.5 S. cerevisiae
S. cerevisiae Ethanol Red 0.8
8.1
Yalçin
27
and Özbas, 2004
Prescott and Dunn, 2002
6 1 1 3 1
S. Saf-Instant, Ethanol Red
20 cerevisiae
8.4 8.7 53.42 150
Mukhtar et al., 2010
7 1 1 3 1
200 350 300 250 200
4 29 500
. Mariam et al., 2009 12.92
8 1 1 3 1
Uden, 1989
28
(Rehm and Reed, 1995)
15
Uden, 1989 25
(Margaritis and Bajpai, 1982
Casey and Ingledew, 1986
40 95
Casey and Ingledew, 1986
Candida Saccharomyces
Moulin et al., 1980, Jiménez and Benítez, 1986 10
25
.(Jiménez and Benitez, 1986)
Duvnjak et al., 1987
Tubb, 1983) 60 40
14
Rehm and Reed, 1995 Uden, 1989
29
(Palmqvist et al., 1999
Dombek and Ingram, 1986; Rosa et al., 1987
15 Jiménez and Benítez, 1986
Ingram and Buttke, 1985
25 Saccharomyces
Ingram and Buttke, 1985
Jiménez and
Benítez, 1988
Benitez et al., 1983 Park and Sato, 1982)
35
Sharma et al., 70 0
1996
Sharma, 1997
30
9-1-1-3 1
16 12
5 2
D'Amore and Stewart, 1987 7.5
-10-1-1-3-1
Dale, 1987
Zymomonas 2 3 1
Zymomonas
Alphaproteobacteria Proteobacteria
Zymomonas Sphingomonadaceae Sphingomonadales
Z.mobilis
31
6 2
Z. 1.5 1
Bochner et al., mobilis
Panesar et al., 2010
2006
Rogers et al., 2007; Zhao et al.,
Z. mobilis 2009; Behera et al., 2010
Ruanglek
et al., 2006; Bochner et al., 2010
Lawford and Rousseau, 2002; Altintas et al., 2006;
Z. mobilis He et al., 2009
Z. mobilis Rogers et al., 2007
S. cerevisiae
Lin S.cerevisiae
and Tanaka, 2006; Rogers et al., 2007
Z. mobilis
S.cerevisiae
32
Bai et al., 2008
Z. mobilis
120
Bochner et al., 2010
Z.mobilis
Gunasekaran and Raj, 1999
S.cerevisiae
0.73 0.87 S.cerevisiae Z.mobilis
1.38 2.75
1.75 1.66
Nellaiah et al., 1988 5 3.34
Z.mobilis Rye
Lee et S.cerevisiae 5
Z.mobilis al., 1983
Sahm et al., 1994
33
Z.mobilis 1 2 3 1
Z.mobilis
1 1 2 3 1
° 31 25 Z.mobili
15
Lawford and Rousseau, 2002 ° 42 25
38
36 30 40
Deanda 80
Z.mobilis et al., 1996
Panesar et al., 2001b
°30
Panesar et al., 2001a 40 35
2 1 2 3 1
pH
buffer
pH Diez and Yokoya,1996
34
5 3 pH
7.5 3.5 pH Z.mobilis 7 6
De Moraes et al.,1981 7 5
6 5
(Buzato, 1984 Z. mobilis
7.5 3.4 pH Z.anaerobia
7.9 3.5 pH
Sprenger, 1996 3.4 2.5
3 1 2 3 1
10 2 Zymomonas
Rogers et al., 1982, 5.5
Rogers et al., 1997
10 5
Entner-Doudoroff
Lee and Huang ,
2000
Montenecourt, 1985
35
4 1 2 3 1
Zymomonas 4
mobilis
ATCC 12526 ATCC 109888 15
20 15
20 IFO 13756, NRRLB 4286
25
Loos et al.,1994
°30 200 6.5 pH
Cazetta et al., 2007 180
Z.mobilis
2 4 14
Ruijter et al., 2003 40
CP4
Z.mobilis
110 34
135
36
(Diez and Yokoya, 1996)
Z.mobilis BL4 25 20
Ahila et al., 25
1992
18.5
0.39 0.5
Singh and Jain,1994
5 1 2 3 1
Z.mobilis
Doelle et
1 5 al., 1990
0.39 500
500 1 5
(Tiwari et al., 2011) 0.55
6 1 2 3 1
Zymomonas 10 7.5 5
10 mobilis
10.56 11.36
37
(Sulfahri et al., 2011) 7.5 5
48
11.36 10
10.80 72
Zhang
and Feng , 2011
4 1
Batch 1 4 1
Amorim et al., 2009
38 semi- Fed- batch 2 4 1
batch
Hewitt and Nienow, 2007
Continuous 3 4 1
Liden, 2002)
5 1
39
Madigan et al., 2000
40
الفصل الثاني أهنية وأهداف البخث
41
- 2
Saccharomyces sp. 1
Zymomonas 2
mobilis
3
4
5
42
الفصل الثالث مواد وطرائم العنل MATERIALS AND METHODS
43
-3
2 1
(1
YGS
YPS PDA HPLC Anti foam
2
Zymomonas mobilis ITS 4 ITS1 DNA- - DNA (ExoSAP-IT Ladder MgSO4 dNTP – Taq- polymerase- Standard Standard
44
2
BIOTECH- 0.45 4GBR-5 KNAUER HPLC 1.5 ependorff EU SELECTA New Brunswick USA Scientific pH mini spin ependorff DO SHIVAKI 30 SHIVAKI 37 EU BIO AIR HANNA pH meter SCO EU JRAD Bio (merieux API
GENE Amp. PCR PCR system 9700 BIO RAD ABI DNA 310-Gendic Analyzer PRISM innu PREP 0.2µm FG Millipore Life science PCR Pure Kit BOECO METLER TOLEDO JSAC HITACHI U-2900
45
1 3
2008
2009
2 3
5-3
25-20
̊4
Safdistil C-7
FERMENTIS
Bio S1929
Springer
Ethanol Red®
FERMENTIS
46
CWBI Zymomonas mobilis LMG404
10 10 (YPS)
°30 1000 20
3 3
9 1
4-10 3-10
30
Nahvi et al., 2002 48
9 1
5 3 10
1
47
1.5 (Merck
̊4
4 3
1 4 3
(Barnet et al., 2002)
8
10
25
21 14 7 3
4
48
21 14 7 3 ̊25
2 4 3
Bio merieux API
5
3 5 suspension medium
2
100 24
7 c medium
100
100 API
72 24
API
Covadonga et al., 2002)
12 Bx)
0.34 0.12
2N 2N
49
10 5
meter pH 3
12
4.5
500 300
2.5 15 °121
Mariam et al., ̊30 24
̊30 2009
5 200
24
Nahvi et al., 2002
̊20
50
Saccharomyces 3-4-3 cerevisiae
DNA
10 750
Sodium Dodecyl Sulfate 150 20 K
37.5 1 mercaptoethanol 7.7 2 (SDS)
Ethylenediaminetetraacetic 15 1 CTAB
50 Tris 529.8 10 EDTA Acid
1 24 25
70 100 3
Bakri et al., DNA
48 1.5 2010
750
30 60 20
10 60 10 80
10 80
3
3
51
30 400
13000 5
10 1 3
4 1 3
13000 5 15
DNA
15 70
50 DNA
260 DNA
260 DNA
280
U-2900 HITACHI Spectrophotometer
PCR
ITS4 ITS1
5.8 S rDNA
5` TCC GTA GGT GAA CCT GCG G 3` ITS1
5` TCC TCC GCT TAT TGA TAT GC 3` ITS4
Bakri et al., 2010 CWBI
52
3 1.5
7 50
Taq-polymerase 0.4 5 DNA
32.1 2.5 MgSo4 2 dNTP 1
Amplification
3 °95
Denaturation DNA
30 94
Annealing
50 DNA
45
Extension
DNA
1 72
35
5 72
53
20 1.5 PCR
4
30 DNA-Ladder
DNA
USB ExoSAP-IT
PCR DNA
Taq ABI
ABI
DNA
PCR
2 PCR 1 ExoSAP-IT
15 37
15 80
54
1 6
1
1 1
10 96 PCR
30 60 5 50 innu
PREP PCR Pure Kit
Life science
DNA
DNA
5 3
0.45 1.5 ependorff mini spin ependorff
NH2 HPLC
EH- (RI Refractive Index Detector
55
254 UV 002
1
1 5 4 3 batch 2 1 AC AK pH 7 6 rpm 9 C H ̊C 8 ml 11 ml 10 ml 13 ml 12
56
750
2.5 15 121
500 5
50 1.2
200 24
T24 T0 30
ependorff
HPLC
Davis et al., 2.4 30 15 HPLC 85
57
0.4 2006
2
Raposo et al., 2009
LMG 404 6 3
Zymomonas mobilis
1 6 3
pH -1 1 6 3
14 750 4
0 12
0 34
5.5 5 4.5 4
15 ̊121
2.5
pH
5.5 5 4.5 4
5 °30
58
50 1.2 500
200 24
Davis et al., 2006 30
3000 ependorff T24 T0
10
pH
-2 1 6 3
°20 5 pH
°35 °30 °25
Bx 3 1 6 3
5 pH
28 24 20 16
°30
.T24 20 16 Bx
28 24 20 Bx
59
T48
Bx
4 1 6 3
5 pH
0.06 16 Bx
0.34 0.12 0.17
0.68 0.24
1.36 0.48
̊30
24
5 1 6 3
16 Bx 5 pH
0.34 0.12
1.25
7.5 5 2.5
60
̊30
24
2 6 3
16 5.5 5 4.5 4 pH 1-2 6 3
35 30 25 20 2-2 6 3
16
28 24 20 16 Bx -3-2 6 3
0.34 0.12 -4-2 6 3
0.68 0.24
1.36 0.48
2.72 0.96
7.5 5 2.5 1 25 -5-2 6 3
0.68 0.24
61
3-6-3
7.5 7 6.5 6 pH 1 3-6-3
16 750 4
0.17 0.06
7 6.5 6
7.5
15 ̊121
10
5
24 1000 3 10 pH 37
7.5 7 6 6
5 30
50 1.2 500
Davis et al., 30 200 24
T24 T0 2006
62
10 3000 ependorff
pH
2 3-6-3
6.5 pH
̊40 ̊35 ̊30 ̊25
Bx 3 3-6-3
6.5 pH
28 24 20 16
̊30
T24 20 %16 Bx
T48 28 24 20 Bx
Bx
63
4 3-6-3
6.5 pH
0.06 16 Bx
0.34 0.12 0.17
0.68 0.24
1.36 0.48
°30 LMG 404
24
5 3-6-3
6.5 pH
16 Bx
0 17 0 06
17.5 15 12.5 10
24 ̊30
64
4-6-3
7.5 7 6.5 6 pH -1 4-6-3
̊40 ̊35 ̊30 ̊25 -2 4-6-3
28 24 20 16 Bx -3 4-6-3
0.12 -4 4-6-3
0.68 0.24 0.34
1.36 0.48
2.72 0.96
17.5 15 12.5 10 -5 4-6-3
0.34 0.12
7 3
1 7 3
65
750 3
5 16
0.34 1.2
5 2.5
1.2 500
50
30 200 24
T30 T24 T12 T6 T0
2 7 3
0.24
0.68
3 7 3
66
750 3
0.06 6.5 16
0 17
5 10
50 1.2 500
24
°30 200
T30 T24 T12 T6 T0
4 7 3
0 12
0 34
67
8 3
ANOVA
LSD General Linear Model
(P≤ 0.05 5
Correlation Coeficient
SPSS 18
68
الفصل الثالث النتائج و املنـاقـشـة RESULTS and DISCUSSION
69
- 4
ICUMSA Bartens, 2005 GS4-13 1994 ICUMSA
. (3 Bartens, 2005 GS4/7-1 1994
100 3
70.63 79.5 Brix 57.1 47.4 27.4 17.5 7.6 15.1 0.93 0.61 Ca 0.57 1.8 Na 0.58 2.7 K 0.11 1.43 N 0.004 0.004 P 173 112 ppm Fe) 3 7 ppm Mn) 4 13 ppm Zn) 3 3 ppm Cu) 3
1 4 56
70
Saccharomyces 20
API
2 4
1 2 4
Barnet
Barnet, 2002 Saccharomyces
API 2 2 4
API
2
S.cerevisiae API
2
71
API 2
72
API 4
4
GLU : D- Glucose MDG- Methyl-a D- Glucopyranoside GLY: Glycerol NAG: N-Acetyl-Glucosamine 2KG: Calcium 2 Keto-Gluconate CEL: D- Cellobiose ARA: L- Arabinose LAC: D- Lactose XYL: D-Xylose MAL: D-Maltose GAL: D- Galactose SAC: D- Sacchrose(sucrose) ADO: Adonito TRE: Trihalose XLT: Xylitol MLZ: D-Melezitose SOR:D- Sorbitol RAF: D-Raeeinose API 4
RAF MLZ TRE SAC MAL LAC CEL NAG MDG SOR INO GAL XLT ADO XYL ARA 2KG GLY GLU
- - + -
Saccharomyces cerevisiae
73
5
24 5
4 SA1 1 4.5 BR3 2 4 SG1 3 4.2 SH1 4 3.7 SR5 5 4.3 BR2 6 4 SR6 7 4.5 BR1 8 3.9 SR4 9 3.8 SB1 10 3.9 NR7 11 3.8 CD1 12 4.1 CC1 13 4 JE1 14 4.1 JF1 15 3.8 JN2 16 3.7 JN4 17 3.5 JN5 18 4 JN6 19 4.1 JN7 20
74
24 5
4.5 BR3 BR2 BR1
3.5 JN5
Elander and Chang,1979
Wang et al.,1979) Phenotype
BR3 BR2 BR1 DNA 3 2 4
DNA
280 260
280 260
DNA
6 260
75
DNA 6
611 1.04 BR1 90.5 1.53 BR2 206.5 1.56 BR3
PCR
PCR
76
a
b
ITS1 PCR :a 3 ITS4 PCR b BP100DNA marker BP100 DNA marker
77
DNA
PCR DNA
R BRI: Saccharomyces cerevisiae 92% CCTCCTGATTTGTTGTCAACTTTAAGACATTGTTCGCCTAGACGCTCTCTTCTT ATCGATAACGTTCCAATACGCTCAGTATAAAAAAGATTAGCCGCAGTTGGTA AAACCTAAAACGACCGTACTTGCATTATACCTCAAGCACGCAGAGAAACCTC TCTTTGGAAAAAAAAAATATCCAATGGGAAAGGCCAGCCAATTTCAAGTTAA CTCCAAAAAGTTTCCCCCCCTACCAAACAAAAGGTTTGAAAAGGAAATGACC CTCAAACAGGGATGCCCCCGGGAAAACCAAGGGGGGCAAGGGGGGTTCAAA AATTCCATGATTCCCGGAATTCTTGCAATTCCCATTACGTATCCCATTTCCCTG GGTTCTTCATCGAATGCGAGAAACCAAGAAAATCCGTTGTTGAAAGTTTTTAA AATTTTAAAATTTCCAGTTACAAAAATTCTTGTTTTTGACAAAAATTTAAGGA ATAAATAAAATTG F BR1:Saccharomyces cerevisiae 99% TTTATAATTTTGAAATGTTTTTTTTTGTTTTGGCAAGAGCATGAGAGCTTTTAC TGGGCAAGAAGACAAGAGATGGAGAGTCCAGCCGGGCCTGCGCTTAAGTGC GCGGTCTTGCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGA TTTCTGTGCTTTTGTTATAGGACAATTAAAACCGTTTCAATTACAACACACTG TGGAGTTTTCATATCTTTGCAACTTTTTCTTTGGGCATTCGAGCAATCGGGGCC CAGAGGTAACAAACACAAACAATTTTATTTATTCATTAAATTTTTGTCAAAAA CAAGAATTTTCGTAACTGGAAATTTTAAAATATTAAAAACTTTCAACAACGG ATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATG TGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCCGCCCC TTGGTATTCCAGGGGGCAT CONTIG BR1: ATGCCCCCGGGAAAACCAAGGGGCGGCAAGGGGCGTTCAAAAATTCCATGAT TCACGGAATTCTTGCAATTCACATTACGTATCCCATTTCCCTGCGTTCTTCATC GAATGCGAGAAACCAAGAAAATCCGTTGTTGAAAGTTTTTAAAATTTTAAAA TTTCCAGTTACAAAAATTCTTGTTTTTGACAAAAATTTAAGGAATAAATAAAA TTG
R BR2: Saccharomyces cerevisiae 99% TTTCAACTTTAAGACATTGTTCGCCTAGACGCTCTCTTCTTATCGATAACGTTC CAATACGCTCAGTATAAAAAAAGATTAGCCGCAGTTGGTAAAACCTAAAACG ACCGTACTTGCATTATACCTCAAGCACGCAGAGAAACCTCTCTTTGGAAAAA AAACATCCAATGAAAAGGCCCAGCAATTTCAAGTTAACTCCAAAGAGTATCA CTCACTACCAAACAGAATGTTTGAGAAGGAAATGACGCTCAAACAGGCATGC CCCCTGGAATACCAAGGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACG GAATTCTGCAATTCACATTACGTATCGCATTTCGCTGCGTTCTTCATCGATGC
78
GAGAACCAAGAGATCCGTTGTTGAAAGTTTTTAATATTTTAAAATTTCCAGTT ACGAAAATTCTTGTTTTTGACAAAAATTTAATGAATAGATAAAATTGTTTGTG TTT F BR2: Saccharomyces cerevisiae 99% TTTTGAAATGTTTTTTTTTGTTTTGGCAAGAGCATGAGAGCTTTTACTGGGCAA GAAGACAAGAGATGGAGAGTCCAGCCGGGCCTGCGCTTAAGTGCGCGGTCTT GCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTG CTTTTGTTATAGGACAATTAAAACCGTTTCAATTACAACACACTGTGGAGTTT TCATATCTTTGCAACTTTTTCTTTGGGCATTCGAGCAATCGGGGCCCAGAGGT AACAAACACAAACAATTTTATCTATTCATTAAATTTTTGTCAAAAACAAGAAT TTTCGTAACTGGAAATTTTAAAATATTAAAAACTTTCAACAACGGATCTCTTG GTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGC AGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGGCCCCTTGGTATT CCAGGGGGCATG
CONTIG BR2: Saccharomyces cerevisiae 99% CATGCCCCCTGGAATACCAAGGGGCCGCAATGTGCGTTCAAAGATTCGATGA TTCACGGAATTCTGCAATTCACATTACGTATCGCATTTCGCTGCGTTCTTCATC GATGCGAGAACCAAGAGATCCGTTGTTGAAAGTTTTTAATATTTTAAAATTTC CAGTTACGAAAATTCTTGTTTTTGACAAAAATTTAATGAATAGATAAAATTGT TTGTGTTT
R BR3: Saccharomyces cerevisiae 94% CTGATTTGTTGCAACTTTAAGACATTGTTCGCCTAGACGCTCTCTTCTTATCGA TAACGTTCCAATACGCTCAGTATAAAAAAGATTAGCCGCAGTTGGTAAAACC TAAAACGACCGTACTTGCATTATACCTCAAGCACGCAGAGAAACCTCTCTTTG GAAAAAAAAACATTCCAAGGAAAAGGGCCAGCAATTTCAAGTTAACCCCAA AGGAGTTTCCCCCCCTACCAAACAAAAGGTTTGAAAAGGAAATGACCCTCAA ACAGGCATGCCCCCGGGAAAACCAAGGGGGGCAAGGGGGGTTCAAAAATTC GATGATTCCCGGAATTCTGCAATTCCCATTACGTATCGCATTTCCCTGGGTTCT TCATCGATGCGAGAACCAAAAAATCCGTTGTTGAAGGTTTTTAATATTTTAAA ATTTCCAGTTACGAAAATTCTTGTTTTTGACAAAAATTTATGAATAAATAAAA TTG F BR3: Saccharomyces cerevisiae 99% TGTTTTTTTTTGTTTTGGCAAGAGCATGAGAGCTTTTACTGGGCAAGAAGACA AGAGATGGAGAGTCCAGCCGGGCCTGCGCTTAAGTGCGCGGTCTTGCTAGGC TTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTGCTTTTGTT ATAGGACAATTAAAACCGTTTCCAATACAACACACTGTGGAGTTTTCATATCT TTGCAACTTTTTCTTTGGGCATTCGAGCAATCGGGGCCCAGAGGTAACAAACA CAAACAATTTTATTTATTCATTAAATTTTTGTCAAAAACAAGAATTTTCGTAA CTGGAAATTTTAAAATATTAAAAACTTTCAACAACGGATCTCTTGGTTCTCGC
79
ATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCC GTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCC CONTIG BR3: Saccharomyces cerevisiae 97% GGAAAACCAAGGGGCGCAAGGGGCGTTCAAAAATTCGATGATTCACGGAATT CTGCAATTCACATTACGTATCGCATTTCCCTGCGTTCTTCATCGATGCGAGAA CCAAAAAATCCGTTGTTGAAAGTTTTTAATATTTTAAAATTTCCAGTTACGAA AATTCTTGTTTTTGACAAAAATTTAATGAATAAATAAAATTG
DNA F Forward DNA R Reverse 3 4
1 3 4
7
7
T24 T24 T0 T24 55 3.6 6.5 0.6 7.1 1 53 3.5 6.6 0.5 7.1 2 58.7 3.7 6.3 0.8 7.1 3
3.7 24
100 6.3
100 58.7
80
2 3 4
8
8
T24 T24 T24 59.8 4.4 7.36 0.9 8.26 BR1 55.4 4 7.22 1.04 8.26 BR2 55.1 4 7.26 1 8.26 BR3
BR1
24
6.5 S. cerevisiae 43
%59.8 15
7.36
Amutha and 15
Monte et al., 2003) Gunasekaran, 2001
BR1 3 3 4
9
BR1 9
T24 T24 T24 T0 53.4 3.9 7.3 1 8.3 57.7 4.2 7.28 1.02 8.3 3 60 4.5 7.5 0.8 8.3 BR1
81
BR1
BR1
S. cerevisiaeBR1 4 4
4 10 pH 1 4 4
BR1
BR1 pH 10
30
pH 100 100 100 100 100
1.45 ± 0.19b 35.33 ± 2.90c 4.43 ± 0.58b 12.53 ± 0.75 1.5 ± 0.1a 14.033 ± 0.15 4
2.49 ± 0.23a 57.61 ± 1.96a 7.6 ± 0.7a 13.19 ± 0.7 0.94 ± 0.05d 14.13 ± 0.15 4.5
2.63 ± 0.19a 61.66 ± 1.19a 8.0 ± 0.6a 13.03 ± 0.51 1.05 ± 0.08c 14.1 ± 0.1 5
2.25 ± 0.14a 52.66 ± 0.37b 6.86 ± 0.45a 13.0 ± 0.52 1.26 ± 0.05b 14.26 ± 0.05 5.5 %5 0.365 3.503 1.112 0.81 0.144 0.172 LSD
5
82
BR1 pH 4
100 13.03 5 pH
5 pH 100 1.5 4 pH
5.5 5 4.5 0.84
5 pH
4.43 4 8
5 4.5 100
100 61.66 5 pH
83
100 35.33 4 pH
5 pH 5.5 5 4.5 pH
4 2.63
1.45
pH
6.39 4.5 5.5-4
Amutha and Kourkoutas et al.,2004 100/ 13.59
Gunasekaran, 2001
(Nigam,1999 Vasconcelos et al.,1998 4.5 4.2 pH
4 S.cerevisiae
4.5 4 5.5 5.5
Mariam et al., 2009 6.39
5 pH
S.cerevisiae BR1
84
BR1 5 11 2 4 4
5 pH BR1 11
100 100 100 100 100 0.90 ± 0.15d 29.5 ± 0.96d 2.76 ± 0.47d 9.4 ± 0.7b 4.73 ± 0.55a 14.13 ± 0.7 20 1.77 ± 0.06c 40.7 ± 2.61c 5.4 ± 0.2c 13.3 ± 0.55a 0.76 ± 0.28b 14.06 ± 0.61 25 2.58 ± 0.11a 59.43 ± 1.26a 7.86 ± 0.35a 13.23 ± 0.41a 0.93 ± 0.15b 14.16 ± 0.3 30 1.99 ± 0.05b 46.86 ± 2.12b 6.06 ± 0.15b 12.96 ± 0.6a 0.98 ± 0.18b 13.93 ± 0.40 35 0.198 3.506 0.602 1.088 0.627 0.31 %5 LSD
5
85
BR1 (5
º25 100 4.73 20
100 0.76
º 35 30 25
º20 100 13.23 º30
100 9.4
7.86 º30
100 2.76 º20 100
86
59.43 º30 100
29.5 º20 100
º30 100
0.90 º20 2.58
(40-25
6.42 30
Mariam et al.,2009 /100 14.79
7.86 BR1 º30
6 12 Bx 3 4 4
BR1
87
30 5 pH BR1 Bx 12
(Bx 100 100 100 100 100 2.14 ± 0.13a 60.33 ± 2.15a 6.53 ± 0.40c 11.03 ± 0.30c 0.63 ± 0.03b 11.8 ± 0.62d 16-24h 2.33 ± 0.21a 50.3 ± 2.05a 7.1 ± 0.65b 14.36 ± 0.87bc 1.13 ± 0.05a 15.46 ± 0.86bc 20-24h 1.31 ± 0.09b 58.76 ± 2.51a 8.64± 0.60ab 14.7 ± 0.41bc 0.76 ± 0.15b 15.46 ± 0.65bc 20-48h 1.38 ± 0.1b 50.93 ±0.94a 8.4 ± 1.08a 16.26 ± 0.97b 0.96 ± 0.11a 17.23 ± 1.95ab 24-48h 1.26 ± 0.07b 40.00 ± 2.1b 7.7 ± 0.31b 19.83 ± 1.85a 1.33 ± 0.23a 21.17 ± 1.63a 28-48h 0.330 16.125 0.530 3.520 0.470 2.958 %5 LSD
5
88
BR1 Bx (6
100 21.17 28
100 11.8 16
100 1.33 48 28
BR1
100 0.63 24 16
48 28
100 19.83
89
100 11.03 24 16
48 24
24 16 8.4
6.53
100 60.33 24 16
100 40 28
24 20
48 28 2.33
1.26
40 %24 Bx 60.83
100/ 23.52 /100 1.28
Laopaiboon et al., 2009
BR1
12
15 21
Mariam et al., 2009 14.92 6.49
6.53
11.03
7.1 14.36
90
BR1 7 13 4 4 4 16 30 5 pH BR1 13
100 100 100 100 100 %0.17 0.06 2.34 ± 0.09ab 60.63 ± 2.10 7.13 ± 0.30ab 11.76 ± 0.55ab 0.9 ±0.1b 12.66 ± 0.65
%0.34 0.12 2.45 ± 0.02a 61.23 ± 1.07 7.46 ± 0.05a 12.2 ± 0.26 a 0.36 ± 0.11c 12.56 ± 0.25
0.68 0.24 2.30 ± 0.0b 59.66 ± 1.42 7 ± 0.2b 11.73 ± 0.25ab 0.43 ± 0.11c 12.16 ± 0.35
1.36 0.48 2.17 ± 0.05c 58.6 ± 0.86 6.6 ± 0.17c 11.26 ± 0.46b 1.16 ± 0.15a 12.26 ± 0.25
0.124 3.2 0.384 0.759 0.230 1.3 %5 LSD
5
91
BR1 7
100 61.23
100 0.36
100 1.16 1.36 0.48
12.2
100
7.46
92
2.45
2.17
Saf-Instant S. cerevisiae
8.3 150
8.5
Red-Ethanol 59.15
8.1
.)Prescott and Dunn, 2002)
93
BR1 (8 14 5 4 4
0.34 0.12 16 Bx 30 5 pH BR1 14
100 100 100 100 100 b a 2.39 ± 0.01 59.6 ± 2.40 7.26 ± 0.05 12.2 ± 0.4 0.4 ± 0.1 12.6 ± 0.50 1.25% a a 2.45 ± 0.02 61.23 ± 0.25 7.46 ± 0.05 12.2 ± 0.1 0.4 ± 0.1 12.6 ± 0.1 2.50% ab a 2.44 ± 0.04 60.93 ± 1.60 7.43 ± 0.11 12.2 ± 0.17 0.53 ± 0.15 12.73 ± 0.25 5% c b 2.29 ± 0.04 57.56 ± 0.65 6.96 ± 0.15 12.1 ± 0.26 0.66 ± 0.15 12.76 ± 0.40 7.50% 15.35 3.78 14.46 0.11 2.93 0.19 F 0.001 0.059 0.001 0.951 0.099 0.901 Sig. %5 0.063 3.72 0.195 0.42 0.61 0.64 LSD
5
94
BR1 8
7.5
100 0.4 2.5 1.25) 100 0.66
100 0.4
12.2 100 12.2 100 12.2 5 2.5 1.25
100 12.1 7.5 100
100 61.23) 2.5
100 45.4 7.5
95
7.5 7.46 2.5
2.5 6.96
2.29 7.5 2.45
Ethanol-Red Saf-Instant 20
8.7 8.4 S. cerevisiae
Mukhtar et al.,
2010
S. cerevisiae BR1 5 4
9 15 pH 1 5 4
BR1
BR1 pH 15
pH 100 100 100 100 100 c c c 2 ± 0 48.17 ± 1.93 6.1 ± .01 12.33 ± 0.35 0.5 ± 0.46 12.83 ± 0.15 4 b b b 2.29 ± 0.01 55.07 ± 1.5 6.93 ± 0.05 12.6 ± 0.26 0.26 ± 0.15 12.85 ± 0.13 4.5 a a a 2.5 ± 0 63.2 ± 0.17 7.57 ± 0.05 11.96 ± 0.05 0.47 ± 0.05 12.43 ± 0.05 5 b b b 2.33 ± 0.05 57.33 ± 1.5 7.07 ± 0.11 12.33 ± 0.51 0.46 ± 0.35 12.78 ± 0.14 5.5 %5 0.056 2.713 0.163 1.2 0.75 0.9 LSD
5
96
BR1 pH (9
100 0.5 4 pH
100 0.26 4.5 pH
100 12.6 4.5 pH
100 11.96 5 pH
6.1 4 pH 7.57 5 pH
5 pH
97
100 48.17 4 pH 100 63.2
2.5 5 pH
2 4 pH
5 pH 59.1
24 100 10.4
Alkabbashi et al., 6.15
2011
(10 16 2 5 4
BR1
5 pH BR1 16
100 100 100 100 100
c c c c a 1.03 ± 0.05 44.56 ± 2.02 3.1 ± 0.17 6.96 ± 0.41 5.46 ±0.41 12.43 ±0.42 20 b b b b b 1.93 ± 0.25 59.5 ± 2.36 5.9 ± 0.65 9.9 ± 0.72 2.7 ± 0.65 12.6 ± 0.1 25 a a a a c 2.5 ± 0 63.36 ± 1.17 7.59 ± 0.1 12.00 ± 0.35 0.83 ± 0.21 12.83 ±0.15 30 a b a a c 2.36 ± 0.0 59.13 ± 1.46 7.13 ± .11 12.07 ± 0.38 0.66 ± 0.32 12.73 ±0.06 35 %5 0.248 3.417 0.654 0.920 0.815 0.31 LSD
5
98
BR1 (10
̊20
0.66 º35 100 5.46
º35 100
100 6.96 º20 100 12.07
7.59 º30
%3.1 º20
63.36 ̊30
º30 100
99
1.03 ̊20 2.5
40 25
6.42 30
100/ 14.79
12 7.59
24
(Mariam et al.,2009)
64.9 6.5
36 30 10.6
(Alkabbashi et al., 2011)
Bx 3 5 4
(11 17
30 5 pH BR1
100
5 pH BR1 17
30
100 100 100 100 100 a a c d 2.53 ± 0.03 62.43 ± 1.06 7.7 ± 0.1 12.33 ± 0.06 0.78 ± 0.27 13.1 ± 0.2 16-24h a b c c 2.33 ± 0.28 52.23 ± 6.04 7.06 ±0.92 13.76 ± 3.16 1.87 ± 2.29 15.63 ± 1.0 20-24h b b bc c 1.23 ± 0.01 50.13 ± 4.10 7.53 ±0.05 15.16 ± 1.35 0.46 ± 0.38 15.63 ± 1.01 20-48h b c b b 1.25 ± 0.0 43.13 ± 0.60 7.6 ± 0.00 17.63 ± 0.25 0.77 ± 0.23 18.4 ± 0.5 24-48h b d a a 1.25 ± 0.0 36.83 ± 1.89 7.6 ± 0.00 20.66 ± 1.02 1.96 ± 1.06 22.63 ± 0.1 28-48h %5 0.236 6.219 0.757 2.922 2.106 1.242 LSD
5
BR1 (11
101
48 28
20 100 1.96
100 0.46 48
24 16
24 20 7.7
7.06
16 100 22.63 28
100 13.1
100 20.66 48 28
100 12.33 24 16
24 16
100 62.43
100 29.03 48 28
24 16
48 20 2.53
1.23
Ethanol (Saf-instant) S. cerevisiae
102
15 13 7.5 7.7 Red
72 100 0.90 0.99
(Mukhtar et al., 2010) 24
72 30 53
24 100 52.23
Bai et al., 2004 100 13.76
(12 18 4 5 4 pH BR1
16 Bx 30 5
103
BR1 18
16 Bx 30 5 pH
100 100 100 100 100 0.12 a ab 2.47 ± 0.00 58.4 ± 2.26 7.5 ± 0.0 12.85 ± 0.49 0.12 ± 0.0 12.95 ±0.4 0.34
0.24 ab a 2.43 ± 0.11 63.86 ± 4.02 7.4 ± 0.34 11.64 ± 1.23 0.10 ±0.01 11.74 ±1.23 0.68
0.48 a ab 2.51 ± 0.04 60.23 ± 1.2 7.53 ±0.25 12.5 ± 0.34 0.24 ±0.22 12.73 ±0.11 1.36
0.96 b b 2.3 ± 0.00 55.6 ± 2.25 7 ± 0.0 13.63 ± 0.49 0.23 ±0.11 12.83 ± 0.4 2.72
0.143 5.796 0.98 1.92 0.42 0.83 %5 LSD
5
104
BR1 (12
100 0.24
100 0.10
100 13.63
7.53
7
100 63.86
105
100 55.6
2.51
2.3
S. cerevisiae Ethanol-Red Saf-Instant
32.5
7.1 7.5
7.9 42.74
7.5
Vasconcelos et al.,2004
(13 19 5 5 4
BR1
5 pH BR1 19 0.68 0.24 16 Bx 30
100 100 100 100 100 a b a c 2.45 ± 0.04 59.23 ± 0.92 7.46 ±0.11 12.6 ± 0.00 0.21 ± 0.15 12.8 ± 0.00 1.25% a a a b 2.4 ± 0.03 61.56 ± 1.41 7.3 ± 0.1 11.96 ± 0.25 0.18 ± 0.02 12.13 ± 0.3 2.50% a ab a a 2.46 ± 0.06 60.16 ± 0.35 7.5 ± 0.2 12.46 ± 0.3 0.2 ± 0.00 12.65 ±0.28 5% b c b a 2.27 ± 0.03 55.2 ± 0.1 6.9 ± 0.1 12.5 ± 0.2 0.26 ± 0.05 12.76 ± 0.25 7.50% %5 0.082 1.628 0.255 0.417 0.062 0.459 LSD
5
106
BR1 (13
2.5 100 0.26 7.5
100 0.18
1.25
100 11.96 2.5 100 12.6
7.5 5
%6.9 7.5
100 61.56 2.5
100 55.2 7.5
107
2.27 7.5 2.46 5
15 10 5
8.1 7.4 7 S. cerevisiae
(Arshad,2005) 5
Z. mobilis LMG 404 6 4
(14 20 pH 1 6 4
LMG 404
LMG 404 20
pH 100 100 100 100 100 b b b a b 2.23 ± 0.06 67.53 ± 0.4 6.8 ± 0.2 10.06 ± 0.35 0.8 ± 0.2 10.86 ± 0.55 6 a a a a b 2.37 ± 0.04 72.73 ± 0.92 7.2 ± 0.1 9.9 ± 0.1 0.66 ± 0.05 10.56 ± 0.11 6.5 ab b ab a b 2.28 ±0.04 68.66 ± 1.25 6.93 ±0.15 10.1 ± 0.43 0.7 ± 0.17 10.73 ± 0.66 7 c b c b a 1.94 ± 0.05 64.45 ± 0.42 5.93 ± 0.11 9.2 ± 0.2 1.7 ± 0.2 10.9 ± 0.2 7.5
0.097 4.260 0.277 0.567 0.317 0.842 %5 LSD
5
108
LMG 404 (14
7.5 pH
6.5 pH 100 3.3
6 pH 100 0.66
100 7.9 7.5 pH 100 10.06
7.2 6.5 pH
5.93 7.5 pH
100 72.73 6.5 pH
100 67.53 6 pH
109
7.5 pH 2.37 6.5 pH
7.3 1.97
40 21 5 pH
Karuppaiya et al., 2009)
(15 21 2 6 4
6.5 pH LMG 404
LMG 404 21
100 100 100 100 100 b b b a 2.23 ± 0.06 63.4 ± 2.95 6.8 ± 0.2 10.73 ± 0.25 0.86 ± 0.057 11.4 ± 0.2 25 a a a ab 2.45 ± 0.04 70.56 ± 1.04 7.43 ± 0.11 10.53 ± 0.25 0.8 ± 0.17 11.33 ± 0.15 30 b b b ab 2.16 ± 0.06 62.3 ± 0.43 6.66 ± 0.15 10.7 ± 0.3 0.76 ± 0.05 11.46 ± 0.25 35 b b b b 2.20 ± 0.06 62.43 ± 1.53 6.7 ± 0.17 10.73 ± 0.05 0.56 ± 0.15 11.3 ± 0.1 40 %5 0.112 3.312 0.307 0.442 0.231 0.348 LSD
5
110
LMG 404 (15
100 10.73 ̊25
º30
º40 100 0.86 º25
º30 100 0.56
6.66 º35 7.43
º30
62.3 º30 100 70.56
111
º30 100
2.16 º35 2.45
̊30 7.6
45 22
Panesar et al., 2006
Bx 3 6 4
16 22
LMG 404
LMG 404 %BX 22
30 6.5 pH
100 100 100 100 100 24
a a b d b d 2.31 ± 0.05 70.33 ± 0.55 7.03 ± 0.15 10 ± 0.17 0.76 ± 0.05 10.76 ± 0.11 16-24h
a c b c b c 2.32 ± 0.04 56.23 ± 1.02 7.06 ± 0.11 12.56 ± 0.15 0.96 ± 0.2 13.53 ± 0.11 20-24h c b b c b c 1.15 ± 0.0 55.1 ± 1.44 7.03 ± 0.2 12.76 ± 0.05 0.76 ± 0.15 13.53 ± 0.11 20-48h b c a b b b 1.24 ± 0.01 49.13 ± 0.51 7.56 ± 0.05 15.4 ± 0.1 0.96 ± 0.05 16.36 ± 0.11 24-48h b d a a a a 1.25 ± 0.03 43.8 ± 2.02 7.6 ± 0.2 17.36 ± 0.37 1.33 ± 0.3 18.7 ± 0.17 28-48h 0.065 2.275 0.286 0.373 0.332 0.235 %5 LSD
5
112
LMG 404 %BX (16
100 18.7 28
100 10.76 16
48 28
24 16 100 1.33
100 0.76
100 17.36 48 28
24 16
28 100 10
113
7.6 48
7.03 24 16
24 16
48 28 100 70.33
100 34.56
24 20
24 24 2.32
7.07 1.15
48 15
7.6
24 100 10.76
. (Behera et al., 2012)
17 23 4 6 4
LMG 404
16 Bx 30 5 pH
114
100 100 100 100 100 0.06 a a a 2.45 ± 0.02 69.56 ± .47 7.46 ± 0.05 10.73 ± 0.05 0.73 ± 0.32 11.46 ± 0.28 0.17
0.12 ab b ab 2.31 ±0.07 64.73 ± 0.87 7.03 ± 0.23 10.83 ± 0.49 0.93 ± 0.47 11.76 ± 0.05 0.34
0.24 b b b 2.13 ±0.13b 62.66 ± 3.52 6.46 ± 0.45 10.36 ± 1.2 1.3 ± 1.3 11.66 ± 0.15 0.68
0.48 b b b 2.15 ± 0.1 61.66 ± 2.3 6.56 ± 0.32 10.66 ± 0.85 1 ± 0.86 11.66 ± 0.15 1.36
0.176 4.070 0.567 1.469 1.572 0.344 %5 LSD
5
115
LMG 404 (17
100 0.73 100 1.3
100 10.83
100 10.36
7.46
6.46
116
100 69.56
100 61.66
2.45
2.13 1
5
500 1
1 10 0.39
0.28 500
Tiwari et al., 2011
(18 24 5 6 4
LMG 404
LMG 404 24 16 Bx 30 6.5 pH
100 100 100 100 100 a a a b 2.69 ± 0.01 70.83 ± 0.4 8.18 ± 0.05 11.56 ± 0.05 0.46 ± 0.05 12.03 ± 0.11 10.00% b b b b 2.58 ± 0.03 67.6 ± 0.6 7.86 ± 0.1 11.63 ± 0.05 0.46 ± 0.15 12.1 ± 0.1 12.50% c c c a 2.46 ± 0.01 66.26 ± 0.85 7.48 ± 0.05 11.3 ± 0.17 0.93 ± 0.25 12.23 ± 0.11 15.00% c d c b 2.45 ± 0.06 64.86 ± 0.65 7.45 ± 0.2 11.5 ± 0.2 0.6 ± 0.17 12.1 ± 0.1 17.50% 0.073 1.227 0.224 0.260 0.326 0.203 %5 LSD
5
117
LMG 404 (18
15 100 11.63 12.5
100 11.3
7.73 10
7 17.5
100 66.83 10
100 60.86 17.5
2.54 10
8.61 2.3 17.5
118
10 9.9
12.03 7.73
et al., 2011) 10
(Sulfahri
Z. mobilis LMG 404 7 4
19 25 pH 1 7 4
LMG 404
LMG 404 25
pH 100 100 100 100 100 b b b ab 2.43 ± 0.0 66.65 ± 2.22 7.39 ± 0.15 11.1 ± 0.6 1.46 ± 0.73 12.56 ± 0.15 6 a a a a 2.62 ± 0.03 70.25 ± 0.28 7.96 ± 0.11 11.33 ± 0.11 0.9 ± 0.26 12.4 ± 0.26 6.5 b b b a 2.45 ± 0.02 66.01 ± 0.51 7.46 ± 0.05 11.3 ± 0.01 1.1 ± 0.3 12.4 ± 0.3 7 c b c b 2.28 ± 0.03 65.78 ± 0.49 6.93 ± 0.1 10.5 ± 0.17 1.46 ± 0.25 11.96 ± 0.15 7.5 0.067 2.219 0.210 0.605 0.824 0.427 %5 LSD
5
119
LMG 404 19
100 0.9 6.5 pH 100 1.46 6 pH
pH
100 10.5 7.5 pH 100 11.33 6.5
7.96 6.5 pH
6.93 7.5 pH
100 70.25 6.5 pH
120
100 65.78 7.5 pH
7.5 pH 2.62 6.5 pH
pH 7.4 2.28
44 31 5.13
6.5
Maiti et al., 2011
(20 26 2 7 4 LMG 404
LMG 404 26
6.5 pH
100 100 100 100 100 b b b b 2.18 ± 0.12 66.2 ± 0.55 6.23 ± 0.37 10.03 ± 0.63 1.93 ± 0.68 11.96 ± 0.48 25 a a a a 2.61 ± 0.01 70.89.3 ± 0.79 7.53 ± 0.05 11.2 ± 0.1 0.9 ± 0.1 12.1 ± 0.1 30 b bc b a 2.31 ± 0.03 64.14 ± 0.81 6.6 ± 0.1 10.93 ± 0.3 1.13 ± 0.15 12.06 ± 0.2 35 b c b ab 2.24 ± 0.03 62.71 ± 2.11 6.4 ± 0.1 10.86 ± 0.55 1.26 ± 0.51 12.13 ± 0.15 40 0.125 2.318 0.384 0.847 0.821 6.114 %5 LSD
5
121
LMG 404 20
100 1.93 ̊25
100 0.9 ̊30
100 11.2 º30
100 10.03 º25
7.53 º30
6.23 º25
100 67.3 º30
122
100 58.96 º40
º25 2.48 º30
6.95 2.05
Cazetta et al., 2007) 48 º30
17 º32 7
24 7.53 º30
Bandaru et al., 2006)
21 27 Bx 3 7 4
LMG 404
LMG 404 %Bx 27
30 6.5 pH
100 100 100 100 100 b a b d c d 2.32 ± 0.04 69.96 ± 0.65 7.06 ± 0.11 10.1 ± 0.1 0.73 ± 0.2 10.83 ± 0.11 16-24h a c b bc b c 2.38 ± 0.05 60.56 ± 0.23 7.26 ± 0.15 12 ± 0.25 1.56 ± 0.4 b 13.56 ± 0.15 20-24h c c a b bc c 1.24 ± 0.02 61.03 ± 0.5 7.56 ± 0.11 12.4 ± 0.2 1.16 ± 0.28 13.56 ± 0.15 20-48h c b a c a b 1.24 ± 0.01 64.23 ± 1.69 7.53 ± 0.11 11.73 ± 0.32 3.23 ± 0.15 14.96 ± 0.25 24-48h c d a a a a 1.26 ± 0.01 49.8 ± 1.15 7.66 ± 0.05 15.4 ± 0.36 3.46 ± 0.25 18.86 ± 0.11 28-48h 0.059 1.805 0.210 0.484 0.499 0.301 %5 LSD
5
123
LMG 404 %Bx (21
28
100 10.83 16 100 18.86
48 28
16 100 3.46
100 0.73 24
48 28
24 16 100 15.4
124
100 10.1
7.66 48 28
24 16
7.06
100 69.96 24 16
39.26 48 28
24 20 100
2.38
1.24 24 24
20 5.07
Panesar et al., 2006
125
(22 28 4 7 4
LMG 404
28
100 100 100 100 100 0.12 a a a 2.31 ± 0.01 73.56 ± 1.98 7.03 ± 0.05 9.56 ± 0.25 0.9 ± 0.1 10.46 ± 0.15 0.34
0.24 b b b 2.02 ± 0.02 64.13 ± 1.36 6.13 ± 0.11 9.56 ± 0.35 1.1 ± 0.2 10.66 ± 0.15 0.68
0.48 bc bc bc 1.97 ± 0.05 62.63 ± 1.43 5.96 ± 0.15 9.53 ± 0.45 1 ± 0.45 10.53 ± 0.11 1.36
0.96 c c c 1.93 ± 0.05 60.96 ± 0.32 5.83 ± 0.15 9.56 ± 0.2 1.13 ± 0.15 10.7 ± 0.1 2.72
0.076 2.656 0.236 0.619 0.501 0.249 %5 LSD
5
126
LMG 404 (22
100 1.13
100 0.9
100 9.56
100 9.53
7.03
127
5.83
73.56
100
100 60.96
2.31
0.05 1.93
500 0.55 0.39
Pradeep and Reddy,2010
(23 29 5 7 4 LMG 404
pH LMG 404 29 16 Bx 30 6.5
100 100 100 100 100 a a a ab b 2.62 ± 0.03 70.13 ± 0.73 7.96 ± 0.05 11.35 ± 0.2 0.96 ± 0.05 12.66 ± 0.15 10.0% a ab a a b 2.58 ± 0.03 68.35 ± 0.49 7.86 ± 0.15 11.5 ± 0.17 0.93 ± 0.25 12.83 ± 0.11 12.5% b b b b a 2.39 ± 0.01 66.85 ± 0.23 7.26 ± 0.05 10.86 ± 0.05 1.6 ± 0.2 12.96 ± 0.15 15.0% c ab c c a 2.3 ± 0.03 67.66 ± 0.15 7.01 ± 0.1 10.36 ± 0.35 1.8 ± 0.3 12.66 ± 0.05 17.5% 0.056 3.094 0.188 0.417 0.417 0.236 %5 LSD
5
128
LMG 404 (23
17.5
100 0.93 12.5 100 1.8
11.5 12.5
100 10.36 17.5 100
7.96 10
7.01 17.5
15 100 70.13 10
100 66.85
129
2.3 17.5 2.62 10
10 11 6.84
°30
Puspita et al., 2010 11.35 %7.96
8 4 S.cerevisiae BR1 1 8 4
BR1 24 30 S. cerevisiae
S. cerevisiae BR1 30
%5 LSD 30 24 12 6 0 BR1 Mean ± SD Mean ± SD Mean ±SD Mean ± SD Mean ± SD
c c b a a 1.663 1.2 ± 0.6 1.32 ± 0.73 5.02 ± 1.37 12.31 ± 0.83 12.91 ± 0.85 100
c c b a a 1.080 11.71 ± 0.56 11.59 ± 0.7 7.29 ± 0.98 0.6 ± 0.05 0 ± 0
a a b c c 0.519 7.2 ± 0.4 7.2 ± 0.4 2.1 ± 0.2 0 ± 0 0 ± 0
a a b c c 2.462 61.46 ± 1.87 62.13 ± 1.23 29.57 ± 2.03 0 ± 0 0 ± 0 a a b c c 0.173 2.37 ± 0.15 2.37 ± 0.15 0.7 ± 0.06 0 ± 0 0 ± 0
5
130
S. cerevisiae BR1 (24
100 11.71 11.59 30 24
24
7.2 30
61.46 62.13 30 24
2.37 30 24
S. cerevisiae
131
24
100 46.9 15
0.25 24 58.3 20
0.31
Raposo et 30 30
al., 2009
BR1 31
100
Pearson Correlation 1 -.939-** .957** .960** .969** .960**
Pearson Correlation .957** -.989-** 1 .943** .980** .943**
Pearson Correlation .960** -.916-** .943** 1 .979** 1.000**
Pearson Correlation .969** -.964-** .980** .979** 1 .979**
Pearson Correlation .960** -.916-** .943** 1.000** .979** 1
31
0.943
. 0.980
0.943
132
S. cerevisiae BR1 2 8 4 25 32 S.cerevisiae BR1
S. cerevisiae BR1 32
%5 LSD 30 24 12 6 0
Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD
a a b c c 0.791 0.867 ± 0.06 1.167 ± 0.38 4.8± 0.51 12.72 ± 0.5 13.4 ± 0.49 100 a a b c d 0.461 12.54 ± 0.44 12.24 ± 0.25 7.91 ± 0.25 0.68 ± 0.06 0 ± 0 a a b c c 0.067 7.67 ± 0.06 7.67 ± 0.06 2.29 ± 0.01 0 ± 0 0 ± 0 a a b c c 2.457 61.21 ± 2.53 62.67 ± 1.4 28.99 ± 0.89 0 ± 0 0 ± 0 a a b c c 0.02 2.52 ± 0.02 2.52 ± 0.02 0.757 ± 0.01 0 ± 0 0 ± 0
5
133
S. cerevisiae BR1 (25
100 12.54 12.24 30 24
24 7.67 30 61.21 62.67 30 24
2.52 30 24
Saccharomyces cerevisiae 22 24 Caylak and 1.01 100 55.8
134
15 Sukan,1998 24 0.26 100 43.1 )Raposo et al., 2009(
BR1 33
100
Pearson Correlation 1 -.944-** .959** .962** .970** .962** Pearson Correlation .959** -.995-** 1 .939** .982** .939** Pearson Correlation .962** -.917-** .939** 1 .985** 1.000** Pearson Correlation .970** -.970-** .982** .985** 1 .985** Pearson Correlation .962** -.918-** .939** 1.000** .985** 1
33
0.939
0.982
0.939
135
Z. LMG 404 3 8 4 26 34 mobilis Z. mobilis LMG 404
Z. mobilis LMG404 34
انزمن بكتريا مىالس %5 LSD شىندر 0 6 12 24 30 Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD كميةانسكر 0.414 e d c b a )غ/100 مم( 0.29 ± 12.06 0.29 ± 11.46 0.26 ± 4.8 0.12 ± 1.36 0.1 ± 0.9 a a b c d انسكرانمستههك 0 ± 0 0.04 ± 0.6 0.41 ± 6.66 0.36 ± 10.7 0.38 ± 11.17 0.541 a a b c c نسبةاإليتانىل 0 ± 0 0 ± 0 0.03 ± 2.27 0.1 ± 7.6 0.1 ± 7.6 0.116 b a c d d انمردود 0 ± 0 0 ± 0 2.17 ± 34.27 1.88 ± 71.06 1.67 ± 68.09 2.699 a a b c c اإلنتاجية 0 ± 0 0 ± 0 0.01 ± 0.75 0.03 ± 2.5 0.03 ± 2.5 0.035
5
Z. mobilis LMG 404 (26
136
100 11.17 10.7 30 24
24
7.6 30
71.06 24
30 24
2.5
LMG 404 35
100
Pearson Correlation 1 -.952-** .963** .962** .969** .962** Pearson Correlation .963** -.997-** 1 .946** .986** .946** Pearson Correlation .962** -.927-** .946** 1 .981** 1.000** Pearson Correlation .969** -.979-** .986** .981** 1 .981** Pearson Correlation .962** -.927-** .946** 1.000** .981** 1
35
0.946
0.986
0.946
137
Z. LMG 404 4 8 4 27 36 mobilis Zymomonas mobilis LMG 404
Z. mobilis LMG404 36
%5 LSD 30 24 12 6 0 Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD d d c b a 0.347 0.50 ± 0.10 0.80 ± 0.00 3.87 ± 0.16 10.57 ± 0.21 12.13 ± 0.32 100 a b c d 0.468 11.63 ± 0.32 11.33 ± 0.32 a 6.70 ± 0.32 1.57 ± 0.15 0.00 ± 0.00 a a b c c 0.066 7.73 ± 0.06 7.73 ± 0.06 2.30 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 a b c c 2.051 66.50 ± 1.33 68.26 ± 1.41 a 34.44 ± 1.61 0.00 ± 0.00 0.00 ± 0.00 a a b c c 0.019 2.54 ± 0.02 2.54 ± 0.02 0.76 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
5
138
Z. mobilis LMG 404 (27
100 11.63 11.33 30 24
24 7.73 30 66.5 68.26 30 24
2.54 30 24
139
LMG 404 37
100
Pearson Correlation 1 -.955-** .983** .962** .968** .962** Pearson Correlation .983** -.989-** 1 .955** .988** .955** Pearson Correlation .962** -.907-** .955** 1 .976** 1.000** Pearson Correlation .968** -.972-** .988** .976** 1 .976** Pearson Correlation .962** -.908-** .955** 1.000** .976** 1
37
0.955
0.988
0.955
9 4
LMG 404 BR1 38
%5 LSD
24 Mean ± SD Mean ± SD Mean ± SD Mean ± SD
0.96 0.80 ± 0.00 1.37 ± 0.12 1.17 ± 0.38 1.32 ± 0.73 100 bc c a ab 0.836 11.33 ± 0.32 10.70 ± 0.36 12.24 ± 0.25 11.59 ± 0.70 0.91 7.73 ± 0.06 7.60 ± 0.10 7.67 ± 0.06 7.20 ± 0.40 a a b b 2.822 68.26 ± 1.41 71.07 ± 1.88 62.67 ± 1.40 62.13 ± 1.23 100 0.08 2.54 ± 0.02 2.50 ± 0.03 2.52 ± 0.02 2.37 ± 0.15
5
140
28
141
29
LMG 404 BR1
29 28 38
100 1.37
100 1.32
100 1.17
100 0.80
142
100 11.59 100 12.24
100 11.33
100 10.70
7.73
7.20
62.67 68.26 71 07
100 62.13
2.54
2.37
143
الفصل اخلامس االستنتاجات واملكرتحات CONCLUSION AND RECOMMENDATIONS
144
1 5
1
Saccharomyces cerevisiae 2
API 3
4
Zymomonas mobilis 5
Saccharomyces cerevisiae
ITS4 ITS1 6
7
2.5 16 ̊30 5 pH
0.34 0.12
0.68 0.24
8
10 16 ̊30 6.5 pH
145
0.17 0.06
0.34 0.12
9
24 10
146
2 5
147
املراجــــــــع References
148
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157
Saccharomyces sp. Batch
API 20
BR1,BR2,BR3
PCR
(Ethanol Red® S1929 Safdistil C
BR1
BR1
5 4.5 4 pH 35 30 25 20
28 24 20 16 BX 5 5
0.48 0.24 0.12 0.06
1.36 0.34 0.17
62.13 7.5 5 2.5 1.25
158
º30 5 pH
0.34 0.12 16 Bx
2.37 24 2.5
0.68 0.24
2.52 62.67
Zymomonas mobilis LMG 404
7 6 6 pH º 40 35 30 25
0.06 28 4 0 16 BX 7
1.36 0.68 0.34 0.17 0.48 0.24 0.12
17.5 15 12.5 10
2.5 71.06
0 06 16 Bx ̊30 6.5 pH
24 10 0 17
0 34 0 12
68.26
2.54
159
F spss
3
0.05
Zymomonas mobilis
Saccharomyces cerevisiae
BR1
160
Abstract: Optimization of bioethanol production from molasses using strains of Saccharomyces sp. yeast and Zymomonas mobilis bacteria
The production of bioethanol from sugar beet molasses (produced in AL-raqa sugar company) and from the molasses by-produced by raw sugar refining(Homs Sugar Company), by batch method using local isolates of Saccharomyces sp., was studied. Twenty local isolates were obtained and screened to test their ability to produce ethanol. They were classified by morphological and biochemical (API) methods. Three isolates named BR1, BR2 and BR3 were superior over the others in ethanol production.Their classification was confirmed through molecular protocol (PCR). Secondary screening of the last three isolates along with three imported isolates (Safdistil C, S1929 and Ethanol Red ®) and the strain of ethanol factory in Homos, was realized. Isolate named BR1 showed superiority in ethanol production over the other isolates and strains. The isolate BR1 was optimized in bioethanol production on the two molasse substrates with different variables: temperature (20, 25, 30 and 30° C), pH (4, 4.5, 5 and 5.5), total solid concentration (BX) (16, 20, 24 and 28%), nutrient additions: urea (0.06, 0.12, 0.24 and 0.28% W/V) and diammonium phosphate (0.17, 0.34 and 0.68% W/V), and inoculum volume of 1.25, 2.50, 5 and 7.5%. The maximum productivity value was 62.13% (V/W) on beet molasse substrate at pH=5, temperature of 30° C, Bx 16%, nutrient addition: 0.12% urea, 0.34% diammonium phosphate (W/V), inoculum volume of 2.5%,and fermentation time period of 24 h. The productivity value
161 was 2.37 g/l/h. When the molasse of refining raw sugar substrate was used, at the same parameters except for nutrient addition (0.24% urea and 0.68 diammonuim phosphate), the maximum yield was 62.67% (V/W) and the productivity 2.52 g/l/h. The optimization of bioethanol using the strain LMG 404 of the species Zymomonas mobilis was studied to evaluate the productivity and bioethanol yield using two molasse substrates. The studied variables were: temperature (25, 30, 35 and 40° C), pH (6, 6.5, 7 and 7.5), Bx% (16, 20, 24 and 28), nutrient addition: urea ( 0.06, 0.12, 0.24 and 0.48%), and diammonium phosphate( 0.17, 0.34, 0.685 and 1.36%) (W/V), and inoculum volume (10, 12.5, 15 and 17.5%). The maximum yield was 71.06% (V/W) and the maximum productivity 2.5 g/l/h, at pH=6.5, temperature 30° C, Bx 16%, nutrient addition 0.06% urea and 0.17% diammonium phosphate and inoculate volume 10% after 24 h. when sugar refining molasse was used in the same conditions except for the nutrient additions (0.12% urea and 0.34% diammonium phosphate), the maximum yield was 68.26% (V/W), and the productivity value was 2.54 g/l/h . All results were analyzed using SPSS program, where the value of F and confidence in the presence of more than 3 variables, then lesser significant differences(LSD) were calculated at p ≤ 0.05. These results indicate the superiority of Zymomonas mobilis bacteria for bioethanol production in the above-mentioned conditions. Concerning yeast strains of Saccharomyces cerevisiae, our experiments proved the superiority of the local strains BR1.
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املــــلخل
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