I= MICROBIOLOGICAL CONVERSION OF WASTE FRUITS AND VEGETABLES INTO ETHANOL
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Title of Thesis
MICROBIOLOGICAL CONVERSION OF WASTE FRUITS AND VEGETABLES INTO ETHANOL

Author(s)
Javed Iqbal Qazi
Institute/University/Department Details
Department of Zoology/ University of the Punjab
Session
2005
Subject
Zoology
Number of Pages
460
Keywords (Extracted from title, table of contents and abstract of thesis)
ethanol, saccharify, biomass pretreatment, fed-batch fermentation, immobilization, ethanologenic microorganisms, solid state fermentation

Abstract
The present study describes the production of ethanol from decayed fruits and vegetables employing saccharifying and ethanologenic microorganisms. Decayed apples, bananas and potatoes were selected for this research, as they usually take the forms of waste heaps in fruit and vegetables stock markets due to post-harvesting mismanagements in this country. The substrates chemical compositions were analyzed prior to fermentation. Apples, bananas and 2.5% H2 SO4 treated potatoes showed 12.53, 22.50 and 13.08% sugar concentrations, respectively. A total of 55 bacterial and 8 yeast strains were isolated from several habitats. The bacterial isolates, represented by 47 Bacillus spp,2 Proteus spp,1 Beijerincka sp., and the yeasts comprising of 4 Saccharomyces spp. and 1 Issatchenkia orientalis showed good ethanol productivity in synthetic media. The bacteria represented alkalophiles, acidophiles, thermoacidophiles thermotolerants and ethanol tolerant species. Thermophilic bacteria had 50°C as optimum, while some isolates of them could tolerate higher temperatures of 55 and 60°C. Many yeasts on the other hand, were found thermotolerant to 50°C, while growing in synthetic medium (YMP). An important finding was that most of the bacterial isolates are starch hydrolyzers and cellulose degraders. Thus potential of these bacteria is important for both starch and ethanol industries.

The bacterial isolates while growing in MIV medium broths resisted the added ethanol up to 30%. In this respect, they comprised three groups. Group-I (B. cereus- AhAS-1, B. coagulan-AhAS-8, B. coagulan-AhAS-16, B. coagulants -AhAS-21 and B. cerelis-AhAS-25), II (B. circli/alls-AhAS-3, B. circulans-AhAS-4, B. cerelis-AhAS-5, B. coagulans-AhAS-20, B. coagulans-AhAS-27, B. coagulans-AhAS-30 and B. circulans-AhAS-53) and III (R. cereus-AhAS-23 and B. coagulans-AhAS-28) of the bacterial isolates showed resistance up to 15, 20 and 30% ethanol, respectively. The isolates also varied in productivities and they produced ethanol in the range of 0.5 to 7.96%. Maximum ethanol concentration from 1.02 to 8.26% fermented by the isolate S. cerevisiae-AhAS-Y-9 appeared in the synthetic media.

Four thermophilic bacteria; B. circulans-AhAS-7, B. coaglilans-AhAS-8, B.circlllans-AhAS-29 and B. drcuialls-AhAS-43, two mesophilic; B. coagulans-AhAS-30 and B, coagulans-AhAS-31, and four yeasts, i.e. S. cerevisiae-AhAS-Y-1, S. cerevisiae-AhAS- Y-3, S. cerevisiae-AhAS-Y-4 and 1. orientials-AhAS-10 were selected for fermentations experiments on the selected natural substrates. Pure culture fermentations showed maximum ethanol yield of 3.76% produced by B. circulans-AhAS-43, while the yeasts S. cerevisiae-AhAS-Y-1 and S. cerevisiae-AhAS-Y-3 yielded 3.91 and 4.0% ethanol, respectively from apples. Bananas fermentations yielded highest ethanol of 3.15 and 4.49% by the isolates B. coaglilans-AhAS-31 and S. cerevisiae-AhAS-Y-3, respectively, while potatoes fermentations had maximum ethanol yields of 0.60 and 4.23% by R.circulans-AhAS-29 and S. cerevisiae-AhAS-Y-4, respectively.

When the substrates fermentations were carried out under optimum pH of the bacterial isolates at different temperatures, maximum ethanol yield of 1.69 and 3.121% were obtained from apples and bananas, respectively by B. coagulans-AhAS-30 at room temperature (RT 34°C). B. coagulans-AhAS-31 produced 3.16 and 3.19% ethanol from apples and bananas, respectively at 37°C. At 50°C highest ethanol yields (2.13 and 1.41 %) amongst the bacterial isolates were obtained by B. coagulans-AhAS-8 from apples and bananas, respectively. Similar growth conditions were followed for potatoes except that they were treated with 2.5% H2SO4. However, maximum ethanol yield of 2.82 and 2.24% were obtained by B. coagulans-AhAS-30 at RT and, 37°C, -respectively, while at 50°C highest yield(0.92%) was obtained by B. ciruculans-AhAS-29.

Co-culturing the yeast S. cerevisiae-AhAS-Y-3 with B. coagulans-AhAS-8 produced 3.42% ethanol from apples, the yield being higher than co-culturing the yeast with B. circulans-AhAS-29 (2.16%) at 37°C. Fermentations of bananas by the co-culture of B. coagulans-AhAS-8 and S. cerevisiae-AhAS-Y-3 had 3.35% higher ethanol than cu-culture of B coagulans-AhAS-31 and S. cerevisiae-AhAS-Y-3 (2.49%). Cu-culture of the yeast S. cerevisiae-AhAS-Y-3 and B. coagulans-AhAS-30 yielded 1.44% higher ethanol than co-culturing it with B. circulans-AhAS-43 (1.39%) while fermenting sulfuric acid-treated potatoes.

All the bacterial and yeast isolates could grow under anaerobic conditions, while some showed even more growth as compared to aerated conditions. This is one of the advantages that are sought for industrial fermentations. Solid state fermentation (SStF) is a beneficial process. Inoculum concentration of 62.5% was applied in SStF while co-culturing of B. coagulans-AhAS-H and S. cerevisiae-AhAS-Y-3; B. circlllans-AhAS-29 and S. cerevisiae-AhAS-Y-3; B. coagulans-AhAS-30 and S. cerevisiae-AhAS-Y-3; B. coagulans-AhAS-31 and S. cerevisiae-AhAS-Y-3; and B. circulans-AhAS-43 and S. cerevisiae-AhAS-Y-3 in apples, bananas and potatoes homogenized in distilled water. Yeast co-cultured with B. coagulans-AhAS-H on apples at 37°C; with B. circulans-AhAS-29 on apples at 37°C; and 50°C; ,with B. coagulans-AhAS-H on bananas at 37°C and with B. coagulans-AhAS-31 at RT (34°C) revealed more ethanol than fermentations of the same substrates employing a single microorganism. In general, the fermentations proceeded with gradual increase in ethanol yields.

Regarding total soluble carbohydrates at 50°C, growth of co-culture of B. coaglllalls-AhAS-8 and S. cerevisiae-AhAS- Y -3 on apples revealed significantly higher contents at3rd and 12th days indicating 217.99 and 553.14% increases, respectively as compared to the values obtained for the cultures incubated at 37°C. Twelve days co-culturing of B. circulans-AhAS-29 and S. cerevisiae-AhAS- Y-3on apples at 50oC yielded 568.95% significantly higher soluble carbohydrates as compared to the fermentation accomplished at 37°C. In ease of bananas co-culturing of B. coagulans-AhAS-8 and S. cerevisiae-AhAS-Y-3 at the elevated temperature again yielded significantly higher soluble carbohydrate contents at 9thand 12th days with corresponding figures of 399.22 and 750.06% increases, respectively. Fermentation of sulfuric acid-treated potatoes with co-culture of circu/ans-AhAS-43 and S. cerevisiae-AhAS-Y-3 also gave higher total soluble carbohydrate contents at the higher temperature as compared to the experiments accomplished at 37°C. The significant differences were 154.38, 172.48 and 127.44% at 3rd, 6th and 12th days; respectively.

Co-culturing of B. coagulans-AhAS-8 and S. cerevisiae-AhAS-Y-3 on apples at 50°C yielded higher glucose contents as compared to the fermentation accomplished at 37oC. The significant differences were 366.76 and 320.16% at 3rd and 6th days, respectively. Ninth day co-culturing of B. circulans-AhAS-29 and S. cerevisiae-AhAS-Y-3 on apples at the elevated temperature again yielded 774.92% significantly higher glucose contents as compared to the culture incubated at 37°C. In case of bananas, co-culturing of B. coagulans-AhAS-8 and S. cerevisiae-AhAS-Y-3 at 50°C yielded significantly higher glucose contents at second and fourth sampling periods with corresponding values of 597.53 and 3614.70% increases, respectively, as period to the outcomes of the fermentation done at 37°C. Co-culturing of B. coagulans-AhAS-31 and S. cerevisiae-AhAS-Y-3 on bananas yielded 1556.14% significantly higher glucose content at 6th day of incubation as compared to the fermentation at RT (34oC). Fermentation of potatoes with co-culture of B. coagulans-AhAS-30 and S. cerevisiae-AhAS-Y-3 gave 150.24% significantly higher glucose contents at 37°C after 3 days. Co-culturing of B circulans-AhAS-43 and S. cerevisiae-AhAS-Y-3 on potatoes also yielded higher glucose contents at 50°C as compared to the values obtained at 37°C. The increases turned out to be 202.98, 533.61, 1532.07 and 2115. 78% at first through last sampling days, respectively.

Co-culturing of B. coagulans-AhAS-8 and S. cerevisiae-AhAS-Y-3 on apples at 37°C yielded significantly higher ethanol amounts as compared to the results obtained at 50°C at the first three sampling days with corresponding figures of 266.16, 52.97 and 1055.07% increases, respectively. Co-culturing of B. circulans-AhAS-29 and S. cerevisiae-AhAS-Y-3 on apples at 37°C yielded 211.37% significantly higher ethanol as compared to the fermentation accomplished at 50oC at 9th day. Similarly, fermentation of bananas by the co-culture of B. coagulans-AhAS-8 and S. cerevisiae-AhAS- Y -3 yielded significantly higher ethanol at 37°C as compared to the yield at 50°C. The significant elevations were 337.75, 661.68 and 544.04% at first; third. and last sampling periods, respectively. Regarding sulfuric acid-treated potatoes, co-culturing of B. coagulans-AhAS-43 and S. cerevisiae-AhAS-Y-3 at 37°C yielded 224.10% significantly higher ethanol as compared to the higher temperature at 9the day post-inoculation.

In conclusion, different co-cultures of the bacterial isolates and the yeast gave differential outcomes in terms of saccharification and ethanol production on different substrates and at different fermentation temperatures. This diversity can be exploited to select a par1icular pair of the microorganisms for the processing of a given natural substrate under specified conditions.

When results in terms of saccharification and ethanol production of the same substrate by different co-cultures at different temperatures were compared by employing ANOV A, it appeared that, in general, high temperature fermentations liberated more sugar, while the ethanol was yielded significantly higher at fermentations carried out at 37°C as compared to the experiments accomplished at 50°C. In case of apples, the yeast S. cerevisilie-AhAS-Y-3 in combination with bacterial isolate B. coagulans-AhAS-8 proved the best in terms of saccharification of total soluble sugars as at first sampling day, the value turned out to be 11.37%. Regarding the glucose content, the co-culture of S. cerevisiae-AhAS- Y -3 and B. circulans-AhAS-29 yielded the highest amount so that at 12th day of fermentation at 50°C, 10.59mg/g were obtained. Concerning the ethanologenic potential, the co-culture of S. cerevisiae-AhAS-Y-3 and B. coagulans-AhAS-8 yielded 3.42% ethanol at third sampling day at fermentation temperature of 37°C. This value was the highest for the fermentations of apples.

In case of bananas, the S. cerevisiae-AhAS-Y-3 in combination with the bacterial isolate B. coagulans-AhAS-8 proved the best for saccharification in terms or total soluble carbohydrates as at first sampling day the value turned out to be 8.522%. Regarding the glucose content, the co-culture of S. cerevisiae-AhAS-Y-3 and B. coagulans-AhAS-8 again yielded the highest content so that at 12th day of fermentation at 50°C, 12.89mg/g were obtained. Concerning the ethanologenic potential, the same co-culture yielded 3.35% ethanol at the last sampling period at. fermentation temperature of 37°C. This value was the highest for the fermentations or bananas.

Regarding sulfuric acid-treated potatoes, the S. cerevisiae-AhAS-Y-3 in combination with the bacterial isolate B. coagltlcllls-AhAS-30 proved the best in terms of saccharification of total soluble sugars as at third sampling day the value appeared K60%1. For glucose content, the co-culture of S. cerevisiae-AhAS- Y-3 and B. cinulans-AhAS-43 yielded the highest amount so that at 12th day of fermentation at 50°C, 15.44mg/g were obtained. Concerning the ethanologenic potential, the co-culture of S. cerevisiae-AhAS- Y -3 and B. coagulans-AhAS-30 yielded 1.44% ethanol at fourth sampling day at fermentation temperature of 37°C This value was the highest for the fermentations of sulfuric acid-treated potatoes.

Ultraviolet (256nm) irradiations were found to improve ethanol productivity of some of the mutants. Microscopically, cells€™ morphologies were changed to some extent, either in diameter or length. However, size of colonies appeared smaller than controls. Some of the mutants of B. coagulans-AhAS-8, B. coagulans-AhAS-30 and S. cerevisiae-AhAS- Y-3 produced higher ethanol concentrations than the wild type strains in the synthetic medium M4F. The microbial diversity preserved as a result of this investigation possesses potential of blofuel, ethanol production form natural agricultural wastes.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
386.64 KB
2 1 Introduction 1
215.27 KB
3 2 Review of literature 16
952.76 KB
  2.1 Ethanol as a Renewable Resource of Energy 16
  2.2 Biomass Pretreatment and its conversion to product 22
  2.3 Emergence of Fermentation Inhibitory substance following pretreatment 28
  2.4 Fed-Batch Fermentation 32
  2.5 Continuous fermentations 33
  2.6 Bacteria versus yeast in the fermentation Technology 34
  2.7 Biotechnology and Bioengineering Aspects 35
  2.8 Simultaneous saccharification and Fermentation( SSF) 39
  2.9 Simultaneous Saccharification and Extraction fermentation (SSEF ) 44
  2.10 Solid State Fermentation ( SStF ) 45
  2.11 Use of Fruits, Vegetables and Agricultural Residues in Biotechnology 48
  2.12 Factors affecting fermentations 51
  2.13 Other factors Influencing Fermentation 63
  2.14 Extremophiles and Thermotolerant / Thermophilic Microorganisms 64
  2.15 Immobilization 68
  2.16 Product recovery 75
  2.17 Consolidated Bioprocessing( CBP) 75
  2.18 Biotechnology applications in Pakistan 78
4 3 Materials and methods 78
290.43 KB
  3.1 Screening of Bacterial and yeast Isolates 78
  3.2 Optimizations of growth conditions of bacteria and yeas isolates 85
  3.3 Assessment of starch Hydrolyzing ability 86
  3.4 Assessment of Cellulose Degrading Ability of Bacteria and Yeasts 87
  3.5 Growth of Bacteria and Yeast on potato Agar 87
  3.6 Facultative Aerobic Experiment 87
  3.7 Assessment of temperature tolerance of the Bacteria Isolates 88
  3.8 Fermentation 88
  3.9 Ultra violet(UV) Irradiation of the Bacteria Isolates AhAS-7, AhAS-29, AhAS-30, AhAS-31 and AhAS-43 and the Yeast AhAS-Y-3 and AhAS-43 and the Yeast AhAs-Y-3 96
  3.10 Identification of the Bacteria and Yeast Isolates 97
5 4 Results 99
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  4.1 Screening of Bacteria and yeast isolates 99
  4.2 Optimizations of Growth conditions of Bacteria and yeast Isolates 111
  4.3 Assessment of starch Hydrolyzing ability 197
  4.4 Assessment of Cellulose Degrading Ability of Bacteria and Yeasts 197
  4.5 Growth of Bacteria and Yeasts on Potato Agar 197
  4.6 Facultative Aerobic experiment 197
  4.7 Assessment of temperature tolerance of the Bacteria Isolates 199
  4.8 Fermentation 199
  4.9 Ultra violet( UV) Irradiation of the Bacteria Isolates AhAS-7, AhAS-29, AhAS-30, AhAS-31 and AhAS-43 and the Yeast AhAS-Y-3 and AhAS-43 and the Yeast AhAs-Y-3 284
  4.10 Identification of the Bacteria and yeast Isolates 291
6 5 Discussion 304
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  5.1 Conclusions and recommendations proposed economically feasible ethnologic Bioreactor for Waste fruits 315
7 6 Annexure 317
150.18 KB
8 7 References 328
934.48 KB