I= STUDIES ON THE PRODUCTION OF RAW STARCH HYDROLYZING AMYLOLYTIC ENZYMES BY VARIOUS MICROBES AND THEIR USE IN ETHANOL FERMENTATION
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Title of Thesis
STUDIES ON THE PRODUCTION OF RAW STARCH HYDROLYZING AMYLOLYTIC ENZYMES BY VARIOUS MICROBES AND THEIR USE IN ETHANOL FERMENTATION

Author(s)
Muhammad Aurangzeb
Institute/University/Department Details
University of the Punjab, Lahore
Session
1993
Subject
Botany
Number of Pages
280
Keywords (Extracted from title, table of contents and abstract of thesis)
ethanol fermentation, raw starch, amylolytic enzymes, mould, rhizopus miqricans, starch hydrolysate, rumen bacterium

Abstract
Thirty one cultures of mould were isolated from different habitats and screened for raw starch digesting activity. Of all the cultures tested, Rhizopus miqricans PCSIR-12 demonstrated maximum raw starch digesting activity. The conditions were optimised for enzyme production by both solid and submerged fermentation methods in the shake flasksas well as in the jstirred fermenter.

Barans of wheat, maize, rice and gram were evaluated as substrates for enzyme production. Wheat bran was found to be an ideal substrate for the synthesis of amylolytic enzymes. The moistening of wheat bran with acetate buffer (PH 4.5) produced best results. The tation of wheat bran to diluent (1:1) was most effective for mould growth was 1.8cm (10gm/250 ml flask). Further imcrease in thicknes resulted in the reduction of amylolytec activity.

Partial replacement of wheat bran with other substrates reduced the enzyme yiesk. The rate of enzyme synthesis was significantly inproved when 1.0 gm of raw wheat starch was added to 20.0gm of wheat bran mash. Similarly the addition of peptone or (NH4)2 so4 at a concentration of 1.0 gm nitrogen/liter of the diluent, enhanced the enzyme formation.am incubation perion of 48 hours was found to be optimum for enzyme production. The maximum amount of enzyme was recoverd from the fermented wheat bran mash when phosphate buffer (PH 6.5) was, for enzyme extraction.

The optimum conditions for the production of amylolytic enzymes by R. nigricans PCSIR-12, in the shake flask were PH 4.5 temperature 30oc and incubation period of 72 hours. The replacement of soluble starch in the basal medium with raw corn starch improved the enzyme yield. However, maximum amount of raw starch digesting activity was observed when 1.0% (w/v) maize starch was added to the medium. Similarly replacement of peptone by corn steep liquor (4.5% v/v) enhanced the enzymic activity. Addition of metal ions such as Ca+2 (1.5 mM) or Mg+2 (2.0mM) and phosphate compounds i.e.KH2PO4 (0.1 % w/v) to the basal medium improved the enzyme synthesis. The rate of production of the enzyme was increased when 10-liter fermenter was employed, during scale-up studies. Partial purification of the cuuke enzyme, resulted in the increase of specific enzyme activity (IU/mg of protein) while total activity units were decreased.

The degradibility of different starches such as corn, wheat, rice, sweet-potato, sago and potato, by the crude enzyme of R. nigricans PCSIR-12 was also studied. These starches were digested in a descending order. Maximum digestibility was recorded when 10 gm of raw maize starch was incubated with 100 m1 of crude enzyme at PH 4.5 and 45oc, for a period of 60 hours, under shalking conditions. The rate of sccharificaiton was further increased to 96.2% in the stirred vessel because of substrate. Glucose was the only product of starch hydrolysis, as revealed by the paper chromatography of the reaction products.

Starch hydrolysate obtained from the enzymatic saccharification of raw corn starch was used for the production of ethanol by saccharomyces cerevesae. The optimum PH, temperature and inoculum size was 4.5, 30oc and 10% (v/v), respectively. The fermentation was almost complete within 96 hours. The replacement jof yeast xtract (0.3% w/v) or (NH4)2 so4 (0.5% w/v) with 3.5% (v/v) corn steep liquor improved the rate jof ethanol production. Maximum ethanol production (47 g/1) was recorded, 60 hours after inculation.

Twelve bacterial cultures were also isolated from the frmen fluid, under anaerobic conditions. Streptococcus bovis PCSIR-7B, produced maximum amount of raw starch digesting activity in the medium M2, at PH 7.0 and 40oc. The replacement of soluble starch by raw corn starch (2.0% w/v) improved the enzyme yield. The replacement of peptone with other nitrogen sources did not improved enzyme formantion. The rate of enzyme production was further improved when scale up studies were careied out in 10-liter glass stainless steel fermenter. The specific activity of the anzyme (IU/mg of protein) increased gradualy during the partial purification of enzyme solution. However, total activity of the enzyme solution was decreased.

The order of digestibility for the starches of waxy corn, wheat, rice, sweet potato, sago and potato was 41%, 32% 25%, 20%, 14%, 11%, respectively. The incubation period was 24 hours. The optimum ph and temperature for starch hdrolysis was 5.5 and 40oc, respectively. The degree of hydrolysis of 2.0% (w/v) corn starch suspension after 72 hours was 86.2% in the shake flask. The rate of saccharification was further increased to 94.2% in the stirred yessel. Paper chromatography of the reaction product showed that glucose and maltose were the main products of enzymatic saccharification of corn starch.

Maltotriose was also produced in significant amounts during early stages of the reactions, but was latter replaced with maltose. Ethanol fermentation of starch hydrolysate was carried out in 10-litre glass stainless steel fermenter using a strain of Saccharomyces cerevisiae. The optimum PH, temperature and inculum size was 4.5, 30oc and 6% (v/v)respectively. Maximum amount of ethanol (9.0gm/L) was produced, 72 hours after inoculation.

Download Full Thesis
2266.02 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
145.95 KB
2 1 Introduction 05-09
44.98 KB
3 2 Review of Literature 10-34
364 KB
  2.1 Chemistry of starch 10
  2.2 Process description 10
  2.3 Starch Hydrolysis 17
  2.4 Ethanol fermentation of raw starch 34
4 3 Materials and methods 35-63
261.15 KB
  3.1 Isolation of raw starch digesting microbes from soil and starch samples 45
  3.2 Screening of isolated culture 47
  3.3 Maintenance of stock cultures 48
  3.4 Identification of selected cultures 49
  3.5 Inoculum Preparation technique 51
  3.6 Partial Purification of Enyme 57
  3.7 Ethanol fermentation of starch hydrolysate 61
5 4 Analytical methods 64-82
910.04 KB
  4.1 Estimation of protein 64
  4.2 Estimation of reducing sugars 67
  4.3 Estimation of alcohol 75
  4.4 Estimation of cell cry weight 78
  4.5 Measurement of enzyme activity 78
  4.6 Paper chromatography 81
6 5 Results 82-223
260.52 KB
  5.1 Isolation and screening of microbes capable of producing raw starch Hydrolyzing enzymes from soil & starch samples 82
  5.2 Identification of selected cultures 86
  5.3 Optimisation of culture conditions for enzymes synthesis 95
  5.4 Enzyme purification 145
  5.5 Starch hydrolysis 147
  5.6 Ethanol fermentation of starch Hydrolysat 158
7 6 Rumen Bacterium 170-202
238.92 KB
  6.1 Isolation screening and selection of raw starch digesting microbes fro rumen fluid 170
  6.2 Nutritional studies 174
  6.3 Scale-up production of amylolytic enzymes in 10-L Glass stainless steel fermenter 184
  6.4 Enzyme purification 186
  6.5 Starch Hydrolysis 189
  6.6 Ethanol fermentation 202
  6.7 Discussion 203-224
  6.8 Mould 203
  6.9 Rumen Bacterium 219
  6.10 References 225