BIOETHANOL PRODUCTION FROM CORN COB USING CO-CULTURE OF ZYMOMONAS MOBILIS AND BACILLUS LICHENIFORMIS
BIOETHANOL PRODUCTION FROM CORN COB USING CO-CULTURE OF ZYMOMONAS MOBILIS AND BACILLUS LICHENIFORMIS
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Date
2023-06
Authors
MIGAP, HELEN HOOMSUK
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Abstract
There have been growing interest about biosynthesis of fuels from renewable biomass
resources due to depleting fossil resources and associated environmental issues in the
past few decades. Bioethanol is considered to be a good choice for an alternative
liquid fuel because it can be produced from a variety of agricultural renewable
materials. Proximate analysis of corn cob was carried out and the results revealed that
the substrate had the following nutritional composition: moisture (13.50%), ash
(14.90%), crude protein (3.50%), lipid (1.00%) and carbohydrate (67.10%). The corn
cob used in this research work had lipid and protein contents that can serve as sources
of energy for microbial growth and replication. The substrate was rich in
carbohydrates that have great potential for bio-ethanol production. In the present
research work, Zymomonas mobilis an ethanologenic bacterium was isolated from
palm sap and characterized molecularly by amplification of the alcohol
dehydrogenase I gene and the 16SrRNA gene. A total of 10 Xylose fermenting
Bacillus species were also isolated from the soil and characterized biochemically and
through sequencing of the 16SrRNA genes. The sequences with their accession
numbers have been deposited in the gene bank. The following Bacillus species were
characterized: Bacillus amyloliquifaciens (20%), Bacillus licheniformis (20%),
Bacillus thuringiensis (30%), Bacillus cereus (10%) and Bacillus subtilis (10%).
Zymomonas mobilis isolates ZM2 and ZM3 were screened for alcohol tolerance and
glucose fermentation to select the best to be used for bioethanol production. Growth
of the two isolates were measured as optical densities at 540nm; Zymomonas mobilis
isolate 3 (ZM3) had optical densities that ranged from 0.66-1.40 while ZM2 had
optical densities that ranged from 0.34-1.36. ZM2 and ZM3 were also screened for
glucose fermentation; ZM3 had ethanol yields that ranged from 30–80g/l while ZM2
had ethanol yields that ranged from 24-60g/l. ZM3 had higher optical densities in the
presence of varying alcohol concentrations compared to ZM2 although it was not
significantly different (P≥0.05). Similarly, ZM3 had higher ethanol yields compared
to ZM2 which were significantly different (P≤0.05).The Bacillus isolates were
screened for ethanol production from fermentation of xylose and cellulase production to select the best isolate for bioethanol production. The isolates had ethanol yields
from the fermentation of xylose that ranged from 18.50-35.5g/l. Bacillus
licheniformis (SCFB22) had the highest ethanol yield of 35.5g/l while Bacillus
thuringiensis (SBX8) had the lowest ethanol yield of 18.50g/l. The cellulase activities
of all the isolates ranged from 1.35-2.98FPU/ml. Bacillus licheniformis (SCFB22)
also had the highest cellulase activity of 2.98FPU/ml while Bacillus thuringiensis
(SBX3) had the lowest cellulase activity of 1.38FPU/ml. Acetic acid adaptation of
Zymomonas mobilis isolate ZM3 was carried out by treating the isolate with varying
amounts of acetic acid that ranged from 0.2-1.6 % to enhance the isolates‟ ability to
withstand high concentrations of inhibitors like acetic acid during fermentation.
Zymomonas mobilis isolate ZMA with a slightly enhanced tolerance to relatively high
acetic acid concentrations was obtained. Submerged fermentation of corn cob
hydrolysate was carried out using acetate adapted ZMA isolate, the parent
Zymomonas mobilis isolate (ZM3) and xylose fermenting Bacillus licheniformis in set
ups that contained co-culture of the two bacteria and those containing the single
strains. High bioethanol yields of 38.6g/l and 35.3g/l were obtained from the coculture
submerged fermentation of corn cob hydrolysate using acetate adapted
Zymomonas mobilis (ZMA) and xylose fermenting Bacillus licheniformis (SCFB22)
as well as co-culture submerged fermentation using the parent Zymomonas mobilis
isolate (ZM3) and Bacillus licheniformis (SCFB22). Submerged fermentation using
single isolates yielded relatively lower bioethanol contents. Isolates ZMA,
Zymomonas mobilis ATCC 29191, ZM3, SCFB22 and Bacillus licheniformis ATCC
14580 had bioethanol yields of 32.1, 31.8, 31.9, 12.8 and 14.5g/l respectively. The
acetate adapted isolate (ZMA) also had a higher ethanol yield of 32.1g/l than the
parent isolate (ZM3) which had ethanol yield of 31.8g/ml. The results of the one way
ANOVA that was carried out showed that the ethanol yields obtained from the coculture
fermentation using different isolates were significantly different (P≤ 0.05).
Co-culture fermentation using Zymomonas mobilis isolate ZMA, ZM3 and Bacillus
licheniformis isolate SCFB22 have great potential for industrial bioethanol production
using agricultural waste (corn cob). The physicochemical parameters of the bioethanol produced were all similar to that of the commercial 98% ethanol used in
this study but with some variation in some of the parameters. All the bioethanol
produced were colourless, the relative densities of the bioethanol produced ranged
from 0.77-0.84g/cm3 while that of the commercial ethanol was 0.76 g/cm3.The
variation in the relative densities of the bioethanol produced could be due to the
different types of bacteria used in the set ups or the different bioethanol yields. The
boiling points of the bioethanol produced were similar to that of the commercial
ethanol. The boiling points of the bioethanol produced ranged from 78.6-78.9ºC while
that of the commercial ethanol was 78.6ºC. The flash points of the bioethanol
produced ranged from 14-16ºC while that of the commercial ethanol was 13ºC. The
variation observed in the flash point of the bioethanol produced could be due to the
fact that the bioethanol produced contained more water that the commercial ethanol.
All the bioethanol produced as well as the commercial ethanol burned with a blue
flame. The result of the physicochemical parameters of the bioethanol produced
suggests that the bioethanol produced are of good quality and could serve as
substitutes to conventional ethanol.
Description
A DISSERTATION SUBMITTED TO THE SCHOOL OF POSTGRADUATE
STUDIES, AHMADU BELLO UNIVERSITY, ZARIA
NIGERIA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF
DOCTOR OF PHYLOSOPHY (PhD) IN MICROBIOLOGY
DEPARTMENT OF MICROBIOLOGY,
FACULTY OF LIFE SCIENCES,
AHMADU BELLO UNIVERSITY,
ZARIA, NIGERIA