Post on 12-Aug-2021
transcript
NMHS Progress Report
(Period from July 2019 to March 2020)
1. Project Information
Project ID: NMHS/2017-18/SG22/03 Sanction Date:
23-02-2018
Project Title: Bioconversion of pine needles: a challenging waste of Himalayan forest to second generation biofuel
BTA: Livelihood Options and Employment Generation
PI and Affiliation (Institution):
Dr Nivedita Sharma, Professor, Dr Y S Parmar University of Horticulture & Forestry, Nauni - Solan (H.P. –India
Name & Address of the Co-PI, if any:
Not Applicable
Abstract -detailing thecurrent yearprogress [WordLimit 250 words]:
In continuation to previous year report, this year (2nd year) major activities successfully
accomplished in the project were saccharification of pine needles biomass using purified
hydrolytic enzymes i.e. cellulase and xylanase. Two hyper hydrolytic enzyme producer
strains i.e. B. stratosphericus N12 (M) and B. altitudinis Kd1 (M) have been used for
inhouse enzyme production. Optimization of process parameters using classical one
factor at a time (OFAT) and statistical model- RSM to enhance the yield of sugars
production has been done. Different process parameters in OFAT had enhanced
saccharification of pine needles and maximum reducing sugars yield achieved was 28.05
mg/g of biomass at enzyme dosage of 12.5 ml/g in the ratio of 7.75 : 4.75 (cellulase:
xylanase) after 72 h of enzymatic hydrolysis at 45 ◦C temperature with purified enzymes.
The optimized conditions of OFAT were further subjected to optimization using
statistical approach i.e. Response surface Methodology (RSM). Further an appreciable
increase in reducing sugars i.e. 33.21 mg/g with overall 453.50 % through RSM was
achieved. Quantitative analysis of sugars obtained during saccharification of biomass by
crude, partially purified and purified enzymes by using HPLC technique has also been
done.
Further fermentation of reducing sugars into bioethanol was done by using monoculture
of Saccharomyces cerevisiae and Pichia stipitis as well as co-culture combinations of
ethanologens (Saccharomyces cerevisiae + Pichia stipitis) in shake flask at 25 ◦C for 72 h
and the maximum ethanol i.e. 11.06 g/l was observed in co-culture of Saccharomyces
cerevisiae and Pichia stipitis which was selected for further studies.
Fermentation process was devised by using a co-culture combination of Ethanologens
(Saccharomyces cerevisiae and Pichia stipitis) with maximum ethanol (16.44 g/l) and
fermentation efficiency of 69.47 %. The optimized conditions were further subjected to
the scale up process in a stirred tank bioreactor (7.5 litres). Standardization of scale up
process parameters i.e. fermentation time, temperature and agitation rate have been
accomplished in a bioreactor to maximize the bioethanol production. The best
conditions for scale up of bioethanol production in stirred tank bioreactor were 30 h of
fermentation time, 25◦C temperature and 200 rpm at which the maximum of bioethanol
i.e. 18.96 g/l with fermentation efficiency of 72.54 %. Increased saccharification and
ethanol yield, higher fermentation efficiency and considerable reduction in fermentation
time are the main highlighting features of the present study.
Project Partner Name
Affiliations Role & Responsibilities
Partner 1Dr Nivedita Sharma, Professor, Dr Y S Parmar University of Horticulture & Forestry, Nauni-Solan (H.P. –India
All Research Work has been done in the same institute
Partner 2 Kasturba Seva Samiti, Solan (H.P.)-India Collection of pine needles from forests.
2. Project site details
Project Site Himachal Pradesh and Uttarakhand
IHR States Covered Chir forests of Himachal Pradesh
Long. & Lat. Himachal Pradesh (Latitude: 33°12'40" N & Longitude: 75°45' 55" E) and Uttarakhand (Latitude: 29.594189 & Longitude: 79.653893)
Site Maps
Site Photographs -
3. Project Activities Chart w.r.t. Timeframe [Gantt or PERT]
Project Activities
WORK UNDERTAKEN AND OUTPUTYear 2018-19
Qtr 1 Qtr 2 Qtr 3 Qtr 4Project Activity 1
Optimization of process
parameters for enhanced
enzymatic hydrolysis of
pine needles biomass:
Following parameters were
optimized by using one
factor at a time approach
(OFAT)
Optimization of microwave
irradiation dose
The optimized microwave
dose was 600 W for 4 min at
which highest amount of
reducing sugars was
observed i.e. 20.31 mg/g of
biomass after 72 h of
Standardization of
process parameters for
complete hydrolysis of
pine needles biomass
using Response Surface
Methodology (RSM) at
pilot plant scale (7.5
litre bioreactor):
Following parameters
were optimized by using
a statistical approach i.e.
Response Surface
Methodology (RSM)
1. Incubation time
2. Enzyme dose
3. Temperature
Conversion of reducing
sugars into bioethanol:
Hydrolytic enzymes i.e.
cellulase and xylanase
production and
purification from B.
stratosphericus N12 (M)
and B. altitudinis Kd1 (M)
respectively has been
done under the conditions
standardized in first year.
Enzymatic saccharification
of pine needles biomass
using inhouse enzymes
Scale up of bioethanol
production in a 7.5 litre
stirred tank bioreactor:
Fermentation process for
bioethanol production was
shifted to bench scale in a
7.5 litre capacity bioreactor
from shake flask experiment.
Different process
parameters viz.
fermentation time,
temperature and agitation
rate were optimized to
maximize the bioethanol
production in the bioreactor.
Collection sites for pine needles from coniferous Himalayan forests
enzymatic hydrolysis over
the control i.e. 6.0 mg/g in
untreated needles.
Optimization of incubation period
At 72 h, the highest amount
of sugars was produced as
20.31 mg/g in pretreated
biomass as compared to
12.46 mg/g in untreated
biomass.
Three independent
variables were chosen
for optimization studies
by employing Central
Composite Design (CCD)
of Response Surface
Methodology (RSM). The
experiment contained 20
runs. The design
involved 6 centre points,
14 non centre points.
The mathematical
relationship of response
(reducing sugars) and
variables i.e. A, B and C
was approximated by a
quadratic model
equation.
Conditions optimized for
maximum
saccharification of
biomass were 16.70 ml/g
of enzyme dose at 45◦C
temperature for 72 h for
pretreated biomass and
reducing sugars obtained
under these conditions
were 33.21 mg/g of
biomass with an overall
453.50 percent increase.
cocktail (crude, partially
purified and purified
enzymes) by applying
previously optimized
conditions i.e. 16.70 ml/g
enzyme dose at 45◦C
temperature for 72 h has
been done and maximum
reducing sugars i.e. 28.05
mg/g were achieved using
purified enzymes.
Fermentation of reducing
sugars into bioethanol by
using monoculture of
Saccharomyces cerevisiae
and Pichia stipitis as well
as co culture
(Saccharomyces cerevisiae
+ Pichia stipitis) has been
done at 25◦C temperature
for 72 h and maximum
ethanol i.e. 11.06 g/l was
produced by co culture
combination of
(Saccharomyces cerevisiae
+ Pichia stipitis) which was
selected for further
studies.
Fermentation of
saccharified solution with
purified enzymes has
been done and maximum
ethanol of 16.44g/l
biomass with
fermentation efficiency of
69.47% was achieved by
using pretreated pine
needles biomass.
Optimization of
fermentation time
Fermentation time was
standardized by observing
bioethanol production at
different time intervals i.e. 6,
12, 18, 24, 30, 36 and 42
hours. Sampling was done at
mentioned different time
interval.
Maximum bioethanol
production of 14.22 g/l with
fermentation efficiency of
54.79% was observed at 30 h
of fermentation time with
appreciable reduction in
fermentation time as
compared to shake flask.
Project Activity 2
Optimization of enzyme dose
Maximum reducing sugar
yield (mg/g) was observed
at enzymatic dosage of 12.5
ml/g in untreated biomass
and pretreated biomass as
19.06 and 20.56 mg/g
respectively.
Quantification of 6C
and 5C sugars in
saccharified solution by
High Performance
Technique (HPLC)
Quantitative analysis of
sugars obtained during
saccharification of
biomass by crude,
partially purified and
purified enzymes by
using HPLC technique
has been done and
found that the biomass
broken down to form 6 C
(29.96 mg/g) sugars
(glucose) and 5 C (1.50
mg/g xylose).
Research paper
published:
Nivedita Sharma, Nisha
Sharma and Dimple
Tanwar. 2020. Enhanced
biodegradation of pine
needles by optimizing
temperature for different
degrading fungi under
solid state fermentation.
Chemical Science and
Review Letters. 9(34):
374-381.
Optimization of
fermentation temperature
Different temperatures i.e.
20 0C, 25 0C and 30 0C were
used for the fermentation by
co cultures (S. cerevisiae + P.
stipitis).
Maximum bioethanol
production of 18.17 g/l with
fermentation efficiency of
70.58 % was observed at 25 0C.
Project Activity 3
Optimization of enzyme
ratio
Enzymatic cocktail of
7.75:4.75 (cellulase:
xylanase) revealed highest
amount of reducing sugars
i.e. 18.97 mg/g and 21.46
mg/g biomass in untreated
and pretreated biomass
respectively
Papers presented in
conferences:
Nisha Sharma and
Nivedita Sharma. 2019.
“Cost effective
enzymatic hydrolysis of
pine needles by
optimizing different
process parameters by
one factor at a time
approach for reducing
sugar production” in
International
Conference on “Recent
Trends in
Biotechnology and
Bioinformatics” on
dated 01-03 August,
2019.
Nisha Sharma and
Research paper
published:
Dimple Tanwar, Nivedita
Sharma and Nisha
Sharma. 2019. Evaluation
of different process
parameters for enhanced
enzymes production using
pine needles as substrate
by Trichoderma
guizhouense S5 [Accession
No. MN170570] isolated
from rotten wood under
solid state fermentation.
Journal of Chemical,
Biological and Physical
Sciences. 9 (4): 617-636.
Optimization of Agitation
rate
Different agitation rates i.e.
100 rpm, 200 rpm and 300
rpm were used for maximum
ethanol production by co
cultures (S. cerevisiae + P.
stipitis).
Maximum bioethanol
production of 18.96 g/l with
fermentation efficiency of
72.54 % was observed at 25 0C of fermentation
temperature with 33.33 %
increase.
Nivedita Sharma. 2019.
“Standardization of
process parameters for
enzymatic hydrolysis of
pine needles by using a
statistical approach:
Response Surface
methodology (RSM)” in
60th Annual conference
of association of
Microbiologists of India
(AMI-2019) and
International
symposium on
Microbial Technologies
in Sustainable
Development of
Energy, Environment,
Agriculture and Health
at “ Central University
of Haryana on dated
15-18th November,
2019.
Project Activity 4
Optimization of
Temperature
Different temperatures i.e.
35◦C, 40◦C, 45◦C, 50◦C and
55◦C were used for the
saccharification of pine
needles biomass and the
maximum of reducing
sugars i.e. 23.35 mg/g were
obtained at 45◦C in
pretreated biomass.
Saccharification of pine
needles biomass using
crude, partially purified and
Research paper
published:
Nivedita Sharma, Nisha
Sharma and Dimple
Tanwar. 2019. An
evaluation study of
different white rot fungi
for degradation of pine
needles under solid state
fermentation.
International Journal of
Current Microbiology
and Applied Sciences.
8(6): 588-601.
Research paper published:
Dimple Tanwar, Nivedita
Sharma and Nisha Sharma.
2020. Isolation and
screening of fungi from
rotten wood for various
hydrolytic enzymes
production. Annals of
Phytomedicine. 9(1):1-7.
.
purified enzymes
Optimization of reducing
sugars production during
saccharification of biomass
with crude, partially purified
and purified enzymes has
been done and the
maximum sugars i.e. 28.05
mg/g were achieved with
purified enzymes.
4. Financial and Resource InformationNote: A separate bank account is expected to be opened for NMHS Project as per the provision of Direct Beneficiary Account (DBA) as laid out by the Govt. of India and also facilitate the audit of accounts. The interest earned out of the NMHS project funds should be reported clearly in the utilization certificate.
Total Grant: 16,25,523.00 Grant Received Date: 23-07-2019
ProjectPartner(s)
Affiliations/ Institution
Budget Allocated to Work Done
Dr Nivedita
Sharma
Dr Y S Parmar
university of
Horticulture &
Forestry, Nauni-
Solan (H.P.)
Dr Y S Parmar university of
Horticulture & Forestry, Nauni-Solan
(H.P.)
At present work is going on
at Dr Y S P university of
Horticulture & Forestry,
Nauni-Solan (H.P.)
Kasturba seva
samiti- Solan
Kasturba seva
samiti- Solan - -
Project Staff Information:
S. No. Name Qualification Designation Fellowship/ Wages paid Remarks1. Nisha Sharma PhD Microbiology Research
Associate -II
49000/-PM
(8, 06, 000.00 rupees)
5. Equipment and Asset InformationS.
No.EquipmentName (Qty)
Details (Make/Model)
Cost Date ofInstallation
Photographsof Equipment
LowestQuotation,
IFNOT
purchased1. Spectrophotom
eter-20DSpectrometer 106/ Systronics
48,000.00 rupees
24/01/2019
Nil
2. Digital autoclave
SLEFA-SS7441/Equitron
2,92,000.00 rupees
29/03/2019
Nil3. Deep freezer Celfrost/GN650 1,50,000.00
rupees24/04/2019
Nil4. High speed
CentrifugeEppendorf/5810R 7,35,000.00
rupees03/04/2019
Nil
6. Expenditure Statement and Utilization CertificatePlease update the annual Expenditure Statement and Utilization Certificate (UC) periodically.
Expenditure Information (on August, 2020):
S. No. Financial Position/Budget Head Funds Sanctioned Expenditure % of Total cost
I Salaries/Manpower cost Rs. 7,99,484.00 Rs. 8,06,000.00 100.81
II Travel Rs. 50,000.00 12.807.00 25.61
III Expendables &Consumables Rs. 4,17,152.00 Rs. 4,15,061.00 99.49
IV Contingencies Rs. 83,887.00 Rs. 83,306.00 99.30
V Activities & Other Project cost - - -
VI Institutional Charges 2,00000.00 Rs. 2,00000.00 100.00
VII Equipments Rs. 75,000.00* - 0.0
Total 16,25,523.00 Rs. 15,17,174.00 93.33
Interest earned Rs. 23,143.00
Grand Total Rs. 16,48,666.00
*Funds left after necessary equipment purchase - request is submitted permit to purchase a new water bath as
old one available in our laboratory has gone out of repair mode
Period (2019-2020) Expenditure Statement Utilization Certificate (UC)Annual [Attached] [Attached]
7. Project Beneficiary Groups
Beneficiary Groups [CapacityBuilding]
Target andAchieved
No. of Beneficiaries with incomeN.A.generation:
No. of stakeholders trained,N.A.particularly women:
No. of capacity building Workshops/N.A.trainings:
No. of Awareness & outreachN.A.programmes:
No. of Research/ ManpowerN.A.developed:
8. Project Progress Summary (as applicable to the project)Description Total (Numeric) Description
IHR States CoveredProject Site/ Field Stations Developed: Pine needles has been collected
from different forests of Himachal Pradesh and Uttarakhand
Fig: Sites for pine needles collectionNo. of Patents filed (Description): N.A. RESEARCH PAPERS:
Nivedita Sharma, Nisha Sharma
and Dimple Tanwar. 2019. An
evaluation study of different
white rot fungi for degradation
of pine needles under solid state
fermentation. International
Journal of Current Microbiology
and Applied Sciences. 8(6): 588-
601.
Nivedita Sharma, Nisha Sharma
and Dimple Tanwar. 2020.
Enhanced biodegradation of
pine needles by optimizing
temperature for different
degrading fungi under solid state
fermentation. Chemical Science
and Review Letters. 9(34): 374-
381.
Dimple Tanwar, Nivedita
Sharma and Nisha Sharma.
2019. Evaluation of different
process parameters for
enhanced enzymes production
using pine needles as substrate
by Trichoderma guizhouense S5
[Accession No. MN170570]
isolated from rotten wood under
solid state fermentation. Journal
of Chemical, Biological and
Physical Sciences. 9 (4): 617-636.
Dimple Tanwar, Nivedita
Sharma and Nisha Sharma.
2020. Isolation and screening of
fungi from rotten wood for
various hydrolytic enzymes
production. Annals of
Phytomedicine. 9(1):1-7.
Book chapter
Nisha Sharma and Nivedita
Sharma. 2020. Industrially
important xylanases and their
commercial applications. Recent
progress in Microbiology and
Biotechnology. Science Domain
International Publisher, London.
Article/ Review/ Research Paper/Publication: 4 Research papers
1 Book chapter
New Methods/ Modellings Developed(description in 250 words):
No. of Trainings N.A. (No. of Beneficiaries):
Workshop:Demonstration Models (Site):
Livelihood Options:
Training Manuals Manual in writing process
Processing Units: -
Species Collection: More than hundred
Species identified: Hyper enzyme producer bacteria: 4
Hyper enzyme producer fungi: 5
Database/ Images/ GIS Maps: Annexure- 1 attached
Note: Photos/ maps should be attached in high quality in compatible formats viz., JPEG, .JPG, .PNG, .SHP, etc. along with a suitable figure legend/ caption.
9. Project Linkages (with nearby Institutions/ State Agencies)S. No. Institute/ Organization Type of Linkages Brief Description
Not applicable
10. Additional (publication, recommendations, etc.)Time Period Publications
(Research Papers, Information Material, Policy drafts, Patents, etc.)Annual [Year 2019-2020
] RESEARCH PAPERS:
1. Nivedita Sharma, Nisha Sharma and Dimple Tanwar. 2019. An evaluation
study of different white rot fungi for degradation of pine needles under
solid state fermentation. International Journal of Current Microbiology
and Applied Sciences. 8(6): 588-601.(soft copy attached)
2. Nivedita Sharma, Nisha Sharma and Dimple Tanwar. 2020. Enhanced
biodegradation of pine needles by optimizing temperature for different
degrading fungi under solid state fermentation. Chemical Science and
Review Letters. 9(34): 374-381. .(soft copy attached)
3. Dimple Tanwar, Nivedita Sharma and Nisha Sharma. 2019. Evaluation of
different process parameters for enhanced enzymes production using
pine needles as substrate by Trichoderma guizhouense S5 [Accession No.
MN170570] isolated from rotten wood under solid state fermentation.
Journal of Chemical, Biological and Physical Sciences. 9 (4): 617-636. .
(soft copy attached)
4. Dimple Tanwar, Nivedita Sharma and Nisha Sharma. 2020. Isolation and
screening of fungi from rotten wood for various hydrolytic enzymes
production. Annals of Phytomedicine. 9(1):1-7. .(soft copy attached)
Book chapter
Nisha Sharma and Nivedita Sharma. 2020. Industrially important xylanases and
their commercial applications. Recent progress in Microbiology and
Biotechnology. Science Domain International Publisher, London.
11. Project Concluding Remark
Kindly update the following Progress Parameters for the Reporting Period:
ProjectObjectives
Project Outputagainst each objective
Progress made againstMonitoring Indicators and Remarks
(specified in Sanction Letter)Bioconversion of reducing sugars into bioethanol and its scale up in a 7.5 litre stirred tank bioreactor
1. Extracellular
hydrolytic enzymes
production and
Purification from
potential bacterial
strains under
submerged
fermentation.
2. Optimization of
different process
parameters to
enhance enzymatic
hydrolysis of
biomass by using
one factor at a time
approach (OFAT).
Objective 1:
The purified cellulase showed 8.58 and
3.68 fold increases in cellulase activity
with the specific activity of 78.20 %.
The purified xylanase showed 4.83 fold
increases in xylanase activity with the
specific activity of 481.14 % yield.
Objective 2:
After the optimization of process
parameters viz. microwave dose,
incubation period, enzyme dose, enzyme
ratio and temperature a good
appreciable increase was observed in
reducing sugars with overall maximum
of percent increase i.e. 272.50 from
microwave pretreated biomass over the
untreated biomass by optimizing process
parameters by one factor at a time
approach.
No. Of new Database/ Datasets generated on the identified dynamics
Genomic Sequences of B. stratosphericus
N12 (W), B. stratosphericus N12 (M), B.
altitudinis Kd1 (W) and B. altitudinis Kd1
(M) have been submitted to NCBI, US
with their respective accession numbers.
B. stratosphericus N12 (W) Accession no. [KC995116]
B. stratosphericus N12 (M) Accession no. [KC995118]
B. altitudinis Kd1 (W) Accession no. [KC995115]
B. altitudinis Kd1 (M) Accession no. [KC995117]
Periodic updates on region specific
best practice/ model developed
(No.) along with the supporting
manual (No.) published;
Manual writing in process
3. Saccharification of pine needles biomass under best optimized conditions by OFAT
4. Optimization of
different process
parameters to
enhance enzymatic
hydrolysis of
biomass by
statistical model-
Response Surface
Methodology (RSM)
5. Quantification of
saccharified sugars
(5 and 6 C sugars)
through HPLC
Objective 3:
Saccharification of pine needle biomass
and production of reducing sugars was
done under the previously optimized
conditions under OFAT approach i.e.12.5
ml/g enzyme dose at 45◦C temperature
for 72 h with crude, partially purified and
purified enzymes. The maximum
reducing sugars of 28.05 mg/g were
found in pretreated pine needles
biomass.
Objective 4:
Optimization of enzymatic hydrolysis of
pine needles biomass using a three
levels three factors using Central
composite design of RSM. This
experimental design reduced the
number of experiments in comparison to
others, yielded precision and it is more
efficient and easier to arrange and to
interpret. Maximum reducing sugars
(33.21 mg/g) were observed under the
saccharification conditions i.e. 16.70
ml/g enzyme dose at 45 ◦C temperature
for 72 h.
Objective 5:
Quantitative analysis of sugars obtained
during saccharification of biomass by
crude, partially purified and purified
enzymes by using HPLC technique has
been done and found that the biomass
broken down to form maximum of 6 C
(29.96 mg/g) sugars (glucose) and 5 C
(1.50 mg/g xylose).
Policy framework/ draft (no.) for assisting in scaling up the process for commercialization
Applicable in third year
Other publications and knowledge products (no.)
Four publications
1. Nivedita Sharma, Nisha Sharma and
Dimple Tanwar. 2019. An evaluation
study of different white rot fungi for
degradation of pine needles under
solid state fermentation. International
Journal of Current Microbiology and
Applied Sciences. 8(6): 588-601.
2. Nivedita Sharma, Nisha Sharma and
Dimple Tanwar. 2020. Enhanced
biodegradation of pine needles by
optimizing temperature for different
degrading fungi under solid state
fermentation. Chemical Science and
Review Letters. 9(34): 374-381.
3. Dimple Tanwar, Nivedita Sharma and
Nisha Sharma. 2019. Evaluation of
different process parameters for
enhanced enzymes production using
pine needles as substrate by
Trichoderma guizhouense S5
[Accession No. MN170570] isolated
from rotten wood under solid state
fermentation. Journal of Chemical,
Biological and Physical Sciences. 9 (4):
617-636.
4. Dimple Tanwar, Nivedita Sharma and
Nisha Sharma. 2020. Isolation and
screening of fungi from rotten wood
6. Fermentation of
reducing sugars
into bioethanol by
optimization of
monoculture and
co-cultures
ethanologens for
maximum
bioethanol
production
7. Optimization of
process parameters
for enhanced
bioethanol
production in a
stirred tank
bioreactor
Objective 6:
Fermentation of reducing sugars into
bioethanol by using co-culture
combination of Saccharomyces
cerevisiae + Pichia stipitis has been done
and maximum ethanol production of
16.44 g/l biomass with fermentation
efficiency of 69.47 % was observed
under pretreated pine needle biomass
Objective 7:
Fermentation process for bioethanol
production was shifted to bench scale in
a 7.5 litre capacity bioreactor from shake
flask experiment. Different process
parameters viz. fermentation time,
temperature and agitation rate were
optimized to maximize the bioethanol
production in the bioreactor. After
optimization of process parameters in a
stirred tank bioreactor maximum of
ethanol 18.96 g/l with fermentation
efficiency of 72.54% was observed.
for various hydrolytic enzymes
production. Annals of Phytomedicine.
9(1):1-7.
Book chapter
Nisha Sharma and Nivedita Sharma. 2020.
Industrially important xylanases and their
commercial applications. Recent progress
in Microbiology and Biotechnology.
Science Domain International Publisher,
London.
PROGRESS REPORT IN DETAILS
Project objectives: Bioconversion of reducing sugars into bioethanol and its scale up in a 7.5 litre stirred tank bioreactor
1. Extracellular hydrolytic enzymes production and Purification from potential bacterial
strains under submerged fermentation.
2. Optimization of different process parameters to enhance enzymatic hydrolysis of
biomass by using one factor at a time approach.
3. Optimization of different process parameters to enhance enzymatic hydrolysis of
biomass by Response Surface Methodology
4. Fermentation of reducing sugars into bioethanol
5. Optimization of process parameters for enhanced bioethanol production in a stirred
tank bioreactor
Project output against each objective:Objective1. Extracellular hydrolytic enzymes production and Purification from potential
bacterial strains under submerged fermentation.
Two potential bacterial strains i.e. Bacillus stratosphericus N12 (M) and Bacillus altitudinis
Kd1 (M) were used for the production and purification of hydrolytic enzymes (cellulase and
xylanase) under submerged fermentation.
Fig 1.1 Morphology of enzyme producer strains
Cellulase production and purification under SmF
As table 1.1 depicted the results of cellulase purification from Bacillus stratosphericus N12
(M) under submerged fermentation (SmF), crude cellulase units of 0.918 IU were enhanced
to 3.910 IU after gel filtration column chromatography. After column chromatography the
fractions from 5 to 21 showed the maximum of enzyme activity and were pooled (fig 1.2) for
Bacillus stratosphericus N12 (M). The purified cellulase showed 8.58 and 3.68 fold increases
in cellulase activity with the specific activity of 78.20 %.
Table1.1 Cellulase activities of crude and purified enzyme
Sr No.
Strain Cellulase (IU/ml) Specific activity (U/mg)Crude Partially
purifiedPurified
1. Bacillus stratosphericus N12 (M)
0.918 2.550 3.910 78.20
(a) B. stratosphericus N12 (M) (b) B. altitudinis Kd1(M)
Fig 1.2 Sephadex G75 chromatography of purified cellulase from Bacillus stratosphericus N12 (M)
Xylanase Production and purification: Xylanase production and its purification from
Bacillus altitudinis Kd1 (M) had been performed under submerged fermentation
(SmF). As the data pertaining in the table 2, crude xylanase units of 20.42 IU were
enhanced to 41.86 IU after gel filtration column chromatography and fractions from
3-25, 9-18 and 8-19 were pooled for Bacillus altitudinis Kd1 (fig 1.3). The purified
xylanase showed 4.83 fold increases in xylanase activity with the specific activity of
481.14 % yield.
Table 1.2 Xylanase activities of crude and purified enzyme
Sr No. Strain Xylanase (IU/ml) Specific activity (U/mg) Crude Partially
purified Purified
1. Bacillus altitudinis Kd1 (M) 20.42 24.58 41.86 481.14
Fig 1.3 Sephadex G75 chromatography of purified cellulase from Bacillus altitudinis Kd1
Molecular weight determination of cellulase and xylanase by SDS-PAGE
Molecular weight was determined by comparing the relative mobility of standard protein
molecular weight marker of 14.3 kDa–97.4 kDa (L1). Multiple bands ranging of 14 kDa to
97.4 kDa of purified cellulase was observed for Bacillus stratosphericus N12 (M) and it was
found approximately 35.0 KDa (L2). Similarly the molecular weight of xylanase enzyme from
Bacillus altitudinis Kd1 (M) (L3) 33.0 KDa was found approximately between as shown in the
fig 1.4.
Fig 1.4 Purification of cellulase from B. stratosphericus N12 (M) (L2), xylanase from B. altitudinis Kd1 (M) (L3) by SDS-PAGE
Objective 2: Optimization of process parameters for enhanced enzymatic hydrolysis of pine needles biomass Following parameters were optimized by using one factor at a time approach (OFAT)
A mixture of bacterial inhouse enzymes (cellulase: xylanase) were used for enzymatic
hydrolysis of untreated and pretreated biomass.
97.4 KDa
L1-Marker
L2
L3
40.0 KDa20.0 KDa14.3
KDa
80.0 KDa
The process parameters optimized for enhanced hydrolysis were microwave
irradiation dose, incubation period, enzymatic dosage, enzymatic ratio and
temperature.
1.1 Optimization of microwave irradiation dose
The effect of microwave irradiation pretreatment on hydrolysis of lignocellulosic
biomass along with selected enzymatic ratio of 3:2 (cellulase: xylanase) @ 5ml/g
dose depicted in table 2.1.
The highest amount of reducing sugars observed was 20.31 mg/g of biomass at
microwave irradiation dosage of 600 W for 4 min after 72 h of enzymatic hydrolysis.
The sugar yield after hydrolysis of pretreated biomass when compared with
untreated biomass, it showed a significant increase from 6.00 mg/g to 20.31 mg/g of
reducing sugars.
Table 2.1 Optimization of microwave irradiation dose for enhanced hydrolysis of lignocellulosic
biomass
100 W 300 W 600 W 900 W0
20
40
60
80
100
120
140
160
5 min4 min
Microwave dose irradiation (Watts)
Perc
ent i
ncre
ase
in re
ducin
g su
gars
Fig 2.1 Percent increase in reducing sugars in microwave pretreated biomass over untreated biomass
Sr No. Microwave doses
(Watts)
Reducing sugars
(mg/g)
4 min
Reducing sugars (mg/g)
5 min
1. 100 6.63 7.08
2. 300 15.12 9.94
3. 600 20.31 9.76
4. 900 10.77 8.65
5. Untreated 6.00 6.78
CD 0.05 0.16 0.72
S.E. (m) 0.09 0.23
2 .2 Optimization of incubation period
Table 2.2 shows the different time intervals employed for hydrolysis of biomass. At
72 h, the highest amount of sugars was produced as 20.31 mg/g in pretreated
biomass as compared to 6.00 mg/g in untreated biomass.
A continuous trend of increase in reducing sugars was observed from 24 h to 72 h;
afterwards reducing sugars started decreasing significantly both in untreated and
pretreated biomass. The least amount of reducing sugars was observed at 120 h i.e.
4.17 and 16.35 mg/g at 24 h and 120 h in untreated and pretreated biomass
respectively.
Table 2.2 Optimization of incubation period for enzymatic hydrolysis of lignocellulosic biomass
Sr. No. Incubation time
(h)
Reducing sugars
(mg/g)
Untreated
Reducing sugars (mg/g)
Pretreated
1. 24 h 4.17 16.54
2. 48 h 5.10 16.60
3. 72 h 6.00 20.31
4. 96 h 4.31 17.68
5. 120 h 4.27 16.35
C.D0.05 0.007 0.01S.E. (m) 0.10 0.21
24 h 48 h 72 h 96 h 120 h0
50
100
150
200
250
300
350
102.44
49.54 63
279.35314.05
Incubation period (h)
Perc
ent i
ncre
ase
in re
ducin
g su
gars
Fig 2.2 Percent increase in reducing sugars after optimization of incubation period
2.3 Optimization of enzyme dose
Table 2.3 depicted the range of different enzymatic dosage (ml/g biomass) used for
enzymatic hydrolysis of untreated and pretreated lignocellulosic biomass by inhouse
enzymes.
It also showed significant variation in the yield of reducing sugars. An increase in
reducing sugars was observed with consistent increase in enzymatic dosage from 5.0
to 12.5 ml/g biomass and afterwards a significant decrease in its amount was
observed.
The pattern of reducing sugars increase and decrease was followed both in
untreated and pretreated biomass. Maximum reducing sugar yield (mg/g) was
observed at enzymatic dosage of 12.5 ml/g in untreated biomass and pretreated
biomass as 18.97 and 20.56 mg/g respectively.
The least amount of reducing sugars was observed at @ 5.0 ml/g i.e. 7.59 in
untreated and 10.48 mg/g in pretreated biomass @ 15.0 ml/g dose.
Table 2.3 Optimization of enzyme doses for enhanced hydrolysis of lignocellulosic biomass
Enzyme dose (ml/g) Untreated Pretreated
1. 5.0 7.59 10.53
2. 7.5 10.74 17.38
3. 10.0 14.90 18.12
4. 12.5 18.97 20.56
5. 15.0 9.48 10.48
C.D. 0.05 0.43 0.70S.E.(m) 0.09 0.28
5 7.5 10 12.5 150
10
20
30
40
50
60
70
38.73
61.82
21.61
7.86 10.54
Enzyme dose (ml/mg)
Perc
ent i
ncre
ase
in re
ducin
g su
gars
Fig 2.3 Percent increase in reducing sugars after optimization of enzyme dose
2.4 Optimization of enzyme ratio Five different ratios were selected i.e. 6.25:6.25, 6.75:5.75, 7.25:5.25, 7.75:4.75,
8.25:4.25 and investigated for its effect on hydrolysis of biomass.
Enzymatic cocktail of 7.75:4.75 revealed highest amount of reducing sugars i.e.
18.97 mg/g and 21.46 mg/g biomass in untreated and pretreated biomass
respectively as shown in table 2.4.
The enzymatic mixture of 7.75:4.75 was vital for extraction of maximum amount of
monomeric sugars, as these enzymatic cocktail hydrolyzed maximum carbohydrates
into monomeric sugars.
Table 2.4. Optimization of enzyme ratio for enhanced hydrolysis of lignocellulosic biomass
Sr No. Enzyme ratio
(cellulase: xylanase)
Untreated Pretreated
1. 6.25:6.25 17.97 21.00
2. 6.75:5.75 16.67 20.00
3. 7.25:5.25 17.99 21.30
4. 7.75:4.75 18.97 21.46
5. 8.25:4.25 17.56 20.15
C.D0.05 0.40 0.64S.E. (m) 0.10 0.15
6.25:6.25 6.75:5.75 7.25:5.25 7.75:4.75 8.25:4.250
5
10
15
20
25
16.8619.97 18.39
13.12 14.74
Enzyme ratio (cellulase: xylanase)
Perc
ent i
ncre
ase
in re
ducin
g su
gars
Fig 2.4 Percent increase in reducing sugars after optimization of enzyme ratio
2.5 Optimization of temperature A regime of 35oC, 40oC, 45oC, 50oC and 55oC was employed for the optimization of
hydrolysis temperature to produce maximum amount of reducing sugars.
The highest amount of reducing sugar of 19.06 and 22.35 mg/g biomass was observed
in untreated and pretreated lignocellulosic biomass at temperature 45oC respectively. A
slight decrease was observed in sugar at 55oC i.e. 9.74 and 19.85 mg/g in untreated and
pretreated biomass.
A gradual increase was observed in reducing sugar yield with increase in temperature
from 35 to 45oC beyond that, a decrease in reducing sugar was obtained in pretreated
biomass. This could be attributed to performance of enzymes at optimal temperature as
they need an optimum temperature for proper catalytic function.
Table 2.5 Optimization of temperature for enhanced hydrolysis of lignocellulosic biomass
Sr No. Temperature Untreated Pretreated
1. 35◦C 18.57 20.00
2. 40◦C 15.79 22.01
3. 45◦C 19.06 22.35
4. 50◦C 10.01 20.10
5. 55◦C 9.74 19.85
C.D0.05 0.36 0.74
S.E. (m) 0.11 0.25
22
35◦C 40◦C 45◦C 50◦C 55◦C0
20
40
60
80
100
120
7.7
39.39 40.92
100.79 103.79
Temperature
Perc
ent i
ncre
ase
in re
ducin
g su
gars
Fig 2.5 Percent increase in reducing sugars after optimization of temperature
Microwav
e dose
Incubati
on period
Enzym
e dose
Enzym
e rati
o
Tempera
ture0
50
100
150
200
250
300
350
400
82.5
239 242.66257.66 272.5
Process parameters
Perc
ent i
ncre
ase
in re
ducin
g su
gars
Fig 2.6 Overall percent increase in reducing sugars in pretreated biomass over untreated biomass after
optimization of process parameters
After the optimization of process parameters viz. microwave dose, incubation period,
enzyme dose, enzyme ratio and temperature a good appreciable increase was observed
in reducing sugars with overall maximum of percent increase i.e. 272.50 from
microwave pretreated biomass over the untreated biomass by optimizing process
parameters by one factor at a time approach (fig2.6).
23
Objective3. Saccharification of pine needle biomass using crude, partially purified and purified enzymes
Hydrolytic enzymes i.e. cellulase and xylanase production from B. stratosphericus
N12 (M) and B. altitudinis Kd1 (M) respectively has been done.
Enzymes were partially purified by ammonium sulphate precipitation approach and
purified by column chromatography.
Enzymatic saccharification of untreated and pre treated pine needles biomass using
crude, partially purified and purified inhouse enzymes cocktail by applying
previously optimized conditions i.e. enzyme dosage of 12.5 ml/g in the ratio of
7.75:4.75 (cellulase: xylanase) for 72 h of enzymatic hydrolysis at 45◦C has been
done.
Maximum reducing sugars of 24.22 mg/g and 28.05 mg/g were obtained from
untreated and pretreated pine needles biomass respectively.
Table 3.1 Reducing sugar production during hydrolysis of biomass using crude, partially purified and purified enzymes
Sr No. In house enzymes used Reducing sugars (mg/g)(in untreated biomass)
Reducing sugars (mg/g)(in pretreated biomass)
1. Crude 19.21 22.21
2. Partially purified 23.36 24.31
3. Purified 24.22 28.05
Objective 4. Standardization of process parameters for complete hydrolysis of pine needles
biomass using Response Surface Methodology (RSM) at pilot plant scale (7.5 litre bioreactor):
In this method, prior knowledge of significant conditions obtained from previous One Variable
at a Time (OVAT) approach experiment had been necessary for achieving a more realistic
model. RSM based on Central Composite Design was used for the optimization of independent
variables for reducing sugar production in untreated and pretreated pine needles biomass.
24
Following parameters were optimized by using a statistical approach i.e. Response Surface
Methodology (RSM)
1. Incubation time
2. Enzyme dose
3. Temperature
Three independent variables were chosen for optimization studies by employing Central
Composite Design (CCD) of Response Surface Methodology (RSM). The experiment
contained 20 runs. The design involved 6 centre points, 14 non centre points. The
mathematical relationship of response (reducing sugars) and variables i.e. A, B and C was
approximated by a quadratic model equation. The optimization of enzymatic hydrolysis of
biomass was carried out for three independent variables (A) incubation time (low-36, high-
60 hours), (B) enzymatic dose (low-10, high-15 ml/g) and (C) temperature (low- 40, high-
50◦C) following the CCD of Response Surface Methodology (RSM) experimental design.
Table 4.1 and 4.2 for untreated and pretreated pine needles biomass respectively, which
had shown a considerable variation in the amount of reducing sugars production depending
upon the interaction of various levels of four independent variables in the medium.
Maximum reducing sugar yield 27.52 mg/g of biomass was observed at enzyme dosage of
10.0 ml/g in the ratio of 7.75:4.75 (cellulase: xylanase) using untreated pine needles as
substrate after 96 h of enzymatic hydrolysis at 30◦C. On the other hand, in pretreated pine
needle biomass maximum reducing sugars i.e. 33.21 mg/g were achieved at 16.70 ml/g of
enzyme dose at 45◦C temperature for 72 h of incubation.
An attempt was made to optimize enzymatic hydrolysis of pine needles biomass using a
three levels three factors using Central composite design of RSM and achieved overall
percent increase of 453.50 % in reducing sugars (fig 4.3). This experimental design reduced
the number of experiments in comparison to others, so it is more efficient and easier to
arrange and to interpret.
25
Table 4.1 Optimization of process parameters for enzymatic saccharification of untreated pine needles biomass
by Response surface methodology
Std Run Incubation
time (h)
Enzyme dose
(ml/g)
Temperature
(◦C)
Reducing sugars
(mg/g)
19 1 72 12.50 35 26.55
6 2 96 10.00 40 26.39
1 3 48 10.00 30 27.44
14 4 72 12.50 43 21.54
8 5 96 15.00 40 27.45
17 6 72 12.50 35 24.36
15 7 72 12.50 35 25.49
16 8 72 12.50 35 27.35
9 9 31 12.50 35 26.56
11 10 72 8.30 35 22.80
5 11 48 10.00 40 27.39
13 12 72 12.50 26 27.47
12 13 72 16.70 35 24.39
2 14 96 10.00 30 27.52
7 15 48 15.00 40 25.40
18 16 72 12.50 35 27.50
3 17 48 15.00 30 22.53
20 18 72 12.50 35 23.67
10 19 112 12.50 35 22.19
4 20 96 15.00 30 27.10
Table 4.2 Optimization of process parameters for enzymatic saccharification of pretreated pine needles biomass
by Response surface methodology
Std Run Incubation time
(h)
Enzyme dose
(ml/g)
Temperature
(◦C)
Reducing
sugars (mg/g)
1 1 48 10.00 40 33.15
2 2 96 10.00 40 28.80
8 3 96 15.00 50 33.05
15 4 72 12.50 4 31.48
26
13 5 72 12.50 36 31.39
19 6 72 12.50 45 31.54
16 7 72 12.50 45 29.78
10 8 112 12.50 45 31.85
9 9 31 12.50 45 33.09
6 10 96 10.00 50 30.01
4 11 96 15.00 40 32.96
14 12 72 12.50 53 29.80
3 13 48 15.00 40 32.87
17 14 72 12.50 45 33.03
5 15 48 10.00 50 33.02
11 16 72 8.30 45 33.11
18 17 72 12.50 45 33.08
20 18 72 12.50 45 31.85
12 19 72 16.70 45 33.21
7 20 48 15.00 50 32.30
27
Fig 4.1 Response surface curves for enzymatic hydrolysis of untreated pine needles biomass showing interactions between a) enzyme dose and temperature b) temperature and incubation period c) enzyme dose and incubation period
Fig 4.2 Response surface curves for enzymatic hydrolysis of microwave pretreated pine needles biomass showing interactions between a) enzyme dose and temperature b) temperature and incubation period c) enzyme dose and incubation period
28
Microwave
Incubation time
Enzym
e dose
Ezyme r
atio
Tempera
tureRSM
0
50
100
150
200
250
300
350
400
450
500
untreatedPretreated
Parameters
Perc
ent i
ncre
ase
(%)
Fig 4.3 Step wise increase in reducing sugars after optimization of process parameters by OFAT and RSM
Objectives 5. Quantification of sugars through HPLC technique
5.1 Quantification of glucose and xylose in untreted pine needles biomass through High
Performance Liquid Chromatography (HPLC)
In this experiment, sugars obtained after the ezymatic saccharification of untreated and
microwave pretreated pine needles biomass by crude, partially purified and purified
ezymes were quatified by using High performance liquid chromatography technique
(HPLC). Both 6 C (glucose) and 5 C (xylose) sugars were estimated and maximum
reducing sugars i.e. 26.59 mg/g (25.18 mg/g glucose and xylose i.e. 1.41 mg/g) were
found in case of saccharification of untreated biomass with purified enzymes (table 5.1).
On the other hand in case of microwave pretreated pine needles biomass maximum of
reducing sugars i.e 31.46 mg/g (29.96 mg/g glucose and xylose i.e. 1.50 mg/g) were
obtained which is highest as compared to untreated biomass (table 5.2) . Fig 5.1 and 5.2
are the standards used for the quantification of glucose and xylose. Fig 5.3 (a, b and c),
5.4 (a, b and c) showing the chromatogram for glucose and xylose estimated in
29
saccharified solution prepared by crude, partially purified and purified enzymes from
untreated and pretreated pine needles biomass respectively. Maximum reducing sugars
were obtained from microwave pretreated biomass using purified inhouse enzymes for
saccharification.
Table 5.1 Quantification of sugars in untreated pine needles biomass
Sr No. Enzymes used
Glucose (mg/g)
Xylose (mg/g)
Total sugars (mg/g)
1. Crude 1.96 1.13 3.09
2. Partially Purified
12.37 1.21 13.58
3. Purified 25.18 1.41 26.59
Table 5.2 Quantification of sugars in pretreated pine needles biomass
Sr No. Enzymes used Glucose (mg/g)
Xylose (mg/g)
Total sugars (mg/g)
1. Crude 18.90 4.20 23.10
2. Partially Purified 18.88 9.36 28.24
3. Purified 29.96 1.50 31.46
Fig 5.1 Chromatogram for Standard of glucose Fig 5.2 Chromatogram for Standard of xylose
30
Fig 5.3 Chromatogram for sugars from untreated biomass with saccharification of (a) crude (b) partially purified (c) purified enzymes
Fig 5.4 Chromatogram for sugars from pretreated biomass with saccharification of (a) crude (b) partially purified (c) purified enzymes
Objective 6. Fermentation of reducing sugars into bioethanol
Sugars prepared under the already optimized conditions during Response surface
methodology i.e. 16.70 ml/g enzyme dose, 45◦C temperature and 72 h of incubation
were further subjected to the fermentation process.
Fermentation of reducing sugars into bioethanol by using monoculture of
Saccharomyces cerevisiae and Pichia stipitis as well as co-culture combination of
Saccharomyces cerevisiae + Pichia stipitis (fig 5.1) has been done and the maximum
ethanol i.e. 11.06 g/l (table 6.1) was obtained from co-culture combination of
Saccharomyces cerevisiae + Pichia stipitis which was selected for further study.
Further ethanol production was done using saccharified solutions obtained from
untreated and pretreated biomass using crude, partially purified and purified
31
enzymes with fermentation of co-culture combination of Saccharomyces cerevisiae +
Pichia stipitis. Maximum ethanol production of 16.44 g/l with fermentation
efficiency of 69.47 % was observed under pretreated pine needle biomass (table 6.2
and 6.3).
Fig 6.1 Colonies of Saccharomyces cerevisiae and Pichia stipitis
Table 6.1 Ethanol production by using monoculture and co-culture combination
Sr No. Ethanologens Ethanol (g/l) Ethanol(g/g)
Fermentation Efficiency
1. Saccharomyces cerevisiae 10.27 0.20 39.13
2. Pichia stipitis 7.90 0.15 29.35
3. Saccharomyces cerevisiae +Pichia stipitis
11.06 0.22 43.05
Table 6.2 Ethanol production using untreated pine needle biomass
Sr No. Reducing sugars produced from enzymes
Ethanol (g/l) Ethanol(g/g)
Fermentation Efficiency
1. Crude 9.80 0.197 38.55
2. Partially purified 12.60 0.246 48.14
3. Purified 14.22 0.286 55.96
Table 6.3 Ethanol productions using pretreated pine needle biomass
Sr No. Reducing sugars produced Ethanol (g/l) Ethanol Fermentation
32
from enzymes (g/g) Efficiency
1. Crude 9.80 0.197 38.55
2. Partially purified 15.70 0.296 57.92
3. Purified 16.44 0.355 69.47
Fig 6.2 Bioethanol production from lignocellulosic waste – pine needles
Objective 7. Optimization of scale up process parameters for bioethanol production in stirred tank bioreactor
Fermentation process for bioethanol production was subjected to scale up process in a 7.5 litre
capacity bioreactor from shake flask experiment. Different process parameters viz.
fermentation time, temperature and agitation rate were optimized to maximize the bioethanol
production in the bioreactor. After optimization of process parameters in a stirred tank
bioreactor maximum of ethanol 18.96 g/l with fermentation efficiency of 72.54% was observed.
7.1 Optimization of fermentation time
Fermentation time was standardized by observing bioethanol production at different
time intervals i.e. 6, 12, 18, 24, 30, 36 and 42 hours. Sampling was done at mentioned
different time interval.
Maximum bioethanol production of 14.22 g/l with fermentation efficiency of 54.79%
was observed at 30 h of fermentation time.
In this scale up experiment we reduced our fermentation time from 72 h (in shake flask)
to 30 h in the bioreactor (table 6.1).
33
Figure 6.1 showing the physiological behaviour of co-cultures (S. cerevisiae + P. stipitis)
in stirred tank bioreactor during optimization of fermentation time.
Table 7.1 Optimization of fermentation time (h)
Sr No. Fermentation time (h) Ethanol (g/l) Ethanol (g/g) Fermentation efficiency (%)
1. 6h 3.16 0.06 11.74
2. 12 h 7.90 0.15 29.35
3. 18 h 10.27 0.20 39.13
4. 24 h 12.64 0.25 48.92
5. 30 h 14.22 0.28 54.79
6. 36 h 11.06 0.22 43.13
7. 42 h 7.90 0.15 29.41
Fig 7.1 Physiological behaviour of co-cultures (S. cerevisiae + P. stipitis) in stirred tank bioreactor during optimization of fermentation time
34
6h 12 h 18 h 24 h 30 h 36 h 42 h0
10
20
30
40
50
60
Fermentation time (h)
Ferm
enta
tion
efficie
ncy
(%)
Fig 7.2 Fermentation efficiency during optimization of fermentation time
7.2 Optimization of fermentation temperature
Different temperatures i.e. 20 0C, 25 0C and 30 0C were used for the fermentation by
co cultures (S. cerevisiae + P. stipitis).
Maximum bioethanol production of 18.17 g/l with fermentation efficiency of 70.58 %
was observed at 25 0C of fermentation temperature.
Figure 6.3 showing the physiological behaviour of co-cultures (S. cerevisiae + P.
stipitis) in stirred tank bioreactor during optimization of temperature.
Table 7.2 Optimization of Temperature (0C)
Sr No. Temperature (0C) Ethanol (g/l) Ethanol (g/g) Fermentation efficiency (%)
1. 20 0C 15.80 0.31 60.78
2. 25 0C 18.17 0.36 70.58
3. 30 0C 12.64 0.25 49.01
35
Fig 7.3 Physiological behaviour of co-cultures (S. cerevisiae + P. stipitis) in stirred tank bioreactor during optimization of temperature
20◦C 25◦C 30◦C0
10
20
30
40
50
60
70
80
Temperature (◦C)
Ferm
enta
tion
efficie
ncy
(%)
Fig 7.4 Fermentation efficiency during optimization of temperature
7.3 Optimization of Agitation rate
Different agitation rates i.e. 100 rpm, 200 rpm and 300 rpm were used for maximum
ethanol production by co cultures (S. cerevisiae + P. stipitis).
Maximum bioethanol production of 18.96 g/l with fermentation efficiency of 72.54 %
was observed at 25 0C of fermentation temperature.
Figure 6.5 showing the physiological behaviour of co-cultures (S. cerevisiae + P.
stipitis) in stirred tank bioreactor during optimization of temperature.
36
Table 7.3 Optimization of Agitation rate
Sr No. Agitation (rpm) Ethanol (g/l) Ethanol (g/g) Fermentation efficiency (%)
1. 100 rpm 14.22 0.28 54.90
2. 200 rpm 18.96 0.37 72.54
3. 300 rpm 16.59 0.3364.70
Fig 7.5 Physiological behaviour of co-cultures (S. cerevisiae + P. stipitis) in stirred tank bioreactor during optimization of agitation rate
37
100 200 3000
10
20
30
40
50
60
70
80
Agitation rate (rpm)
Ferm
enta
tion
efficie
ncy
(%)
Fig 7.6 Fermentation efficiency during optimization of agitation rate
Methodology (in brief)
1. Production and Purification of hydrolytic enzymes
1.1 Collection of Biomass: Pine needles were collected from the forests of adjoining Himalayas
and brought to the laboratory. Biomass was washed with tap water and dried at 600 C
temperatures in the oven. Dried biomass was chopped into small pieces and then grinded
into 2 mm sieve size and stored for the further experiments.
1.2 Microbial strains used for hydrolytic enzymes production
Sr No. Name Accession No. Source
1. Bacillus stratosphericus N12 (M) KC995118 9 [NCBI, US] soil
2.. Bacillus altitudinis Kd1 (M) KC995117 [NCBI, US] soil
1.3 Production of hydrolytic enzymes from potential bacterial strains under submerged
fermentation (SmF)
38
Inoculum Preparation: Each bacterial strain was grown in 100 ml of nutrient broth
at 35±2°C for 24 h. As soon as the substantial growth was observed in the broth, the
optical density was set to 1.0 using autoclaved distilled water.
Enzyme production: 5 ml of inoculum was added to each 45 ml of specific broth
media in 250 ml of Erlenmeyer flasks and the flasks were incubated their optimized
incubation days at 35±2°C. After incubation, the culture contents were centrifuged
at 10,000 rpm for 15 min (4°C). The supernatant was collected and enzyme assays
and protein estimation was done.
Enzyme Assays:
(i) CMCase assay (Reese & Mandel, 1963)
(ii) FPase assay (Reese & Mandel, 1963)
(iii) ß-Glucosidase assay (Berghem and Petterson, 1973)
Xylanase Assay: Dinitrosalicylic acid (DNSA) method (Miller, 1959)
Protein Assays: Lowry’s method (Lowry et al., 1951)
1.4 Partial Purification by Ammonium sulphate precipitation
Different concentrations of ammonium sulphate i.e. 0-10%, 10-20%, 20-30%, 30-40%, 40-50%,
50-60%, 60-70%, 70-80%, 80-90% were evaluated to attain saturation point for each of cellulase
subunits i.e. CMCase, FPase and β-glucosidase. The preparations were kept at 4°C for overnight
and centrifuged which resulted in separation of pellets and supernatants. CMCase and FPase
were precipitated at 30-60%, β-glucosidase at 0-30% and xylanase at 0-70 % level of saturation
of ammonium sulfate. Precipitates of each subunit so obtained were dissolved in phosphate
buffer (0.1 M, pH 6.9) separately.
1.5 Purification of enzymes by Gel Filtration Column chromatography
Sephadex G-100 (5g) was suspended in 500 ml of distilled water for 24 h. It was packed into the
glass column having dimensions of (31x2.5 cm). It was equilibrated with three bed volumes of
0.1M Phosphate buffer (pH 6.9). Partially purified protein sample (2 ml) was loaded on the
Sephadex G-100 column. It was eluted with three bed volumes of 0.1M phosphate buffer (pH
6.9) and 3 ml fractions were collected. A flow rate of 3 ml in 7 min was maintained. The protein
39
content of collected fractions was measured at 280 nm and fractions showing maximum
absorbance were analyzed for enzyme activity. The most active fractions were pooled and
stored at 4oC.
1.6 Sodium Dodecyl Sulphate - Polyacrylamide Gel Electrophoresis
Equipment
Minigel apparatus, Power supply (200 V, 500mA), Boiling water bath, eppendorf centrifuge
(Sigma), Hamilton syringes (50 μl and 100 μl capacity), Small glass or plastic container with lid
Eppendorf tubes, Rocking or rotary shaker
Working Solution
Solution A (Acrylamide stock solution). 100 ml 30 % w/v acrylamide, 0.8 % (w/v)
bisacrylamide
Solution B (4 x separating gel buffer), 100 ml, 75 ml 2 M Tris HCl pH 8.8, 4 ml 10 % SDS
and 21 ml H2O.
Solution C (4 x stacking gel buffer: 50 ml 1 M Tris HCl (pH 6.8), 4 ml 10 % SDS, 46 ml H2O,
10 % ammonium persulfate, 5 ml, 0.5 g ammonium persulfate, 5 ml H2O
Electrophoresis buffer
3 g Tris,14.4 g Glycine, 1g SDS, H2O to make 1 litre and pH should be approximately 8.3
5 x sample buffer: 0.6 ml 1 M Tris -HCl pH 6.8, 50 % glycerol, 10 % SDS,
mercaptoethanol, 1% bromophenol blue, H2O
Assembling of Gel Sandwich
For minigel, bottom of both gel plates and spacer were perfectly flushed against a flat surface
before tightening lamp assembly. Solutions A and B and water was combined in a small
Erlenmeyer flask or a test tube. Ammonium persulfate and TEMED were added and mixed by
swirling or inverting container gently. Gel solution was introduced into gel sandwich using a
pipette. When appropriate amount of separating gel solution had been added, 1 cm of water
was gently layered on top of separating gel solution to keep the gel surface flat. Gel was
allowed to polymerize (30 – 60 min). When the gel has polymerized, a distinct interface
appeared between the separating gel.
Pouring of Stacking Gel
40
Water covering the separating gel was poured off. Solution A, C and water were combined in a
test tube. Ammonium persulfate and TEMED was added and mixed by gently swirling or
inverting the container. Stacking gel solution was pipette onto separating gel until solution
reached top of front plate. The comb was inserted into gel sandwich until bottom of teeth
reached top of front plate. Care was taken in order to avoid making of any bubbles. Staking gel
was allowed to polymerize (about 30 minutes). After staking gel polymerized, the comb was
removed carefully. The gel was placed into electrophoresis chamber. Electrophoresis buffer was
added into the buffer tank making sure that both top and bottom of gel were immersed in
buffer. Air bubbles clinging to bottom of gel were removed to ensure even current flow.
Preparation and Loading of Samples
Protein sample and 5x sample buffer (20μl + 5μl) were combined in an eppendorf tube and
heated at 100oC for 2-10 min. This sample solution was spinned for 1 sec in microfuge. Sample
solution was introduced into well using Hamilton syringe. Molecular weight standards were run
in one side of the well.
Running of Gel
Electrode plugs were attached to proper electrodes. Current was allowed to flow towards
anode. Power supply was turned on to 200 V. The dye front was allowed to migrate to 1 cm
from the bottom of the gel in 30 - 40 min for two 0.75 mm gels. Power supply was turned off.
Electrode plugs were removed from electrodes. Gel plates were removed from electrode
assembly. A spacer was removed carefully and the space inserted in one corner between the
plates gently used to apart the gel plates. The gel sticked to one of the plate.
Staining of a Gel with Coomassie Blue
Method of staining was used to detect as little as 0.1 μg of protein in a single band. Gel was
picked up and transferred to a small container containing coomassie staining (approx. 20 ml). It
was agitated for 5-10 min on slow rotatory shaker. Stain was poured out. Coomassie destain
was added about 50 ml. To destain completely, destain solution was changed 3 times after 20-
20 min and agitated overnight. After destaining clear bands of both the isolates appeared on
the gel indicating homogeneity of protein.
Molecular Weight Determination
41
The molecular weight of partially purified xylanase was determined with the help of molecular
marker ranging between 14.3 kDa- 97.4 kDa.
2. Optimization of process parameters for enzymatic hydrolysis of biomass
The optimization of enzymatic hydrolysis of biomass was carried out for microwave irradiation
dose, incubation period, enzyme dosage, enzymatic ratio and temperature by one factor at a
time approach (OFAT).
2.1 Inhouse enzyme cocktail preparation
The inhouse enzymes which were prepared had been mixed in the ratio of (3:2) i.e. 3.0 ml of
cellulase from cellulase from B. stratosphericus N12 (M) (CMCase: 1.706 IU, FPase: 2.008 IU and
β-glucosidase: 0.196 IU) and 2.0 ml of xylanase from B. altitudinis Kd1 (M) (41.86 IU) and
enzymatic dose was adjusted @ 1ml/g of biomass for hydrolysis.
2.2 Optimization of microwave irradiation dose
1 g untreated dried lignocellulosic biomass was taken in different petriplates and subjected to
different doses of microwave irradiation i.e. 100, 300, 600 and 900 W for different time
intervals of 4 min and 5 min. Sodium citrate buffer (0.05 M, pH 5.5) was added as moistening
agent in 1:4 ratio. Purified enzymatic mixture of different inhouse hydrolytic enzymes (cellulase:
xylanase) in 3:2 @ 5ml/g dose was employed for biomass hydrolysis at 50◦C temperature for 72
h of incubation period. After incubation reducing sugars were estimated (Miller, 1959).
2.3 Optimization of incubation period
To each 1 g untreated and microwave (600 W, 4 min) pretreated biomass was taken and to
these sodium citrate buffer (0.05 M, pH 5.5) was added and autoclaved. Inhouse enzymes in the
ratio of 3:2 (cellulase: xylanase) @ 5.0 ml/g dose was added to each flask under sterile
conditions and incubated at optimum temperature, 50oC. The hydrolysis period was varied from
24 h, 48 h, 72 h, 96 h and 120 h for enzymatic hydrolysis. After incubation period, biomass was
filtered and centrifuged at 10,000 rpm for 10 min. The supernatant was used for estimation of
reducing sugars.
2.4 Optimization of enzymatic dose
42
To each 1 g untreated and pretreated biomass sodium citrate buffer (1:4 ratio) was added and
autoclaved. Inhouse enzymes in the ratio of 3:2 (cellulase: xylanase) was added in different
doses i.e. 5.0 ml/g, 7.5 ml/g, 10.0 ml/g, 12.5 ml/g and 15.0 ml/g were added to each flask under
sterile conditions and incubated at temperature, 50oC for 72 h. After 72 h, saccharified biomass
was filtered and centrifuged at 10,000 rpm for 10 min. The supernatant was used for estimation
of reducing sugars.
2.5 Optimization of enzymatic ratio
To each 1 g untreated and pretreated biomass sodium citrate buffer (0.05M, pH 5.5) was added
and autoclaved. Then enzymatic mixture of inhouse enzymes in different ratio i.e. 6.25: 6.25,
6.75: 5.75, 7.25: 5.25, 7.75: 4.75 and 8.25: 4.25 @ 12.5 ml/g doses were added for hydrolysis
and the flasks were incubated at 45oC for 72 h to undergo enzymatic hydrolysis. After 72 h,
biomass was filtered and centrifuged at 10,000 rpm for 10 min. The supernatant was used for
estimation of reducing sugars.
2.6 Optimization of temperature
To each 1 g untreated and microwave pretreated biomass sodium citrate buffer (0.05 M, pH
5.5) and autoclaved. The best selected enzymatic ratio of 7.75: 4.75 (cellulase: xylanase) with
optimum enzyme dose @ 12.5 ml/g was added to each flask under sterile conditions. The flasks
were incubated at different temperatures i.e. 35oC, 40oC, 45oC, 50oC and 55oC for 72 h to
undergo enzymatic saccharification. After 72 h, saccharified biomass was filtered and
centrifuged at 10,000 rpm for 10 min. The supernatant was used for estimation of reducing
sugars.
3. Enzymatic saccharification of pine needles biomass
3.1 Inhouse enzyme cocktail preparation
As discussed in section 1.3 in the methodology
3.2 Partial purification of enzymes
As discussed in section 1.4 in the methodology
3.3 Purification of enzymes
As discussed in section 1.5 in the methodology
43
3.4 Hydrolysis of pine needles biomass
Enzymatic saccharification of untreated and pre treated pine needles biomass using crude,
partially purified and purified inhouse enzymes cocktail by applying previously optimized
conditions i.e. enzyme dosage of 12.5 ml/g in the ratio of 7.75:4.75 (cellulase: xylanase) for 72 h
of enzymatic hydrolysis at 45◦C has been done and reducing sugars were estimated (Miller,
1959).
4. Optimization of process parameters for saccharification of pine needles biomass using
Response Surface Methodology (RSM) approach
In this method, prior knowledge of significant conditions obtained from previous One Variable
at a Time (OVAT) approach experiment had been necessary for achieving a more realistic
model. RSM based on Central Composite Design was used for the optimization of independent
variables for reducing sugar production in untreated and pretreated pine needles biomass.
Following parameters were optimized by using a statistical approach i.e. Response Surface
Methodology (RSM)
Incubation time
Enzyme dose
Temperature
Three independent variables were chosen for optimization studies by employing Central
Composite Design (CCD) of Response Surface Methodology (RSM). The experiment
contained 20 runs. The design involved 6 centre points, 14 non centre points. The
mathematical relationship of response (reducing sugars) and variables i.e. A, B and C was
approximated by a quadratic model equation. The optimization of enzymatic hydrolysis of
biomass was carried out for three independent variables (A) incubation time (low-36, high-
60 hours), (B) enzymatic dose (low-10, high-15 ml/g) and (C) temperature (low- 40, high-
50◦C) following the CCD of Response Surface Methodology (RSM) experimental design.
5. Quantitative analysis of sugars to estimate 5 and 6 C sugars by High Performance
Liquid Chromatography (HPLC)
44
High Performance Liquid Chromatography was performed for the estimation of 6 C
(glucose) and 5 C (xylose) sugars produced in saccharified solution during enzymatic
saccharification of pine needles biomass. Saccharification of biomass was done by
applying crude, partially purified and purified enzymes and sugars were quantified
through HPLC under the following conditions.
HPLC Conditions
Column: Ultra C18 (Restek Corp.), 250mm × 4.6 mm, 5µm
Mobile Phase A: 90: 10 water: methanol, 10mM ammonium formate
Mobile phase B: 10: 90 water: methanol, 10mM ammonium formate
Gradient: 0-5 min at 100% A, to 100% B at 10 min, 10 min hold
Flow: 0.5 mL /min
Temperature: ambient
Detector: UV@ 280 nm
Injection volume: 10µL
Standard dilution: 100ppm
Formula to calculate the concentration of 5-HMF
Concentration of 5-HMF = Area of Sample X Standard dilution (100 ppm)Area of Standard
6. Fermentation of reducing sugars into bioethanol
6.1 Ethanologens used: co-culture combination of Saccharomyces cerevisiae(MTCC 3089) + Pichia stipitis (NCIM 3498)
Growth Media: For Saccharomyces cerevisiae (MTCC 3089) Yeast extracts- 3.0g
Peptone - 10.0g
Dextrose - 20.0g
Distilled water - 1 litre
For Pichia stipitis (NCIM 3498)
45
Malt extract - 0.3g
Glucose - 1.0g
Yeast extract - 0.3g
Peptone - 0.5g
Distilled water - 1 litre
6.2 Fermentation conditions: 72 h of fermentation time at 25◦C temperature
6.3 Ethanol estimation:
34.0 g of potassium dichromate was dissolved in 500ml of distilled water. To this 375 ml of
concentrated sulphuric acid was added, mixed thoroughly and allowed to cool. Final volume
was made 1000ml by adding distilled water. To the distillation flask 29 ml of distilled water and
1 ml of sample was added. On the other side to the 50 ml volumetric flask 25 ml of potassium
dichromate was added. Distillation was set at 600C and tap water was turned ON. To the 25 ml
of potassium dichromate, 20 ml of distilled sample was collected and it became total 45 ml. To
this 45 ml solution 5 ml of distilled water was added and total volume became 50 ml and was
incubated at 60oC for 20 min. After that O.D. was measure at 600 nm against blank (Caputi et
al., 1969).
Bioethanol was estimated in terms of g/l of fermented liquor and g/g of biomass on dry weight
basis. Fermentation efficiency was calculated using the following formula:
Fermentation efficiency =
ethanol produced (g/g)× 100theoretical yield of
ethanol
Theoretical yield was referred as standard value of 0.511 g/g of sugars.
7. Optimization of process parameters for scale up of bioethanol production in stirred tank bioreactor
Fermentation process for bioethanol production was subjected to scale up process in a 7.5 litre
capacity bioreactor from shake flask experiment. Different process parameters viz.
fermentation time, temperature and agitation rate were optimized to maximize the bioethanol
production in the bioreactor.
46
7.1 Ethanologens used
Co-culture combination of Saccharomyces cerevisiae + Pichia stipitis
7.2 Batch Fermentation
Batch cultivation for the production of ethanol was carried out in a 7.5 L stirred tank bioreactor
(New Brunswick Scientific, New Jersey USA) at temperature 25◦C and pH 5.5. 3.0 litre sugary
syrup prepared after saccharification of pine needles biomass was subjected to the bioreactor
and to this yeast extract (0.5%) and peptone (0.5%) were added. Foaming was controlled with
addition of 2-3 drops of polypropylene glycol. The medium was sterilized in situ for 20 min at
121oC. The bioreactor was inoculated with 10% inoculum of S. cerevisiae II + P. Stipitis. The
fermentation was carried out under anaerobic conditions. The agitation speed 100 rpm and
agitation rate 1.0 vvm respectively used for batch cultivation in bioreactor. The sample was
withdrawn regularly at different intervals ranging from 6, 12………..48 h and ethanol estimation
was done.
7.3 Optimization of Fermentation time
Fermentation time was standardized by observing bioethanol production at different time
intervals i.e. 6, 12, 18, 24, 30, 36 and 42 hours. Sampling was done at mentioned different time
interval.
7.4 Optimization of Temperature
Different temperatures i.e. 20 0C, 25 0C and 30 0C were used for the fermentation.
7.5 Optimization of Agitation rate
Different agitation rates i.e. 100 rpm, 200 rpm and 300 rpm were used for maximum ethanol
production
7.6 Ethanol Estimation
Same as section 5.3
47
Major Research achievements
The purified cellulase showed 8.58 and 3.68 fold increases in cellulase activity with the
specific activity of 78.20 %.The purified xylanase showed 4.83 fold increases in xylanase
activity with the specific activity of 481.14 % yield.
After the optimization of process parameters viz. microwave dose, incubation period,
enzyme dose, enzyme ratio and temperature a good appreciable increase was observed
in reducing sugars with overall maximum of percent increase i.e. 272.50 from
microwave pretreated biomass over the untreated biomass by optimizing process
parameters by one factor at a time approach.
Maximum reducing sugar yield 22.35 mg/g of biomass was observed at enzyme dosage
of 12.5 ml/g in the ratio of 7.75:4.75 (cellulase: xylanase) using pine needles as substrate
after 72 h of enzymatic hydrolysis at 45◦c after optimization of process parameters by
one factor at a time approach.
Enzymatic saccharification of untreated and pre treated pine needles biomass using
crude, partially purified and purified inhouse enzymes cocktail by applying previously
optimized conditions i.e. enzyme dosage of 16.70 ml/g for 72 h of enzymatic hydrolysis
at 45◦C has been done and achieved 28.05 mg/g reducing sugars from purified enzymes.
Maximum reducing sugars of 33.21 mg/g were obtained from pretreated pine needles
biomass. Overall percent increase i.e. 453.50 % in reducing sugars production was
achieved after optimization of process parameters during RSM approach.
Quantitative analysis of sugars obtained during saccharification of biomass by crude,
partially purified and purified enzymes by using HPLC technique has been done and
found that the biomass broken down to form 6 C (29.96 mg/g) sugars (glucose) and 5 C
(1.50 mg/g xylose).
Fermentation of reducing sugars into bioethanol by using co-culture combination of
Saccharomyces cerevisiae + Pichia stipitis has been done and maximum ethanol
production of 16.44 g/l with fermentation efficiency of 69.47 % was observed under
pretreated pine needle biomass in shake flask.
48
Fermentation process for bioethanol production was subjected to scale up process in a
7.5 litre capacity bioreactor from shake flask experiment. Different process parameters
viz. fermentation time, temperature and agitation rate were optimized to maximize the
bioethanol production in the bioreactor. After optimization of process parameters in a
stirred tank bioreactor maximum of ethanol 18.96 g/l with fermentation efficiency of
72.54% was observed.
Brief conclusion (point wise)
Saccharification of pine needles biomass using purified hydrolytic enzymes i.e. cellulase
and xylanase has been done successfully.
The process parameters were optimized using OFAT approach and statistical model-
RSM. Maximum reducing sugars of 33.21 mg/g were obtained from pretreated pine
needles biomass with 453.50 % overall increase during optimization.
Quantitative analysis of sugars i.e. 6 C (glucose) and 5 C (xylose) has been done by
applying High Performance Liquid Chromatography (HPLC).
Fermentation of sugars into bioethanol using co- culture combination of ethanologens
(S. cerevisiae and P. stipitis) has been done.
Different process parameters viz. fermentation time, temperature and agitation rate
were optimized to maximize the bioethanol production in the bioreactor. The best
optimized conditions were 30h of fermentation time, 25 ◦C temperature and 200rpm of
agitation rate. After optimization of process parameters in a stirred tank bioreactor
maximum of ethanol 18.96 g/l with fermentation efficiency of 72.54% has been
obtained.
49
Goal of the present study
50
Pine forests
Forest fires
Collection of pine
needles
Microorganisms for
biological degradation of
pine needles
Biological degradation of pine needles by
potential microbes
Production of enzymes
Fermentation
Bioethanol
Bioethanol plant
Green fuelFuture of
biofuel
51