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