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Utilization of waste pine needles for the production of cellulolytic enzymes in

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fermented residue:...

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DOI: 10.1016/j.renene.2022.06.067

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 Renewable Energy 195 (2022) 1064e1076

Contents lists available at ScienceDirect

Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Utilization of waste pine needles for the production of cellulolytic

enzymes in a solid state fermentation bioreactor and high calorific
value fuel pellets from fermented residue: Towards a biorefinery
approach
Raikamal Bhattacharya a, 1, Sidharth Arora b, 2, Sanjoy Ghosh a, *
a
Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, 247667, India
b
Fermentech Labs Pvt. Ltd, TIDES Business Incubator, Indian Institute of Technology, Roorkee, Uttarakhand, 247667, India

a r t i c l e i n f o a b s t r a c t

Article history: The present study addresses valorisation of abundantly available, low-cost pine needle (PN) waste into
Received 11 February 2022 cellulolytic enzymes and high calorific value (CV) fuel pellets (FP). Initial studies in flasks involving hot
Received in revised form water steam pre-treatment (HWSP) using Aspergillus niger MTCC 9652 for 5 days resulted in an increase
6 June 2022
in Cellulase, Endoglucanase and Xylanase activities compared to the untreated pine needles (UPN).
Accepted 12 June 2022
Available online 19 June 2022
Fermentation experiments were scaled-up in a 6-L Solid State fermentation (SSF) packed bed reactor
(PBR) at different bed heights (BH) using UPN. The maximum cellulase and xylanase yields were ach-
ieved as 9.97 ± 0.27 FPU g-ds
1 and 8276 ± 254 IU g-ds
1, respectively, at 72 h using UPN in the PBR. The
Keywords:
Pine needle
fermented residue showed 53.5% consumption of holocellulose, a decrease in crystallinity index to 29.5%
Solid-state fermentation from 46%, with an increase in total lignin content of 45.9%. The fermented residue was processed for
Bioreactor pellet production, resulting in a higher CV (19.13 ± 0.69 MJ kg-1) than the pellet made from unfermented
Cellulolytic enzymes PN (17.63 ± 0.66 MJ kg
1), along with enhanced compressive strength. Sulphur content (0.14%) was
Lignin-rich fermented residue below the permissible limit of SOx emission for fuels with ash content less than 5%. Therefore, the
Fuel pellets present study represents a novel and sustainable biorefinery approach for PN valorisation.
© 2022 Elsevier Ltd. All rights reserved.

1. Introduction tonnes (MT), while the net yearly yield is around 1.33 MT [2]. Each
year, from the onset of the summer season till the rainy season,
Pine forest covers a vast expanse of the Himalayan region at an millions of tons of dried PN fall on the forest floor, forming a thick
altitude ranging from 450 to 2300 m covering an area of around 8.9 layer of blanket [3]. PN mulching on the forest floor up to an
lakh hectares [1]. In India, the state of Uttarakhand, located in the average height of 50 mm is considered safe and non-inflammable
Central Himalayan region, is one of the richest sources of pine trees. and mitigates soil erosion [4]. However, beyond this, PN presents
The annual gross PN yield has been estimated to be 1.9 million a grave concern to the environment. Higher lignin content in PN
results in slow degradation, limiting other flora's growth, and
cannot even be used as an animal fodder [5].
Moreover, PN, a rich source of resin and fibre, contains 70.03% of
Abbreviations: PN, pine needle; SSF, solid state fermentation; PBR, packed bed
reactor; g-ds, gram dry substrate; FPU, Filter paper unit; HWSP, hot water steam mean volatile matter content, making them highly inflammable,
pre-treatment; UPN, untreated pine needle; TPN, treated pine needle; FPN, fer- often resulting in uncontrollable forest fire [6]. Collection and
mented pine needle; BH, bed height; FESEM, field emission scanning electron mi- transportation of PN, although a significant challenge, provides
croscopy; XRD, x-ray diffraction; CrI, crystallinity index; CV, calorific value; FP, fuel employment opportunities for the rural communities. Unemploy-
pellet; HPLC, high performance liquid chromatography; MTCC, Microbial type
culture collection; MT, million tonnes.
ment is one of the primary concerns (4.1% and 6.8% in rural and
* Corresponding author. urban areas, respectively) in Uttarakhand, especially in the hilly
E-mail addresses: sanjoyiitr@gmail.com, sanjoy.ghosh@bt.iitr.ac.in (S. Ghosh). regions, resulting in a high migration rate [2]. Thus developing a
1
MHRD (160903031), MeitY Startup Hub, TIDES Business Incubator (Entrepre- sustainable technology demonstrating large scale valorisation of PN
neurship in residence fellowship)
2 into industrially important metabolites and products, thereby
Engineers India Limited (EIL) Startup India Initiative

https://doi.org/10.1016/j.renene.2022.06.067
0960-1481/© 2022 Elsevier Ltd. All rights reserved.
R. Bhattacharya, S. Arora and S. Ghosh Renewable Energy 195 (2022) 1064e1076

mitigating environmental damage and strengthening the rural controlled process conditions of temperature and airflow rate.
economy, is an urgent need. Initial experiments were carried out in flasks to study the effect of
Applications like PN briquetting as a source of energy [6], nano- HWSP on PN for the production of cellulolytic enzymes. The find-
fibrillated cellulose and its aerogel [7], or as an internal packaging ings were corroborated with proximate analysis, FESEM micro-
material [1], and most recently, PN lignocellulosic active paper graphs, and XRD results of UPN and HWSP treated pine needles
incorporated with nano zeolite [8] have been studied at lab scale, (TPN). After fermentation, the lignin-rich end residue was dried,
but techno-economic viability remains unanswered. Recent reports characterised, and pelletized to produce high CV FP. Overall, the
on pyrolysis of PN (bio-oil), solid char (bio-char) and non- objective of this study focussed on utilising PN to obtain a high
condensable gases are encouraging only if valorisation includes yield of cellulolytic enzymes without compromising on the lignin
high-value products [9]. In a recent study, anaerobic degradation of fraction.
PN was inefficient due to loss of available organics, formation of
intermediate toxins and partial hydrolysis of lignin products [10]. 2. Materials and methods
Harnessing PN for generating electrical power by gasification
technique to produce fuel gas has also been explored [2,11]. Given 2.1. Microorganism
the significant content of holocellulose (~50%) and lignin (~28%) [4]
along with the wide availability, annual accessibility [2,11] and The fungal strain Aspergillus niger MTCC 9652 was procured
renewability of PN [6], focusing on the production of cellulolytic from Microbial Type culture collection (MTCC) Chandigarh, India.
enzymes offers a novel path for the sustainable valorisation of The culture was routinely maintained on a Potato Dextrose agar
waste PN. plate for 5e6 days at 30  C. The spores formed were harvested by
In the recent past, the growing demand for sustainable energy suspending them in distilled water, achieving a final concentration
resources due to the depletion of fossil fuel reserves and the high of 1  106 spores mL
1. The spores were preserved in 30% glycerol
level of environmental issues associated with it led to the search for solution and stored at
80  C for future use [18].
environmentally friendly and renewable materials for biofuel pro-
duction, especially cellulosic ethanol [12]. However, the high pro- 2.2. Raw material collection and preparation
duction cost of the enzymes is a major hindrance to its
commercialisation, especially for the production of bioethanol from PN used for this study were collected from IIT Roorkee, Uttar-
lignocellulosic biomass [13]. The scope for commercialisation of any akhand, India, from March to May. The PN underwent extensive
enzyme lies in the cost-effectiveness of its large-scale production water washing followed by sun-drying for 4 days and then kept
using a low-cost carbon source, leading to sustainability [12]. overnight in the hot air oven at 60  C [19]. The dried leaves were
Sources of cellulolytic enzymes, particularly fungi, are widespread grounded in a Wiley Mill (JHPL-ML-WM0011, James Healthcare Pvt
in nature, and the most common commercially available cellulases Ltd, India) and further sieved to obtain a particle size of 0.850 e1
are derived from species of Aspergillus and Trichoderma [14]. mm size using an Indian standard (IS) sieve. The final moisture
Further, the most common mode of production is submerged content of grounded PN was 13.41%. Wheat bran (WB) was used as
fermentation involving synthetic carbon and nitrogen source and an additive in the fermentation media. The UPN and WB obtained
genetically modified micro-organisms. Alternatively, SSF is a were kept in airtight sealed polyethylene bags for further experi-
promising technology for enzyme production utilising low cost and mentation work and analysis. Molasses used for this study was
renewable biomass that mimics the natural habitat of microor- collected from a nearby sugar mill in Roorkee. All other chemicals
ganisms, particularly fungi, more often resulting in higher yield and and reagents used in this study were of analytical grade and ob-
productivity, lower wastewater generation, lower catabolic tained from Hi-Media Laboratories Pvt Ltd.
repression and low sterility requirement due to absence of free
water in comparison to submerged fermentation [14,15]. A signif- 2.3. Pre-treatment of the substrate
icant expansion in SSF technology has been observed, owing to its
application in the production of enzymes and several secondary The dried PN or UPN was pre-treated with HWSP at different
metabolites [14]. Efforts are prioritised more toward biorefineries time intervals (15, 30, 45 and 60 min) where 10 g of the PN were
for the production of value-added products [16]. Production in suspended in distilled water, achieving solid to liquid ratio of 1:10
scalable bioreactors that can operate at high substrate loading and and sterilised at 121 ± 1  C in 250 mL Erlenmeyer flask and was
under strict aseptic conditions will further improve the process termed as treated pine needles (TPN). The pressure was released
economics of SSF [16]. Several fungi such as A. niger, T. reesei, T. immediately to disrupt the PN's compact nature and expose the
viridae, P. chrysosporium, and A. nidulans have been reported to cellulose fibres. The TPN were washed with distilled water and then
commercially produce cellulolytic enzymes in SSF using agro- kept for drying overnight at 60  C for further use [19].
wastes [15]. Several studies have assessed the potential of utilis-
ing lignocellulosic biomass like wheat bran, apple pomace, sugar- 2.4. Solid-state fermentation
cane bagasse, soybean hull wheat straw, and oil palm empty fruit
branch to produce cellulolytic enzymes [15]. However, the use of 2.4.1. SSF in the flask: media, cultivation conditions and extraction
PN is not yet explored. In addition, the prospects of re-cycling the The fungal cultivation for enzyme production was performed in
fermented biomass, which is high in lignin content for fuel pro- 250 mL conical flasks with both UPN and HWSP PN mixed with 3%
duction, have not been investigated to the best of the author's (w/w) WB, respectively, achieving a total of 5 g in each flask. 3% (w/
knowledge. Utilization of agro-waste with higher lignin content can w) Urea was supplemented with PN as the nitrogen source. A
stand out as an alternative renewable energy source in the form of mineral salt solution was prepared using Mandels and Weber [20]
FP, which can be used in gasification either as a substitute or in media with slight modifications, the composition of which was in g
combination with coal in power plants [17]. 100 mL
1: MgSO4.7H2O, 0.055 g; CaCL2.2H2O, 0.055 g; KH2PO4,
Thus the present study represents a novel approach of PN val- 0.3 g supplemented with trace elements: FeSO4.7H2O, 0.5 mg;
orisation leading towards an integrated biorefinery strategy, where MnSO4.7H2O, 0.16 mg; ZnSO4.7H2O, 0.14 mg; CoCL2.6H2O, 0.2 mg.
the holo-cellulosic content of PN was hydrolyzed by cellulolytic The solid substrate was subsequently moistened with the mineral
enzymes produced by fungi in a SSF packed bed reactor (PBR) under salt solution mixed with 5.5% molasses (data not shown),
1065
R. Bhattacharya, S. Arora and S. Ghosh Renewable Energy 195 (2022) 1064e1076

maintaining a ratio of Solid: Liquid ¼ 1:2. The pH of the mineral salt conditions. The experiments were conducted with different sub-
solution was adjusted to 5 by adding 1 N HCl or 1 N NaOH as strate loading (100 g, 150 g, 200 g, 300 g and 400 g) corresponding
required. The components in the flasks were thoroughly mixed and to different working BH (0.5 cm, 1 cm, 2 cm, 3 cm and 4 cm). 3 g of
autoclaved for 15 min at 121  C. After cooling down, the solid media fermented sample was taken at an interval of 24 h, and extraction
components were inoculated with 20% inoculum (v/w) with a spore was done by a similar protocol as defined previously. The crude
suspension of 1  106 spores mL
1 .They were mixed thoroughly to mixture thus obtained was analysed for different enzymatic activ-
ensure proper distribution of inoculum throughout the solid media. ities. A comparative performance analysis was done between the 2-
The initial moisture content of the substrate before inoculation was L flask and bioreactor system at similar BH for different enzymatic
63.4%, and the final substrate-inoculum moisture content was activities and was expressed as unit per gram dry substrate (U g-
66.01%. The flasks were then kept in a CO2 static incubator at 30  C ds
1) [18].
for 5 days. The fermented samples were collected on the 5th day.
The extracellular metabolites were extracted using 50 mM citrate 2.5. Proximate analysis of the untreated pine needle, HWSP treated
buffer in the ratio of Solid: Liquid as 1:10 in a magnetic stirrer for pine needle and fermented pine needle
1 h at 600 rpm. The whole fermented content of the flask was then
filtered through muslin cloth followed by centrifugation at The proximate analysis of UPN, HWSP TPN used during the
7000 rpm for 15 min. The clear supernatant thus obtained was fermentation process and fermented pine needle (FPN) obtained
treated as the crude extract and stored at
20  C for future analysis. after fermentation was done following the Standard NREL protocol
Further, fungal cultivation was carried out in 2-L conical flasks published under the Standard laboratory Analytical (LAP) proced-
containing 30, 50, 75, 100 and 125 g of UPN corresponding to 0.5, 1, ure for estimation of total solids (LAP 001) [22], ash (LAP 005) [23],
2, 3 and 4 cm BH respectively where UPN was mixed with 3% (w/w) total carbohydrates [24], acid-soluble and acid-insoluble lignin
WB achieving the desired substrate quantity in each flask. The (LAP 003e004) [25,26].
fermentation process was followed like before and the whole fer-
mented sample of one flask was taken out at an interval of every 2.6. Ultimate analysis of the UPN, TPN and FPN
24 h for extraction and enzyme analysis.
The ultimate analyses (C, H, N, S, O) of the UPN and FPN were
2.4.2. SSF in PBR: operation and extraction done according to the method proposed by Jimenez & Ladha [27]. in
The bioreactor design was similar to the previous study in our a Vario MICRO CHNS analyser, Germany. A combustion and
laboratory by Arora et al. [21] but with slight modifications, the reduction tube was present in the system that operates at 1150  C
details and the schematic diagram of which are provided in (Fig. 1). and 850  C, respectively. Helium was used as the carrier gas during
The bioreactor, with a volume of 6-L, was kept in a temperature- combustion by oxygen. The composition of the elements was
controlled room maintained at 30 ± 2  C. The bioreactor was analysed through a thermal conductivity detector (TCD) at 60  C.
steam sterilised in-situ for 1 h at 121  C with the help of a steam Around 2e5 mg of the dried powdered PN were weighed and
generator, and the substrate was separately autoclaved along with packed in small tin vials loaded in the autosampler. The elemental
the moisture media in an autoclavable bag at 121  C for 15 min. The composition was noted from the instrument's software interface
autoclaved substrate was carefully transferred into the bioreactor after the combustion and reduction process and was expressed in
and inoculated following standard aseptic protocols and proced- terms of percentage dry weight basis.
ures, including sterile N95 masks, nitrile gloves, and lab wear. A
stream of air (ambient temperature 30  C) with a flow rate of 2.7. Field emission scanning electron microscopy
0.75 L min
1 was supplied to the substrate bed through the inlet
opening. After cooling down the substrate and the reactor com- The microstructural differences of the UPN, TPN, and FPN were
ponents to room temperature, the solid media was inoculated with investigated by FESEM (Apreo LoVac, Thermofisher, Netherland) at
20% (v/w) inoculum with the same spore concentration. The an accelerated voltage of 15 kV. All the samples were dried and
fermentation process was continued for 5 days under static mounted on a carbon taped metallic stub for carbon coating inside

Fig. 1. Schematic diagram of the PBR.

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R. Bhattacharya, S. Arora and S. Ghosh Renewable Energy 195 (2022) 1064e1076

a vacuum sputter coater to improve the conductivity of the samples H¼Known CV of benzoic acid (6236 Cal/g), W¼Water equivalent
and viewed under Apreo FESEM with a low vacuum. The machine (KJ/ C or Cal/ C).
had special Energy-dispersive X-ray spectroscopy (EDX) attached to CVT¼CV of thread 2.1/cm (using a 10 cm thread, where
the FESEM unit for analysing the quantitative elemental composi- CVT ¼ 2.1*10 ¼ 21 cal).
tion of the samples. CVW¼CV of wire 2.33/cm (for using 6 cm wire.
CVT ¼ 2.33*6 ¼ 13.98 cal).
2.8. X-ray diffraction The pellets were weighed and attached to the thread and
burned inside the calorimeter, and the temperature rise was noted.
The change in crystallinity of the biomass before and after pre- The energy density of the pellets was determined based on the
treatment and after fermentation was determined by XRD (Bruker, temperature difference wherein the combustion takes place in
D8-Advance) studies using Cu-Ka radiation (@ ¼ 0.179 nm) at excess of oxygen. The following formula calculated the CV
30 mA and 40 kV. The samples were scanned in the range of
2q ¼ 5 e 60 with a scan rate of 1 /min. The percentage crystal- T*W
ðCVT þ CVw Þ
CVs ¼ (2)
linity was calculated from [(I002
Iam)/I002]  100, where M
I002 ¼ maximum peak intensity (2q) between 22 and 23 and
Where M ¼ Mass of the pellet (g).
Iam ¼ minimum peak intensity (2q) between 16 and 18 [28].
T ¼ Final rise in temp ( C).
The physical strength of the pellets was determined by
2.9. Analytical methods compressive strength following the protocol of Williams et al. [33].
The length of the pellets made from UPN and FPN was
Filter paper activity was conducted to determine the total 10.03 ± 0.065 mm and 6.30 ± 0.058 mm, respectively. The diameter
cellulase activity using filter paper as the substrate following NREL of the UPN and FPN made pellets was 13.42 ± 0.068 and
Laboratory Analytical Procedure [29]. The Endoglucanase activity 13.33 ± 0.045 mm, respectively. The pellets were tested in axial
was analysed using 2% carboxymethylcellulose, and the b-glucosi- orientation and performed using the universal testing machine
dase activity was performed using 15 mM cellobiose as the sub- (Instron-5982, Norwood, MA, USA) following the ASTM E9-09
strate, respectively, following the protocol of Ghose. [30]. The standard. Ultimate compressive strength (UCS) was calculated from
Xylanase activity was calculated by Bailey et al. [31] using 1% the stress-strain plots. Crosshead speed during compressive tests
beechwood Xylan as the substrate. The total reducing sugar con- was 0.5 mm min
1 with a 10 kN load for all the tests. The test was
centrations were primarily detected by the DNS assay method us- conducted after two days of pellet production.
ing glucose as the standard [32]. For cellobiase assay, analysis of
sugars (glucose and cellobiose) was done with HPLC Apparatus
2.12. Statistical analysis
(Waters 2487, USA) equipped with a refractive index (RI) detector
and an Aminex HPX-87H chromatography column (Bio-Rad, USA).
All the flask experiments were carried out in triplicates, and the
The mobile phase used in the process was 5 mM H2SO4 with a flow
reactor experiments was carried out in duplicates, and the values
rate of 0.6 mL min
1. The column temperature was maintained at
are expressed as mean SD. Statistical analysis were conducted with
60  C. Samples were diluted accordingly and filter sterilised with a
an ordinary one-way ANOVA technique based on the Tukey test
0.22 mm syringe filter, and 20 ml of the sample was injected for each
(p < 0.05) using GraphPad Prism 9.3.1 (GraphPad Software, San
analysis. The concentration was calculated by extrapolating the
Diego, California, USA).
peak areas against the standard slope values.

2.10. Fermented residue valorisation 3. Results and discussion

The FPN residue was collected after extraction of enzymes and 3.1. Effect of pine needle pre-treatment on cellulolytic enzyme
dried overnight before pellet preparation. In the present study, UPN production
and FPN were weighed, and pellets were produced by using a hy-
draulic press (200513196, Techno-search Instruments, India) by The lignocellulosic matrix of PN is highly resistant to microbes
applying a pressure of 2 ton/cm2 where the pressure roller squee- to hydrolyse cellulose for a high yield of the desired metabolite,
zes the material into the die hole. Five samples were prepared each making pre-treatment an appropriate step. The choice of a suitable
from UPN and FPN, and the length and diameter of each pellet were pre-treatment method is crucial which should ideally result in in-
measured using Vernier Caliper with 0.01 mm accuracy. The weight crease in surface area, reduction in lignin content, decrease in
of each pellet was noted down using a digital electronic balance, crystallinity, increase in porosity, no requirement and formation of
and the bulk density was calculated accordingly. toxic products, low capital equipment, and minimal energy utili-
zation [34,35]. HWSP seems to be an attractive method satisfying
all the above conditions. Operating conditions for pre-treatment
2.11. Gross calorific value estimation and ultimate compressive
involved using hot water at 121  C for different time intervals (15,
strength
30, 45 and 60 min) which ensured low severity factor, low oper-
ating cost, and absence of toxic chemicals, thus lowering the pro-
The pellets made from untreated and fermented biomass were
duction of inhibitory compounds. To further enhance the hydrolytic
estimated using a Digital Oxygen bomb calorimeter (Prime Scien-
efficiency, the sudden release of pressure after HWSP resulted in an
tific Industries, New Delhi). The water equivalent (W) was calcu-
explosion of the lignocellulosic biomass into spitted fibres while
lated by using benzoic acid pellet as the standard in the bomb
maintaining low severity conditions, making the biomass more
calorimeter by the formula
amenable to microbial attack [36].
H*M þ ðCVT þ CVw Þ The TPN used as the substrate for SSF were compared with the
W¼ (1) UPN for the production of Cellulase, Endoglucanase, b-glucosidase
T
and Xylanase. The 5th-day fermented samples were subjected to
Where T ¼ Final rise in temp ( C), M ¼ Mass of the sample. extraction and processed for enzymatic analysis. An increase of
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R. Bhattacharya, S. Arora and S. Ghosh Renewable Energy 195 (2022) 1064e1076

21.7% in Cellulase activity (7.95 ± 0.17 FPU g-ds
1) and 22.2% in activities by Trichoderma reesei CCT 2768 which were further vali-
Endoglucanase activity (56.51 ± 1.21 IU g-ds
1) was observed dated by morphological, X-ray and structural analysis.
(p < 0.05) compared to the UPN with pre-treatment time of 45 min
(Fig. 2a and Fig. 2b) following which a slight decrease in the activity 3.2. Effect of pre-treatment on the chemical composition of pine
was noted (p > 0.05). Increase in activity (p < 0.05) of around 20% needle
was observed for Xylanase (9863 ± 384 IU g-ds
1) production in the
present study with 45 min TPN suggesting that HWSP not only The changes generally observed during different pre-treatment
enriched the holo-cellulosic content with partial removal of lignin conditions include deconstruction of cellulose, hemicellulose sol-
but also altered the organised and compact structure of biomass ubilisation, partial removal and relocation of lignin and reduction in
[37]. Similar findings were also reported by Xin and Geng [38], crystallinity of the substrate [41]. From (Table 1), maximum
where steam pre-treatment of horticultural waste resulted in a enhancement of 57.4% holocellulose (15.8% cellulose þ 41.6%
substantial increase in cellulolytic and hemicellulolytic enzyme hemicellulose) content was observed at 45 min pre-treatment. A
activities. In contrast, studies by Pandya and Gupte [34] showed a decrease in lignin content was observed with an increase in pre-
significant decrease in Xylanase activity from 6887 IU g-ds
1 to 832 treatment time. Thus the present study enriched the holo-
IU g-ds
1 after steam pre-treatment of wheat straw at 121  C for cellulosic content with partial removal of lignin content resulting
2 h. Still, an increase in FPU was observed from 0.673 U/g to 0.966 in higher enzyme activities except for b-glucosidase. Studies re-
U/g by Aspergillus tubingenesis JP-1. ported by Li et al. [41] showed that the lignin content reduced from
However, no significant improvement in case of b-glucosidase 23.7% in the untreated poplar solids to 21.3% in the liquid hot water
activity (Fig. 2c) was found and in fact gradual decrease in activity pre-treated ones with a relative increase in cellulose content by
(p < 0.05) was observed with increase in pre-treatment time. To 28e38%, which were in agreement with the present study. Still,
verify the results, the liquid hydrolysate obtained after pre- unlike the present study, there was substantial dissolution in
treatment was analysed by HPLC which showed that there was hemicellulose content. The chemical composition of the substrate
an increase in concentration of cellobiose in the liquid filtrate with was thus used to correlate HWSP pre-treatments with enzyme
increase in pre-treatment time probably explaining the reason for production under SSF, where an increase in cellulosic and hemi-
decrease in the b-glucosidase activity. The cellobiose released after cellulosic content resulted in an increase in Cellulase and Xyla-
60 min pre-treatment (0.25 mg mL
1) was higher than PN treated nase activity, respectively.
for 15 min (0.19 mg mL
1), 30 min (0.22 mg mL
1) and 45 min
(0.23 mg mL
1) and this may have led to repressor effect of cello- 3.3. X-ray diffraction analysis of untreated and treated pine needle
biose on b-glucosidase. The reason for decline in other enzyme
activities after 45 min could be due to the undesirable severity CrI is a measure of assessing the changes in the structure with an
factor resulting in loss of holo-cellulose or due to recondensation in increase or decrease in the crystallinity of the lignocellulosic
the lignin matrix [39]. Similar findings were reported by da Silva biomass, where crystalline cellulose is considered more recalcitrant
et al. [40] where hydrothermal treatment of carnauba straw residue than the amorphous cellulose [40]. Fig. 3 showed the X-ray
at 121  C for 30 min was efficient in inducing the cellulolytic diffraction analysis of the UPN and TPN at different time intervals.
From (Table 1), a decrease in crystallinity index from 46% (UPN) to
30.1% (TPN) was noticed with an increase in pre-treatment time.
The maximum reduction in crystallinity was observed when the
UPN was subjected to HWSP for 45 min which was reflected in
enhanced enzymatic activity. The decrease in CrI with an increase
in time indicates that HWSP partially disrupted and hydrolyzed the
UPN. With the increase in pre-treatment time, hydrogen bonds of
the crystalline region of the cellulose may have been disturbed,
thus increasing the amount of amorphous cellulose in UPN which
correlated with an increase in enzymatic activity. Present findings
are consistent with the study by da Silva et al. [40] where hot water
treatment decreased the crystallinity index to 26.7% in carnauba
straw residue.

3.4. FESEM studies of the untreated and treated pine needle with
elemental analysis by EDS detector

The effect of HWSP on UPN resulted in swelling and fragmen-

tation with enhanced surface area. There was clear evidence of
perforations in the vascular bundles in HWSP treated PN. Moreover,
the sudden pressure drop after HWSP treatment in the autoclave
caused flash evaporation of the superheated water which in turn
forced an explosion of the substrate causing extensive defibration,
loosening and rupture of fibres which was evident from (Fig. 4b) in
the TPN [42]. The gradual loosening of the fibres and the increased
surface area created a positive environment for the hyphae to
penetrate into the lignocellulosic matrix and target the cellulosic
and hemi-cellulosic regions thus favouring the growth of the
microorganism with enhanced enzyme production. In the EDX
Fig. 2. Effect of hot water steam pre-treatment at different intervals on (a) Cellulase analysis, the atomic weight percentage of carbon and oxygen was
activity (b) Endoglucanase activity (c) b-glucosidase activity (d) Xylanase activity. 61.0% and 38.2% respectively. Post pre-treatment, the weight
1068
R. Bhattacharya, S. Arora and S. Ghosh Renewable Energy 195 (2022) 1064e1076

Table 1
Composition of untreated and pre-treated PN (dry weight basis).

UPN 15 min TPN 30 min TPN 45 min TPN 60 min TPN

Cellulose (%) 23.71 ± 0.7a 25.65 ± 0.48a 26.75 ± 0.48 27.46 ± 0.61 25.75 ± 0.60a
Hemicellulose (%) 6.61 ± 0.13 7.87 ± 0.16 8.52 ± 0.24 9.36 ± 0.26 8.99 ± 0.16
Acid insoluble lignin (%) 34.35 ± 0.91b 32.95 ± 0.35b 30.65 ± 0.9 29.8 ± 0.42 30.1 ± 1.5
Acid soluble lignin (%) 2.86 ± 0.05 1.73 ± 0.07 1.63 ± 0.11 1.71 ± 0.11 1.56 ± 0.08
Moisture content (%) 13.41 ± 0.202 10.4 ± 0.26 10.6 ± 0.12 10.50 ± 0.18 9.11 ± 0.25
Ash content (%) 2.57 ± 0.29 3.79 ± 0.11 3.42 ± 0.12 3.71 ± 0.20 3.73 ± 0.21
Crystallinity index (%) 46 34.3 33 30.1 32.5

n ¼ 3 means that the same letters in the same row do not differ significantly (p > 0.05). Comparisons were made taking UPN as the control with other treated samples.

Cellulase activity decreased by ~45% when the BH was 4 cm

(p < 0.0001). A similar trend was observed in the case of Endo-
glucanase (47.5 ± 0.7 IU g-ds
1), b-glucosidase (5.65 ± 0.07 IU g-
ds
1) and Xylanase (9792 ± 345 IU g-ds
1) production where the
highest activity was attained after 96 h at a BH of 0.5 cm. Increasing
the BH to 4 cm resulted in a significant loss of all the enzymatic
activity at the end of 96 h and 120 h (p < 0.05). Therefore, a trend
was apparent wherein increasing the BH beyond 0.5 cm led to a
decrease in activity for all the four enzymes in the flask study. This
was confirmed by visual monitoring, where the fungal growth was
limited to the outer surface of UPN (for BH > 1 cm) and nominal
growth was observed inside the substrate bed. With the increase in
BH, compaction was observed, which probably created an unfav-
ourable environment for the fungus to penetrate into the depth of
the PN, thus negatively affecting the enzyme production.
Fig. 3. X ray diffraction analysis of the untreated and the pre-treated PN at different A major hindrance in the commercialisation of the SSF process is
time interval.
due to the lack of scalable bioreactors which can operate at high
substrate loading, address heat build-up (attributed to the low
percentage of carbon increased up to 62.6% whereas for oxygen it thermal conductivity of substrate) through mathematical models,
was 36.8%. A slight increase in the atomic percentage of elemental C heterogeneity of heat and mass transfer across the bed and non-
after pre-treatment ensured the separation of the lignin complex sterile operation [43]. Thus further experiments were performed
which was in accordance with the study by Mahajan et al. [5]. with the provision of controlled ambient temperature and airflow
Thus all the characterization and microscopic studies reinforce rate in the bioreactor and a comparative performance analysis for
the result of further increase in Cellulase and Xylanase activity, as enzyme production by A. niger was done with the 2-L flask studies
observed after HWSP. However, a major issue observed during the with an increase in BH.
pre-treatment was the significant loss of biomass (30e35%) during
the washing process. Though very few studies have reported this
3.6. Solid State fermentation in packed bed reactor
drawback, a study by Pandya and Gupte [34] was on a similar line
where the application of mild alkali and steam pre-treatment
Studies from the PBR showed maximum enzyme yield after
resulted in weight loss of the substrate as a result of lignin sol-
72 h, therefore enhancing the productivity significantly than the
ubilisation. Thus, further statistical optimisation of HWSP seems to
flask studies. Also, with the provision of temperature and airflow
be a good option for enhanced enzyme production.
rate control, enzyme production at 2 cm BH in the PBR showed
Although pre-treatment resulted in desirable structural changes
better performance than in flask studies. The Cellulase activity in
in biomass leading to high enzyme yield, significant loss of lignin
the bioreactor increased to 9.97 ± 0.27 FPU g-ds
1 compared to the
content shall not be economical for large scale process. Since the
2-L flasks (7.15 ± 0.35 FPU g-ds
1) (p < 0.05). Maximum Xylanase
focus of the present study was to preserve the lignin fraction for the
activity of 8276 ± 254 IU g-ds
1 was achieved in the bioreactor at a
production of high CV FP from the fermented residue, the biore-
BH of 2 cm which was 18.3% lower than the 2-L flask at 0.5 cm BH
actor studies were performed with UPN. Alternatively, during pre-
(p < 0.05). More favourable conditions of temperature, moisture
treatment, the removal of lignin may be recovered and redirected
and airflow rate in the bioreactor may have led to higher utilization
to be used to produce a higher CV solid fuel. Moreover, statistical
of cellulose by A. niger. The Endoglucanase production fared best at
optimisation studies needs to be undertaken to ensure minimal
BH of 1 cm achieving its maximum activity of 48.03 ± 1.08 IU g-ds
1
holocellulose loss and maximum lignin removal.
which was close to that obtained in 2-L flasks at 0.5 cm BH
(p > 0.05). For b-glucosidase, enzyme yield in the bioreactor at 1 cm
3.5. Solid-state fermentation in 2-L flask and 2 cm BH did not differ significantly (p > 0.05), however, upon
further increasing the BH to 3 cm and 4 cm, the enzyme yield
To assess the effect of BH on enzyme production, SSF was carried declined sharply (p < 0.05). Maximum b-glucosidase activity ob-
out in 2- L conical flask containing 30, 50, 75, 100 and 125 g of UPN tained in the bioreactor (6.25 ± 0.09 IU g-ds
1) at the end of 96 h
corresponding to 0.5, 1, 2, 3 and 4 cm BH respectively. Fig. 5 shows was 9.6% higher than the 2-L flasks studies (p < 0.05). Enzyme yield
the day-wise activity profile for all the four enzymes at different BH. at lower BH (0.5 cm) in PBR was less than that obtained in flasks
The Cellulase activity peaked on the 4th day (7.15 ± 0.35 FPU g- which may be due to high shear stress on A. niger, attributed to high
ds
1) at 0.5 cm BH. However, a gradual decline in Cellulase activity airflow rate. Earlier studies by Arora et al. [21] showed bed
with a further increase in BH was observed. The maximum compaction and air channelling after the initial surge in microbial
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R. Bhattacharya, S. Arora and S. Ghosh Renewable Energy 195 (2022) 1064e1076

Fig. 4. FESEM micrographs of the substrates with EDX analysis for (a) UPN (b) 45 min TPN.

activity by Rhizopus oryzae with WB as the substrate. In the present circulation of moist air in PBR possibly proved beneficial and
study, though the bed temperature reached 35  C during the initial resulted in better performance for the enzyme production. Similar
10e14 h of fermentation, suggesting high microbial activity, how- reports were documented by Hamrouni et al. [45] where aeration
ever, this did not result in significant bed compaction and air induced significant enhancement in Cellulase activity using
channelling resulting in higher enzymatic yields except for Xyla- T. asperellum TF1 and T. asperellum TV104 in Reimbault column.
nase. PBR studies involving WB, and oil cakes for the production of Maximum Cellulase productivity of 3.32 ± 0.09 FPU g-ds
1 d
1
enzymes often report bed compaction and shrinkage issues, was achieved in the bioreactor, at the end of the 3rd day, which was
thereby presenting scale-up challenges. However, the present 1.8-fold more than that obtained in the flasks (p < 0.0001). From
study found PN attributing to enhanced porosity possibly miti- Fig. 6 higher productivity was observed in the bioreactor for all the
gating air channelling. Similar studies were reported by Biz et al. enzyme systems at different BH (p < 0.05). In accordance with the
[44] where shrinkage and compaction along with subsequent heat present study, Cerda et al. [46] reported average Filter paper ac-
removal were avoided using sugarcane bagasse as the substrate. In tivity (3.1 ± 0.5 FPU g
1 DM) and Xylanase activity (45 ± 8 U/g DM)
the present study, though there was a decline in enzymatic activ- in the first 24 h of operation in a pilot-scale reactor. The findings of
ities at 3 cm and 4 cm BH, but the performance of the proposed the present work are consistent with the studies by Rayhane et al.
bioreactor was better than the 2-L flasks at similar BH. Further [47] where single used plastic bioreactor provided with humid air
studies on scale-up shall be addressed by using design equations showed enhanced activity and productivity for cellulase and
involving mathematical models for heat and mass transfer in the amylase production compared to flask. In Table 2, a longer
bioreactor. fermentation time is mostly observed in the case of all the enzyme
Aspergillus niger being a fast-growing fungus, the initial surge in production studies. In this sense, the findings are pretty promising
microbial activity was visibly significant even at the 8 h in the where significant enzyme production is achieved in less time,
bioreactor. The initial surge, however, was not noticed in the flasks resulting in higher productivity, thereby allowing faster valor-
up to 24 h. This probably led to higher productivity in PBR as isation of PN, which improves the overall process economics.
compared to the flasks. Replenishment of O2 and moisture through

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R. Bhattacharya, S. Arora and S. Ghosh Renewable Energy 195 (2022) 1064e1076

Fig. 5. Day wise profile of (a) Cellulase activity in PBR (b) Cellulase activity in Flask (c) Endoglucanase activity in PBR (d) Endoglucanase activity in Flask (e) b-glucosidase activity in
PBR (f) b-glucosidase activity in Flask (g) Xylanase activity in PBR (h) Xylanase activity in Flask. n ¼ 2 for bioreactor studies and n ¼ 3 for flask studies.

3.7. Fermented residue characterization and valorisation studies by Salgado et al. [58] reported a reduction of 28.08% cel-
lulose, 10.78% hemicellulose and 13.3% lignin content in fermented
Microbial fermentation in the PBR showed a pronounced effect olive pomace by Aspergillus uvarum for its further utilization as
on the composition of UPN which were further analysed by prox- animal feed. However, unlike the previous findings, an increase in
imate analysis, crystallinity and scanning micrographs. The solid lignin content by 29.5% in the fermented residue was observed in
residue left after fermentation and extraction was washed and oven the present study. A similar finding by Li et al. [56] also showed that
dried for 24 h. The structure of the UPN and the FPN were char- biological pre-treatment by Pipotene betulinus apparently led to an
acterized by FESEM studies, where fermentation has significantly increase in lignin content because of cellulose and hemicellulose
altered the structure making FPN coarser and more distorted with removal. However, an exact fraction of the increment was not
enhanced porosity compared to the untreated ones (Fig. A.2). The apparent. To the best of our knowledge, a 29.5% increase in lignin
changes observed here are similar to the studies reported by Ezeilo content in fermented residue makes it an ideal platform for pro-
et al. [12] and Qadir et al. [57] wherein the appearance of the ducing FP and is not reported elsewhere.
substrate was found to be highly unstructured, porous and coarser
after fermentation. Moreover, XRD studies revealed that there is a 3.8. Fuel pellet production and characterisation
decrease in crystallinity index from 46% (UPN) to 29.5% in the FPN
(Fig. A.1) EDX analysis of the fermented substrate (Fig. A.3) showed High lignin content is a favourable factor in the pelletizing
the presence of the elements such as Na, Mg. Si, Ca and K which process as it enhances the CV and increases the mechanical dura-
could be used for improving soil fertility [9]. Thus from the above bility of the FP [59]. The fuel pellet derived from UPN in the present
studies, the structure of FPN showing a disorganisation of the fibres study had a CV of 17.63 ± 0.66 MJ kg
1, which was very close
and its reduced crystallinity and enhanced porosity could be (17.56 MJ kg
1) to Mandal et al. [4]. Further, the FP processed from
exploited for enhanced enzymatic digestibility of cellulose. the FPN enhanced the CV to 19.13 ± 0.69 MJ kg
1, corroborating the
Fermented samples were again processed for proximate analysis findings of enhanced lignin content after fermentation. From
to study the degradation of cellulose, hemicellulose and lignin Table 3, the moisture content analysis of UPN and FPN (<10%)
immediately after the extraction of enzymes. A consumption of showed that the latter is more suitable for making pellets. The ash
23.4% of total initial cellulose and 30.1% hemicellulose at the end of content of the pellets made from FPN was lesser than UPN and
fermentation was observed (Table 3). Very few literature reported below 5%. Low ash content signifies that the pellets produced from
characterization and utilization of fermented residue. Earlier FPN could be a valuable source of fuel for gasifiers and other
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R. Bhattacharya, S. Arora and S. Ghosh Renewable Energy 195 (2022) 1064e1076

thermal applications [4]. Fig. 7b, the ultimate compressive strength (UCS) of the pellet made
The bulk density obtained from the UPN made pellets were from FPN was found to be 57.01 MPa whereas from UPN it was
931 ± 9.89 kg m
3 and for FPN made pellets was 974 ± 13.4 kg m
3. 27.8 MPa which was marked by a dotted line. Interestingly, both the
Recent research by Brand and Jacinto [60] for pellet formation using samples showed a post-peak softening response. Fermentation
a mix of apple prunnings with PN resulted in decreased ash content resulted in enhanced stiffness and peak compressive strength in
and increased bulk density and energy density. Replacing UPN with the FPN made pellets compared to untreated ones.
fermented ones will further improve the pellet quality for its usage
in boilers. Therefore, the present work represents an advancement
in prior art for valorisation of PN for both enzyme as well as pellet 3.9. Economic potential of PN bioconversion based biorefinery
production, wherein both the holo-cellulosic and lignin fractions
are being utilized. An innovative eco-friendly bio residue briquet- The cost of cellulase enzyme is one of the prime factors that
ting machine was developed by Joshi et al. [6] which claimed to be determine the commercial success of bioethanol production. This
lightweight with zero electricity requirement but required heating could be achieved by using a low-cost substrate, adopting a circular
PN for its conversion into briquettes. Alternative options like using bio-economy approach and increasing enzyme productivity. Thus,
binders, molasses or cow dung were supposed to mitigate the very low cost of waste PN [62] coupled with its abundant avail-
heating requirements. So, the FPN, with higher lignin content from ability, and the possibility of side co-product like fuel pellets open
the present study could be an interesting alternative to binding up the potential of waste PN for industrial exploitation.
agents and could also reduce the processing cost. Table 3 showed an The total area of pine forest in Uttarakhand is 3.43 lakh hectares,
increase in the concentration of Nitrogen in FPN made pellets where the accessible amount of PN is 2.05 million tonnes per year
indicating an enhancement in the protein content. Nitrogen and [11]. According to the present study, 1 kg of dried PN can produce
Sulphur content were insignificant in both FPN and UPN, indicating up to 9970 FPU of cellulase. Based on the above, considering a 1-ton
lesser emission of harmful gases like SOx and NOx as compared to pilot plant that would work for 330 days/year where 7 days are
conventional fuels like coal. The sulphur content of 0.14% was required for the whole operation to be completed (including
within the permissible limit as values above 0.2% indicate signifi- loading, running, cleaning in place and pellet formation), a total of
cant emission of SOx [59]. The oxygen content was also reduced in 47 reactor runs would be needed to produce 470 kL of cellulase
the FPN indicating its potential to be an efficient fuel with a high containing 4.7  108 FPU per year. This amount of enzyme pro-
heating value compared to UPN. The future scope involves co- duced could be utilized for hydrolysing glucan for ethanol pro-
densification of the FPN with coal to improve the physical, me- duction, where 17.94 kL of ethanol could be produced, considering
chanical, thermal and other combustion properties [61]. From the cellulase loading as 15 FPU/g glucans [63]. Based on the above,
1.53 kg of glucose (which amounts to 1.7 kg of glucan) is required to

Fig. 5. (continued).

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R. Bhattacharya, S. Arora and S. Ghosh Renewable Energy 195 (2022) 1064e1076

Fig. 6. Comparative analysis of productivity between 2-L Flask and Bioreactor for (a) Cellulase (b) Endoglucanase (c) b-glucosidase (d) Xylanase.

produce 1 L of ethanol. Again, considering the loading as 15 FPU/g for enzyme production due to revenue generation in pellet pro-
of glucan, 2.55 kg of PN will be needed to produce 25,500 FPU. As duction to 80%, which is reasonable as additional cost shall only be
per data, the price of PN is INR 1.5 kg
1 (USD 0.020) [62]. So, the raw incurred due to electricity requirement in pelletizer and drying, and
material cost contribution for producing 1 L ethanol shall be INR distribution and marketing of pellets. As a result, the enzyme cost
3.83 (0.051 USD). Thus, the cost of PN required to produce 1 gallon contribution for a gallon of bioethanol shall be further reduced to
of bioethanol is INR 14.47 (USD 0.19), which corresponds to 9.64 kg INR 310.62 (USD 4.08). Data from the industry indicate that the
of PN. present industrial enzyme costs USD 2.24 per gallon of bioethanol
Moreover, considering the cost of raw materials supplements [13]. Thus proof of concept studies for optimisation of pre-
(wheat bran, molasses, urea, calcium chloride, magnesium chlo- treatment method, bioreactor scale-up studies using heat and
ride, potassium di-hydrogen orthophosphate and trace elements) mass transfer design equation and the prospect of genetically
required for the SSF process amounting to INR 18.70 (USD 0.24), the engineered fungi shall pave the way to bring down the cost of
total raw material cost contribution is INR 33.17 (USD 0.43). enzymes for bioethanol production. Comprehensive techno-
Assuming that the raw material contributes around 7e9% of the economic analysis following trials at the pilot-scale bioreactor
total enzyme production cost in SSF [64,65], the contribution of and proof of concept studies shall be taken up in the future. The
enzyme costs for bioethanol production in the present process shall effects of % CO2 emission reduction and carbon taxation positively
amount to ~INR 368.50 per gallon of bioethanol (USD 4.84). Sub- influence the cost of PN (used in the form of FP) compared to coal as
sequently, the fermented residue obtained after enzyme extraction a source of energy [67]. While looking at the environmental
was ~50% of the initial biomass. Assuming the selling price of pel- perspective, the state of Uttarakhand faced a significant forest fire
lets made from the fermented residue with CV 19.13 MJ/kg as INR in 2016 that destroyed around 2,16,600 ha of the total forest cover
15 per kg (USD 0.20) [66], the total revenue from ~4.82 kg pellets resulting in 20,18,712 tonnes of CO2 emission (considering an
produced will be INR 72.35 (USD 0.95). Assuming the cost offsets emission rate of 9.32 tonnes of CO2/hectares) and 1,44,472 tonnes

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R. Bhattacharya, S. Arora and S. Ghosh Renewable Energy 195 (2022) 1064e1076

Table 2
Studies on cellulolytic enzyme production in SSF Flask and Bioreactor using waste/agro-residues.

Microorganism Substrate used Bioreactor Enzyme Production level Scale Fermentation time Reference
type (g)
Cellulase Endoglucanase b- Xylanase
(FPU g- (IU g-ds
1) glucosidase (IU g-
ds
1) (IU g-ds
1) ds
1)

Myceliophthora Sugarcane bagasse and Packed bed ~75 ~400 620 96 h [48]
thermophile wheat bran
Aspergilus Winery and olive mill waste Tray 56.3 10.9 189 50 96 h for cellulase 48 h for b glucosidase [49]
ibericus 168 for Xylanase
Autothonous Biowaste digestate Packed bed 0.5e1.5 120 48 h [50]
microbiome
Specialised Coffee husk Adiabatic 10 90 24e48 h [51]
consortia packed bed
reactor
Aspergillus niger Wheat bran Tray 2919 100 48 h [52]
CCUG33991
Trichoderma Mixture of vine shoots, Singles used 19.25 5500 72 h [47]
asperellum jatropha cake, olive plastic
TF1 pomace, olive oil bioreactor
Aspergillus niger Coffee industry waste Column type 3.03 10 120 h [53]
F12 packed bed
Aspergillus Sorghum straw Flask 5177.23 72 120 h [54]
tubigensis
FDHN1
R.oryzae UC2 Raw oil palm frond leaves Flask 25.46 94.68 145.05 213.99 6 120 h [55]
Piptoporus Rice straw Flask 7.43 58.37 3 288 h (12th day for endoglucanase) and [56]
betulinus 432 h (18th day for Cellulase)
IFP0072
Aspergillus niger Pine needles Packed bed 5.48 40.41 (72 h) 5.01 (96 h) 7809 100 72 h for cellulase, endoglucanase and Present
MTCC 9652 reactor (72 h) (72 h) xylanase activity 96 h for b-glucosidase study
6.43 48.03 (72 h) 6.18 (96 h) 7662 150 activity e
(72 h) (72 h)
9.97 28.91 (72 h) 6.25 (96 h) 8276 200 e
(72 h) (72 h)
7.24 24.87 (72 h) 5.12 (96 h) 5775 300 e
(72 h) (72 h)
5.79 24.65 (96 h) 4.65 (96 h) 4166 400 e
(96 h) (72 h)

Table 3 FPU g-ds
1, obtained in the bioreactor, is an attractive proposition

Substrate characterisation before and after SSF. for techno-economic assessment of enzymatic hydrolysis on
Proximate analysis (%) Raw substrate Fermented substrate lignocellulosic biomass for biofuel production. Analysis of fer-
Cellulose 24.13 ± 0.41 18.46 ± 1.01
mented residue showed significant consumption of holo-cellulosic
Hemicellulose 6.97 ± 0.86 4.87 ± 0.71 content with a 29.5% increase in lignin fraction, leading to high CV
Acid insoluble lignin 34.61 ± 1.33 43.01 ± 1.78 FP (19.13 MJ kg
1) compared to UPN. Future studies will involve
Acid soluble lignin 2.8 ± 0.08 2.95 ± 0.17 optimisation and scaling up the process in a pilot-scale bioreactor
Moisture content 13.67 ± 0.20 9.6 ± 1.94
using these PN, which would facilitate the commercialisation pro-
Ash content 3.53 ± 0.51 1.893 ± 0.66
Ultimate analysis (%) cess at the industrial level. Thus, the aim of this study of valor-
C 45.73 ± 1.32 48.39 ± 1.06 isation of waste PN to high-value products through a novel
H 9.13 ± 0.11 9.51 ± 0.22 approach that can potentially mitigate forest fires and pollution
N 1.33 ± 0.04 1.97 ± 0.22 problems and provide employment opportunities catering to the
S 0.19 ± 0.014 0.14 ± 0.016
O 43.62 ± 1.50 39.99 ± 1.41
region's socio-economic development.

CRediT authorship contribution statement

of CO (0.667 tonnes/hectare) [11]. Hence by utilising the PN, an
attempt could mitigate the amount of CO2 emission. Raikamal Bhattacharya: Conceptualization, Methodology,
Validation, Formal analysis, Experimentation, Writing e original
draft, Writing e review & editing, Visualization. Sidharth Arora:
4. Conclusion
Conceptualization, Writing e review & editing, Supervision. Sanjoy
Ghosh: Supervision, Manuscript proofreading, Project administra-
This study explored the potential of abundantly available, low-
tion, Resources, and, Funding acquisition.
cost PN to produce cellulolytic enzymes and high CV FP. Effect of
steam pre-treatment resulted in reduced recalcitrance of PN, which
enhanced enzyme production and considerable lignin loss. Scale- Declaration of competing interest
up studies in the bioreactor resulted in increased enzyme yields
and productivity compared to the flasks. Cellulase activity of 9.97 The authors declare that they have no known competing

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R. Bhattacharya, S. Arora and S. Ghosh Renewable Energy 195 (2022) 1064e1076

Fig. 7. (a) Pellets made from untreated and fermented pine needle, (b) Ultimate compressive strength of the pellets from stress and strain curve.

financial interests or personal relationships which could have of Pine Needle Biomass Gasification Plants (< 250 kW) for Electricity Gener-
ation in the Western Himalayan Region: Uttarakhand, India, Process Inte-
appeared to influence the work reported in the paper.
gration and Optimization for Sustainability, 2021, https://doi.org/10.1007/
s41660-021-00199-y.
Acknowledgment [12] U.R. Ezeilo, C.T. Lee, F. Huyop, I.I. Zakaria, R.A. Wahab, Raw oil palm frond
leaves as cost-effective substrate for cellulase and xylanase productions by
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work, Government of India (ID: 16903031) and Fermentech Labs [13] S. Acharya, A. Chaudhary, Bioprospecting thermophiles for cellulase produc-
tion: a review, Braz. J. Microbiol. 43 (2012) 844e856, https://doi.org/10.1590/
Pvt. Ltd. under the project scheme EngSUI of Engineers India
S1517-83822012000300001.
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