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org International Journal of Energy and Power (IJEP) Volume 2 Issue 4, November 2013
Dilute Acid Hydrolysis of Parthenium
Hysterophorus L. for the Production of
Ethanol Using Pichia Stipitis
Preeti Singh1, Shouvik Saha1, Shubhaneel Neogi1, Swati Ghosh2, Amit Ganguly1, Subhamay Haldar3 and
Pradip Kumar Chatterjee1*
1 Thermal Engineering Division, CSIR Central Mechanical Engineering Research Institute, Durgapur – 713209,
India.
2 Department of Chemistry, Institute of Engineering and Industrial Technology, Durgapur – 713212, India.
3Department of Chemical Engineering, National Institute of Technology, Durgapur – 713209, India.
* Corresponding author, Tel No 91‐343‐6510236, Fax No 91‐343‐2547375, Email: pradipcmeri@gmail.com
Abstract 25% [Hinman N.D. et al, 1989].
Due to the present scenario of depletion of fossil fuels, a Ethanol has been used as fuel in the United States
promising alternative; the production of bioethanol from since at least 1908. Although early efforts to sustain a
renewable source that is lignocellulosic biomass; Parthenium
U.S. ethanol program failed, oil supply disruptions in
hysterophorus L raises a substitute of petroleum. Parthenium
the Middle East and environmental concerns over the
hysterophorus L. has been defined as pioneer for
consideration of this biomass to be assessed as its high
use of lead as a gasoline octane booster renewed
regeneration capacity, production of huge amounts of seeds, interest in ethanol in the late 1970s. Ethanol production
high seed germinabilty & extreme adaptability to wide range in the United States grew from 175 million gallons in
of ecosystems. Dilute acid hydrolysis of the biomass 1980 to 1.4 billion gallons in 1998, with support from
cooperate an important role in pentose as well as hexose Federal and State ethanol tax subsidies and the
sugar yield. Pretreatment process revealed the presence of mandated use of high‐oxygen gasoline. The transfer of
61.7 mg/g of xylose and 115.5 mg/g of total reducing sugars. crude oil‐based refinery to biomass‐based bio refinery
Among the xylose fermenting yeast Pichia stipitis has shown has attracted strong scientific interest with focus on the
promising capability for industrial use, as it is able to
development of cellulosic ethanol as an alternative
ferment xylose rapidly with a high ethanol yield (26.19 mg/g)
transportation fuel to petroleum fuels. The production
by utilizing 84% available hexose sugars and 53% of xylose.
of fuel ethanol from biomass involves pretreatment,
Keywords hydrolysis, fermentation, and distillation. The
Parthenium; Lignocellulosic; Biomass; Xylose; Pichia Stipitis; Cell hydrolysate resulting from the pretreatment and the
Optical Density; Ethanol. hydrolysis, contain varying amounts of
monosaccharides, both pentoses and hexoses, and a
Introduction broad range of substances either derived from the raw
material or resulting as reaction products from sugar
Global depletion of energy supply due to the
and lignin degradation. Many of these substances may
continuing over‐utilization of petro‐resources is a
have an inhibitory effect on the micro‐organisms in
major problem of the present and future world
subsequent fermentation steps [Nigam J.N. et al, 2002].
community. It is estimated that the fossil fuels will be
running out in the next few decades [Bentley RW., The fermenting microorganisms must be able to
2002], therefore, attention has currently been dedicated ferment all monosaccharaides present and must have
to the conversion of biomass into fuel ethanol. Over tolerance against potential inhibitors produced during
several supplementary resources, lignocellulose is pre‐hydrolysis steps. From the yore of fermentation
considered as the most promising raw material for history, Saccharomyces cerevisiae is most commonly
production of green fuel, ethanol, because of its used as ethanol producer. S. cerevisiae is unable to
availability in large quantities and low cost [Lynd L.R., ferment pentoses, which comprise upto 40% of the raw
1989]. The efficient utilization of hemicellulose biomass. Among the xylose fermenting yeast Pichia
components of lignocellulosic feedstock offers an stipitis has shown promising capability for industrial
opportunity to reduce the cost of fuel production by use; because it is able to ferment xylose rapidly with a
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International Journal of Energy and Power (IJEP) Volume 2 Issue 4, November 2013 www.ijep.org
high ethanol yield and comparatively produces less al, 2013) was carried out at 121 ºC treatment
xylitol [Dominguez H. et al, 1993] and is able to temperature (Roberto Inês C. et al, 2003) against 10%
ferment a wider range of sugars than even Candida loading of dilute (5%) H2SO4 (N. Shubhaneel et al,
shehatae [Preez Du. J.C. et al, 1986]. 2013). Since the chemical composition of Parthenium
Parthenium hysterophorus, the species of flowering plant acid hydrolysate is complex and could be expected to
of Asteraceae family, i.e., native to the American contain unknown, potentially toxic elements,
tropics, is also known as congress grass, carrot weed, especially presence of parthenin toxin, a potential
star weed, feverfew, white top, chatak chandni, bitter MAP kinase inhibitor [Hwang D. Et al, 1996]. This
weed, ramphool, garghas depending on the country study was conducted focusing on whether growth can
and area infested. This weed is well known for its be promoted after pre‐treatment and detoxification,
allergic responses to human being, causing respiratory using sugar content of Parthenium. Detailed studies
problems, contact dermatitis, mutagenicity in human were carried out to establish the correlation between
& livestock. Biodiversity is greatly altered, and crop utilization of pentose and hexose sugar with ethanol
production is remarkably reduced by its allelopathic production and these factors were related with the
properties, characterized by the presence of highly growth of the microorganism.
alkaline toxin perthenin of seqsuiterpene‐lactone
group of compound [Singh H.P. et al, 2003]. Materials and Methods
Parthenium hysterophorus adapts to a wide range of soil Preparation of Substrate
types ranging from sandy to heavy clays but growth is
In this novel approach to utilize a toxic weed as
better in moist type of soil. It occurs in areas with
substrate for zero cost fuel, the fresh plants were
summer rainfall greater than 500 mm per annum.
collected from local areas surrounding CSIR‐CMERI
Germination favours at temperatures between 10 ºC
research laboratory abundant to perform this test. The
and 25 ºC. Parthenium weed colonises new areas
fresh green healthy plants were chopped into pieces
rapidly by means of relatively high numbers of seeds,
and dried in a hot air oven at 106 ºC for 6 hrs. The
producing one lakh seeds per plant. Dispersal of seed
dried mass then carefully grounded to 3‐5 mm size
takes place via vehicle, water, animals, farm
approximately and stored in a dry airtight container
machinery and wind. Disturbed habitats such as
for further work.
roadsides and railway tracks, stockyards, buildings
surroundings and fallow agricultural land are Preparation of Hemicellulose Hydrolysate
particularly suitable for growing P hysterophorus due to
a lack of interspecies competition [Eberts T. J. et al, 60 gm of dried Parthenium (0.95 mm) was mixed with
1979]. 600 ml. of 5% of concentrated H2SO4 in a conical flask.
The acid hydrolysis reaction was catalysed at 121 ºC at
Parthenium weed was first noted in India near Poona 15 lb pressure for 15 min of treatment time. After that,
in Maharashtra State in 1951. By 1972 it had spread the hydrolysate was cooled down to room temperature
into the most of the western States from Kashmir in the and carefully filtered aseptically using Whattman filter
north to Kerala in the south and is now spreaded paper no.1 to remove the unhydrolysed material and
almost throughout the subcontinent with abnormal solid residues. The filtrate was collected and the total
density [Mahadevappa M., 2008]. content of xylose and total reducing sugar was
The main objective of this work is to apply a simple estimated before fermentation.
feasible pre‐treatment process with moist heat dilute
Detoxification of Acid Hydrolysate
acid hydrolysis for the conversion of dry parthenium
to release the xylose and other reducing sugars; also The detoxification of hemicellulose acid hydrolysate
utilizing this sugar hydrolysate for microbial by boiling and over liming to pH 10 with solid
fermentation after detoxification. The Parthenium used Ca(OH)2 and sulphite synergize better fermentibility of
for this study was previously estimated, having a the hydrolysate. Boiling assists to reduce volatile
composition of 27% cellulose, 21% hemicellulose, 12‐ compounds, such as furfural and phenols and over‐
15% lignin as determined in the laboratory. Estimation liming with Ca(OH)2 remove the concentration of
of xylose, a major fermentable pentose sugar, and other acid compounds. Also increased pH to 10.0 helps
other reducing sugars, has been performed by precipitating heavy metals. The furfural is transformed
spectrophotometer. The pre‐treatment process of dried into furfuryl acid and condensed with other
biomass having particle size 0.95 mm (Ghosh Swati et components of pre‐hydrolysate [Strickland. R.J. et al,
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www.ijep.org International Journal of Energy and Power (IJEP) Volume 2 Issue 4, November 2013
1985]. Estimation of Total Reducing Sugar in the Biomass
0.05 gm of xylose was mixed with 10 ml of benzoic 3) Ethanol Stock Solution (10 mg/ml.)
acid to make 10 g/L solution. From this stock solution, a series of standards
3) Phologlucinol Solution ranging from 5 mg/ml was prepared for the
standard graph.
0.5 gm of pholoroglucinol was mixed with 10:1
ratio of acetic acid and HCL mixture to make 100 Fermentation
ml pholoroglucinol solution.
The fermentation was carried out in a closed glass
4) Benzoic Acid Solution vessel having Swan necked tapped outlet, to minimize
aeration effect and outside contamination. The vessel
100 ml of distilled water was heated at 60 ºC and 0.2
containing 200 ml of detoxified parthenium
gm of benzoic acid was mixed thoroughly then
hydrolysate was inoculated with 10 mg. of P. stipitis
cooled and filtered to store the solution.
cell mass suspended in 1 ml of distilled water. The
200 μL of sample was mixed with 5 mL of inoculum (10 mg/ml) was optically evaluated at 600
phloroglucinol solution. The samples were heated at nm, which conferred the absorbance of 3.15. The
100 ºC for 4 mins and cooled to room temperature. The inoculated hydrolysate was taken to shaking incubator
samples were then measured for available xylose with at 30±0.2 ºC in pH 6.0±0.2 for 30 consecutive hours. In
spectrophotometer at 554 nm. every 3 hrs interval, 5 ml sample was taken out
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International Journal of Energy and Power (IJEP) Volume 2 Issue 4, November 2013 www.ijep.org
through Swan necked outlet by aseptically opening the
outlet tap. The collected sample was further
investigated for available free xylose, total reducing
sugar, ethanol and cell optical density (OD) changes
with time and nutrients.
research is to evaluate the potentiality and probability
to utilize this high growth rate having toxic weed, as a
promising resource of zero cost second generation
environment friendly fuel.
The bio‐chemical conversion of hemicellulose to
pentose and hexose monomers was successfully
achieved by moist heat acid hydrolysis at approx. 15 lb
pressure for 15 min treatment time. Auto hydrolysis of
Parthenium by dilute acid yielded mixture of sugars
with xylose as a major component and other hexose FIG 2: CHANGES IN CELL CONCENTRATION IN RESPECT TO
sugars like glucose, galactose, mannose etc. The above ETHANOL PRODUCTION
depicted pretreatment process revealed the presence of
From comparative study of changes in cell
61.7 mg/g of xylose and 115.5 mg/g of total reducing concentration with ethanol production, it was
sugars. observed that hexose mediated log phase was
The fermentation process involves utilization of sugar continued upto 9 hrs and reached stationary phase
by microorganisms and production of ethanol as a by‐ where 17.23 mg/g ethanol was obtained. After
product. Ethanol also has cytotoxicity towards maximum hexose was utilized by microbes when the
microorganism. So in batch culture system, the overall Sugar availability became stressed out for growth,
Pichia started to utilize pentose sugars and growth was
production depends on available sugars, physical
maximized upto 18 hrs before reaching stationary
growth factors, ethanol tolerance of the microorganism
phase, where maximum production of Ethanol was
etc. Pichia stipitis is able to utilize both hexose &
obtained at 24 hrs, 26.19 mg/g after which cytotoxicity
pentose sugars.
by secondary metabolic compounds and limiting
In order to establish the relationship between nutrient source catalysed degradation in total cell
utilization of hexose & pentose, the microbial growth density and diminished ethanol production.
pattern was closely observed via optical density
measurement of the inoculum containing hydrolysate.
After inoculation of P. stipitis into optimized
hydrolysate, maximum hexose was utilized within 9
hrs and the culture system initially reached stationary
phase for hexose. Completing 12 hrs, Pichia stipitis
started utilizing pentose in order to survive and render
further growth via pentose phosphate pathway.
Production From growth curve, it was observed that
maximum growth was attended at 18 hrs utilizing
xylose while maximum secondary metabolite, ethanol
was engendered at 24 hrs, and the culture became
FIG 3: SUGAR UTILIZATION IN RESPECT TO ETHANOL
subsequently decelerated. PRODUCTION AND CELL GROWTH.
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Fig 3 elaborates total percentage of hexose and pentose 1440‐1443.
reduction to support fungal cell growth and ethanol
Ghosh, Swati, Das, Kuntala, Sinha, Sneha, Das, Subhabrata,
production. In the whole tenure of fermentation
Ganguly, Amit, Haldar, Subhamay and Chatterjee,
process, 84% of total hexose was utilized; out of which
51% was utilized within 12 hrs. There was 53% of total Pradip Kumar “Effect of Particle Size on the Hydrolysis
pentose sugar utilisation by the microbes before the of Parthenium Hysterophorus L. for the Production of
growth inhibition due to media toxicity & other Ethanol” International Journal of energy and Power 2(1)
physical factors. (February 2013): 26‐32.
Conclusion Hinman, N.D., Wright, J.D., Hougland, W. and Wyman, C.E.
“Xylose fermentation, an economic analysis. Appl.
Conventional fuels are depleting unprecedentedly by
profuse consumption in human civilization since few Biochem.” Biotechnol. 20/21 (1989): 391‐401.
decades. On the other hand, perpetuating depletion of Hwang, D, Fischer, N.H., Jang, B.C., Tak, H., Kim, J.K. and
forestry and exponential increase of pollutions have Lee, W. “Inhibition of the expression of inducible
astronomical effects on global warming. These factors
cyclooxygenase and proinflammatory cytokines by
necessitate us to find some effective alternative to
sesquiterpene lactones in macrophages correlates with
conventional fuels. Biofuel plays a crucial role as
alternative source of conventional fuel in ameliorating the inhibition of MAP kinases.” Biochem Biophys Res
the conundrums of global warming. Parthenium Commun. 226 (1996): 810‐8.
hysterophorus, a toxic weed with excessive growth rate Johnson, S.L., Bliss, M., Mayersohn and Conrad, K.A.
and high adaptability toward environmental stresses,
“Phloroglucinol‐based colorimetry of xylose in plasma
is a shining option for zero cost fuel. The maximum
and urine compared with a specific gas‐chromatographic
value of ethanol yield was 26.19 mg/g of biomass by
utilizing 84% available hexose sugar and 53% of xylose. procedure.” Clin Chem. 30 (1984): 1571‐1574.
Fermentation was carried out at a pH of 5.6 and 30 ºC Lynd, L. R. “Production of ethanol from lignocellulosic
temperatures to minimize generation of toxic materials using thermophilic bacteria: critical evaluation
components. This preliminary work on use of
of potential and review.” Adv. Biochem. Eng./ Biotechnol.
Parthenium hysterophorus L. will definite pioneer for
consideration of this biomass to be assessed as high 38 (1989): 1‐52.
valued resource. There is also research possibility on Mahadevappa, M. “Parthenium – A Dreaded Weed
different optimization parameter to make this process International Parthenium research News.” 1(1) (June 1,
industrially viable. 2008).
Nigam, J. N. “Bioconversion of water‐hyacinth
ACKNOWLEDGMENT
(Eichhorniacrassipes) hemicellulose acid hydrolysate to
The authors are grateful to Director, CSIR‐Central
motor fuel ethanol by xylose‐fermenting yeast.” J.
Mechanical Engineering Research Institute, Durgapur
for constant support, encouragement and permission Biotechnol. 97 (2002): 107–116.
to publish this paper. N., Shubhaneel, Ghosh, Swati, Haldar, S, Ganguly, A and
Chatterjee, P. K. “Acid catalyzed auto‐hydrolysis of
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