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Energy
EnergyProcedia
Procedia153 (2018) 000–000
00 (2017) 231–236
www.elsevier.com/locate/procedia
5th International Conference on Energy and Environment Research, ICEER 2018
5th International Conference on Energy and Environment Research, ICEER 2018
Butanol production by Clostridium beijerinckii from pineapple
Butanol production by Clostridium beijerinckii from pineapple
The 15th International Symposium waste juice on District Heating and Cooling
waste juice
a, b
Assessing Vorapat
the Sanguanchaipaiwong
feasibility of using *, Noppol
the heatLeksawasdi
demand-outdoor
Vorapat Sanguanchaipaiwong *, Noppol Leksawasdib a,

b temperature
Department
a
of Biology,
function for aFaculty
long-term
King Mongkut’s of
Institute
district
Chiang
of Technology
heat
Mai University
Ladkrabang
demand
(CMU),Chiang
(KMITL),
forecast
Department of Biology, Faculty of science, King Mongkut’s Institute of Technology Ladkrabang (KMITL), Bangkok, 10520, Thailand
a

Bioprocess Research Cluster,Faculty Agro-Industry,

Schoolofofscience, Agro-Industry, Mai,50100,Thailand
Bangkok, 10520, Thailand
b
Bioprocess Research Cluster, School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University (CMU),Chiang Mai,50100,Thailand
I. Andrića,b,c*, A. Pinaa, P. Ferrãoa, J. Fournierb., B. Lacarrièrec, O. Le Correc
Abstract
a
IN+ Center for Innovation, Technology and Policy Research - Instituto Superior Técnico, Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal
Abstract b
Veolia Recherche & Innovation, 291 Avenue Dreyfous Daniel, 78520 Limay, France
c
Renewable energy Département Systèmes
has received Énergétiques
increasing et Environnement
attention, due to global - IMT Atlantique,
energy crisis 4andruelimited
Alfred Kastler,
supply44300 Nantes,
of fossil France
fuels. Butanol is one
Renewable energybiofuels
of the alternative has received
with the increasing attention,
similar energy due to global
properties energysuch
as gasoline, crisis
as,and limited
energy supply
density andof fossil
heat fuels. ButanolIt is
of vaporization. one
could
of
be the alternative
produced biofuels with
by Clostridia the similar energy properties
via acetone-butanol-ethanol as gasoline,
fermentation fromsuch as, energy
various density
renewable and heat
sources. Toofobtain
vaporization.
economical It could
raw
be produced
materials, by Clostridia
pineapple via acetone-butanol-ethanol
waste juice was utilized as a carbonfermentation from various
source for Clostridium renewableTISTR
beijerinckii sources. To The
1461. obtain economical
juice was collectedraw
Abstract
materials,
from pineapple
‘Pattavia’ wastewaste
pineapple juice wasand utilized as a carbonculture
to prepare for Clostridium
sourcemedium. beijerinckii
The maximum viableTISTR 1461. The concentration
C. beijerinckii juice was collected
(2.40
from
± 0.12‘Pattavia’ pineapple
x 108 CFU/mL) was waste and utilized
obtained at 168 htoofprepare culture
cultivation withmedium. Thewaste
pineapple maximum under C.
juice viable beijerinckii
anaerobic concentration
condition at 37 °C.(2.40
The
± District
butanol heating
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10 8 CFU/mL)networks
ofwas are
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± 0.16 g/L 168 addressed
at was in the
h of cultivation
subsequently literature
with
produced. as one
pineapple
A yield waste
of of the
0.08 juice most∙g-1effective
under
g butanol anaerobic
reducing
solutions for atdecreasing
condition
sugars was achieved 37 °C. The
suggesting the
greenhouse
butanol
the gasimprove
emissions
concentration
necessity to of 3.14 from
± 0.16
fermentation theg/L
building sector.
was subsequently
process for These
higher systems
produced.
level Arequire
of butanol yield ofhigh
0.08investments
concentration. whichsugars
g butanol∙g-1 reducing are was
returned through
achieved the heat
suggesting
sales.
the Due to
necessity to improve
the changed climate process
fermentation conditions and building
for higher level of renovation policies, heat demand in the future could decrease,
butanol concentration.
prolonging the investment return
© 2018 The Authors. Published by Elsevier Ltd. period.
©The
2018 Thescope
Authors. Published by Elsevier Ltd.
©
This ismain
2018 The
an Authors.
open of this
access paperunder
Published
article isbytoElsevier
assess
the CC the
Ltd.feasibilitylicense
BY-NC-ND of using the heat demand – outdoor temperature function for heat demand
(https://creativecommons.org/licenses/by-nc-nd/4.0/)
This is
forecast.an open access
Thepeer-review article
districtarticle under
of Alvalade, the CC
located BY-NC-ND
in ofLisbon license (https://creativecommons.org/licenses/by-nc-nd/4.0/)
(Portugal), was used as a5th case study. TheConference
district is consisted ofand
665
This is
Selection
Selectionan open
and
and access
peer-review under
under
under the CC BY-NC-ND
responsibility
responsibility the
ofand license
scientific
the typology. (https://creativecommons.org/licenses/by-nc-nd/4.0/)
committee
scientific Three
committee of
of the
the 5th International
International Conference on
on Energy
Energy and
buildingsand
Selection
Environment that vary in ICEER
peer-review
Research, both
underconstruction
2018. period
responsibility of the scientific committeeweather
of scenarios
the 5th (low, medium,
International high) and
Conference on three district
Energy and
Environment Research, ICEER 2018.
renovation scenarios
Environment Research, were
ICEER developed
2018. (shallow, intermediate, deep). To estimate the error, obtained heat demand values were
compared
Keywords: with results from a dynamic
Acetone-butanol-ethanol heat anaerobe;
fermentation; demand model,
Clostridiumpreviously developed
beijerinckii; pineapple andwaste.
validated by the authors.
The results
Keywords: showed that when only
Acetone-butanol-ethanol weatheranaerobe;
fermentation; change is considered,
Clostridium the margin
beijerinckii; of error
pineapple could be acceptable for some applications
waste.
(the error in annual demand was lower than 20% for all weather scenarios considered). However, after introducing renovation
scenarios, the error value increased up to 59.5% (depending on the weather and renovation scenarios combination considered).
The value of slope coefficient increased on average within the range of 3.8% up to 8% per decade, that corresponds to the
decrease in the number of heating hours of 22-139h during the heating season (depending on the combination of weather and
renovation scenarios considered). On the other hand, function intercept increased for 7.8-12.7% per decade (depending on the
coupled scenarios). The values suggested could be used to modify the function parameters for the scenarios considered, and
improve the accuracy of heat demand estimations.

© 2017 The Authors. Published by Elsevier Ltd.

* Corresponding author. Tel.: +0-662-329-8400; fax: +0-662-329-8427.
Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and
* E-mail address:author.
Corresponding vorapats@hotmail.com; vorapat.sa@kmitl.ac.th
Tel.: +0-662-329-8400; fax: +0-662-329-8427.
Cooling.
E-mail address: vorapats@hotmail.com; vorapat.sa@kmitl.ac.th
1876-6102 © 2018 The Authors. Published by Elsevier Ltd.
Keywords: Heat demand; Forecast; Climate change
This is an open
1876-6102 access
© 2018 Thearticle under
Authors. the CC BY-NC-ND
Published license (https://creativecommons.org/licenses/by-nc-nd/4.0/)
by Elsevier Ltd.
Selection
This is an and
openpeer-review under
access article responsibility
under of the scientific
the CC BY-NC-ND licensecommittee of the 5th International Conference on Energy and Environment
(https://creativecommons.org/licenses/by-nc-nd/4.0/)
Research, and
Selection ICEER 2018. under responsibility of the scientific committee of the 5th International Conference on Energy and Environment
peer-review
Research, ICEER 2018.
1876-6102 © 2017 The Authors. Published by Elsevier Ltd.
1876-6102 © 2018 The Authors. Published by Elsevier Ltd.
Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and Cooling.
This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/)
Selection and peer-review under responsibility of the scientific committee of the 5th International Conference on Energy and
Environment Research, ICEER 2018.
10.1016/j.egypro.2018.10.006
232 Vorapat Sanguanchaipaiwong et al. / Energy Procedia 153 (2018) 231–236
2 Vorapat Sanguanchaipaiwong et al. / Energy Procedia 00 (2018) 000–000

1. Introduction

An increasing concern to discovery of alternative energy has been an issue due to fuel crisis and inadequate resource
of petroleum. The advantage of bioenergy was renewable substrate resulting in enhanced opportunity to produce
sustainable and cost-effective fermentation fuel. Since its resemblance properties to gasoline, butanol has potential to
be an alternative fuel [1]. These similarities include energy level, low vapor pressure, and non-corrosivity. Butanol
was a precursor for many chemicals using in numerous industries [1]. Clostridium sp. could generate butanol using
acetone-butanol-ethanol (ABE) fermentation [2-4]. This metabolism using sugar- and starch-based carbon sources is
broadly recognized, and has been patented and utilized in industrial process [5]. Two of the most identified
solventogenic species for butanol production are C. beijerinckii and C. acetobutylicum.
Although these microorganisms are able to ferment a broad range of sugary substrates [6], few reports on the
utilization of fruit industry waste to produce biogas and bioalcohol were available [7-10]. Pineapple, Ananas comosus,
is one of the important fruits being commercially produced and consumed in Thailand. Pineapple production in this
country reached 1.8 million tons per year in 2016 [11] and increased to 2.5 million tons per year in 2017 [12]. However,
the procedure of the industrial canned pineapple creates approximately 40% (w/w) waste in form of core and peel [8].
Nonetheless, the liquid portion of this waste contained high reducing sugar concentration of 40.40 g/L with 16.75 g/L
sucrose, 19.72 g/L glucose and 20.62 g/L fructose [13]. Raganati et al. utilized commercial high-sugar-content
beverages and, under optimized conditions, the butanol concentration and yield from pineapple juice were 13.3 g/L
and 0.16 g/g, respectively, using C. acetobutylicum DSM 792 [7]. Biobutanol production (ABE concentration of 5.23
g/L and ABE yield of 0.15 g/g) from pineapple peel waste using C. acetobutylicum B527 were also achieved with
acid hydrolysis and detoxification by activated carbon [9].
In this research, pineapple waste juice was considered as an ideal waste of choice since there was no need for any
pretreatment or hydrolysis steps. It was utilized as a supplement for C. beijerinckii growth and the production of ABE.
To investigate its effect, some basic kinetic parameters (productivity and yields) have been compared. Currently, there
was no previous report on ABE fermentation of C. beijerinckii on medium containing pineapple waste.

2. Materials and methods

2.1. Pineapple juice preparation

To prepare pineapple waste juice, peels and cores of ‘Pattavia’ pineapple were obtained from local market in
Ladkrabang district, Bangkok, Thailand. They were blended with blender (Philips® 1400W) for 15 min, squeezed
through muslin cloth, and filtered through Whatman® no. 1 filter paper. The filtrate was then centrifuged at 4,000
rpm (2,258 x g) for 15 min and determination of reducing sugars concentration was then followed [14].

2.2. Bacterium and medium

Clostridium beijerinckii TISTR 1390 has been supplied by Thailand Institute of Scientific and Technological
Research (TISTR) Culture Collection. It was transferred and conserved in Reinforced Clostridial Medium (RCM)
(Difco™) every 4 wk at 4 °C and also reserved in glycerol stock at -70 °C.
P2 medium [15] was employed for C. beijerinckii growth and ABE production which contained 60 g/L glucose, 1
g/L yeast extract, 0.5 g/L KH 2PO4, 0.5 g/L K2HPO4, 2.2 g/L ammonium acetate, 0.2 g/L MgSO 4•7H2O, 0.01 g/L
MnSO4•H2O, 0.01 g/L FeSO4•7H2O, 0.01 g/L NaCl, 0.001 g/L para-amino-benzoic acid, 0.001 g/L thiamine and
0.00001 g/L biotin. Minerals and vitamins have been sterilized using membrane filtration. The other constituents were
sterilized at 121 °C, 15 psi for 15 min. The juice of pineapple cores and peels was used to prepare P2 medium instead
of water and the glucose was added to the equivalent level of 60 g/L reducing sugar concentration.

2.3. Inoculum preparation and ABE fermentation process

Stock culture was subcultured to 5 mL RCM and heat-shocked at 80 °C for 10 min. Subsequently, C. beijerinckii
was incubated at 37 °C for 48 h. To prepare an inoculum for ABE fermentation, the culture was transferred to 45 mL
 Vorapat Sanguanchaipaiwong et al. / Energy Procedia 153 (2018) 231–236 233
Vorapat Sanguanchaipaiwong et al. / Energy Procedia 00 (2018) 000–000 3

RCM and cultivated at 37 °C for 24 h in anaerobic condition. The inoculum was added into P2 medium at 10% (v/v)
and the anaerobic fermentation was performed in triplicates 250-mL flasks at 37 °C for 168 h (glucose) and for 240 h
(pineapple waste juice).

2.4. Chemical analyses

The sampling was conducted every 24 h and measured for viable cell concentration by pour plate technique
(CFU/mL). Afterwards, the culture samples were centrifuged at 4,000 rpm (2,258 x g) for 15 min and the supernatants
were collected to examine the concentration of reducing sugars by 3,5-dinitrosalicylic acid (DNS) method [14] and
the concentration of acetone and butanol (AB) measured by High Performance Liquid Chromatography (HPLC). The
HPLC column was Aminex® HPX-87H Ion Exclusion (7.8 × 300 mm) using refractive index detector and operated
at 37 °C with 0.005 M H2SO4 as a mobile phase at 0.75 mL/min. The injection size was 20 L.

2.5. Kinetic parameters

The evaluated kinetic parameters included specific growth rate (  , h-1), productivity (g/L h), yield coefficients
for cell growth ( Yx s , CFU/g), butanol and acetone-butanol (AB) production ( Y p s , g/g) [16] using following
equations.

ln( x max x 0 )
= (1)
t

p max − p0
Productivity = (2)
t

x max − x0
Yx s = (3)
s0 − s

p max − p 0
Yp s = (4)
s0 − s

where xmax and x0 were the maximum and initial number of viable cell (CFU/mL), s and s0 represented final
and initial substrate concentration (g/L), pmax and p 0 were the maximum and initial concentration of butanol or AB
(g/L) and t was time of cells growth or butanol or AB production (h).

3. Results and discussion

3.1. Pineapple waste juice

Peels and cores of ‘Pattavia’ pineapple were blended, squeezed and centrifuged as described before prior to
determination of reducing sugars [14]. The reducing sugar concentration of pineapple waste juice was 39.11 ± 0.68
g/L. Abdullah and Mat [13] investigated the characteristics of liquid pineapple waste and the reducing sugar of 40.40
g/L was obtained with sucrose (16.75 g/L), glucose (19.72 g/L), fructose (20.62 g/L), proteins and minerals. The
appropriate level of reducing sugars for butanol production by Clostridium beijerinckii was 60 g/L [17], therefore
glucose would be added into P2 medium to prepare equivalent level of 60 g/L reducing sugars.
234 Vorapat Sanguanchaipaiwong et al. / Energy Procedia 153 (2018) 231–236
4 Vorapat Sanguanchaipaiwong et al. / Energy Procedia 00 (2018) 000–000

3.2. Cell growth of C. beijerinckii with pineapple waste juice

The time profiles of the cell growth and pH have been shown in Fig. 1. The cultivation in 60 g/L glucose (Fig. 1a)
provided the maximum viable cell concentration of 9.46 ± 0.43 x 10 6 CFU/mL at 96 h. There were two exponential
growth phases between 0 to 48 h and 120 to 168 h. This phenomenon could be diauxic growth, since there were at
least three fermentable sugars (glucose, fructose, sucrose) for C. beijerinckii [18] in pineapple waste juice. The cell
number in pineapple waste juice has achieved the maximum growth of 2.40 ± 0.12 x 10 8 CFU/mL after 168 h of
cultivation. The explanation of a much slower growth phase could also be influenced by the initial pH level of
pineapple waste juice medium (4.2) which was lower than that of glucose medium (6.2) (Fig. 1b).

a b

Fig. 1. (a) The viable cell concentration and (b) the culture pH of C. beijerinckii TISTR 1390 using glucose (circle) or pineapple waste juice
(square) at 37 °C with anaerobic condition.

3.3. ABE fermentation of C. beijerinckii

Fig. 2. The concentration of acetone (circle) and butanol (square) in P2 medium containing (a) glucose; (b) pineapple waste juice
from C. beijerinckii TISTR 1390 at 37 °C with anaerobic condition.

The concentration of the products from the cultures are shown in Fig. 2. In P2 medium containing 60 g/L glucose,
C. beijerinckii produced maximum concentration levels of acetone (1.58 ± 0.04 g/L) and butanol (8.12 ± 0.65 g/L) at
96 h (Fig. 2a). The maximum solvent concentration (4.17 ± 0.23 g/L with 3.14 ± 0.16 g/L butanol and 1.03 ± 0.07 g/L
acetone) has been achieved at 168 h of the cultivation in pineapple waste juice (Fig. 2b). There was no ethanol obtained
from all cultivations of C. beijerinckii. Previous report also observed that ethanol was barely produced from C.
beijerinckii NRRL B593 [19]. From Table 1, the specific growth rate calculated from pineapple waste juice (0.081 h-
 Vorapat Sanguanchaipaiwong et al. / Energy Procedia 153 (2018) 231–236 235
Vorapat Sanguanchaipaiwong et al. / Energy Procedia 00 (2018) 000–000 5

1
) was not much lower than that of glucose (0.095 h-1). Butanol yield of 0.08 g/g reducing sugars and productivity of
0.019 g/L∙ h were gained from pineapple waste juice. C. acetobutylicum DSM 792 fermented commercial pineapple
juice beverages under optimized conditions with the butanol concentration and yield of 13.3 g/L and 0.16 g/g,
respectively [7]. From acid hydrolyzed pineapple peel (solid) waste, ABE concentration of 5.23 g/L and ABE yield
of 0.15 g/g were obtained using C. acetobutylicum B527 [9]. Since commercial pineapple juice beverages from former
report and pineapple peel (solid) waste from latter report were acid-hydrolyzed before the cultivation, their main
carbon sources were monosaccharides, which bacteria could digest straightforwardly [20].

Table 1. Kinetic parameters of C. beijerinckii TISTR 1390 growth and AB production with glucose or pineapple waste juice as a carbon
source.
Kinetics parameters Glucose Pineapple waste juice
 (h )-1
0.095 0.081
Yx s (CFU/g) 2.12 × 105 6.33 × 106
Yp s of butanol (g/g) 0.182 0.08
Butanol productivity (g/L h) 0.084 0.019
Yp s of AB (g/g) 0.197 0.098
AB productivity (g/L h) 0.091 0.022

4. Conclusion

This research successfully utilized pineapple waste juice as a part of carbon source for C. beijerinckii TISTR 1390.
Even though the relatively lower concentration (3.14 ± 0.16 g/L) and yield (0.08 g/g) of butanol were obtained,
pineapple waste juice which required no pretreatment or hydrolysis process has potential for the fermentation of
economical, feasible and renewable biobutanol.

Acknowledgements

The authors would like to acknowledge the financial support and supports from National Research Council of
Thailand (NRCT) and Faculty of Science, KMITL (V Sanguanchaipaiwong) as well as Chiang Mai University–Co-
Research program under International College of Digital Innovation (ICDI), National Research University (NRU) -
CMU, NRU - Office of Higher Education Commission (OHEC), Ministry of Education, CMU, Thailand (N
Leksawasdi) for this research.

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