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Energy Procedia 68 (2015) 254 – 262

2nd International Conference on Sustainable Energy Engineering and Application, ICSEEA 2014

Decolorization of black liquor wastewater generated from

bioethanol process by using oil palm empty fruit bunches
Ajeng Arum Saria,*, Hendris Hendarsyah Kurniawana,Muhammad Nurdinb,
Haznan Abimanyua
a
Research Center for Chemistry, Indonesian Institute of Sciences, Kawasan Puspiptek Serpong, Tangerang Selatan 15314, Indonesia
b
Faculty of Mathematic and Science, University of Halu Oleo, Kampus Hijau Bumi TridharmaAnduonohu, Kendari, Sulawesi Tenggara 93232,
Indonesia

Abstract

Bioethanol from oil palm empty fruit bunches as a second-generation bioethanol (bioethanol G2) has been developed. It produces
high amounts of black liquor wastewater which also contains alkaline solution (NaOH). Black liquor wastewater is harmful to
aquatic ecosystems if discharge directly into waters. This study aimed to determine decolorization of black liquor wastewater by
using chemical and biological methods. Coagulation-flocculation method by using Poly Aluminium Chloride (PAC) and
alumwith different ratio was also investigated. This study also identified the ability of white-rot fungi to decolorize black liquor
wastewater on agar and liquid medium.The results showed that black liquor wastewater with initial concentration 30,000 ppm
can be decolorized by using two methods, coagulation-flocculation and decolorization by white-rot fungi.The optimum condition
to obtain 96% decolorization was using concentration coagulant 1% combination alum-PAC 3:1 with total retention time 33
minutes.Out of the three fungi studied, Trametes versicolor U97 showed the most degraded fungus onagar medium containing
black liquor. In liquid medium, the percentage of decolorization ranged from 70% to 89% at 30 days depending on the fungal
strain.

© 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
© 2015 The Authors. Published by Elsevier B.V.
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of Scientific Committee of ICSEEA 2014.
Peer-review under responsibility of Scientific Committee of ICSEEA 2014
Keywords: bioethanol from oil palm empty fruit bunches; black liquor; decolorization; coagulation-flocculation; white-rot fungi

* Corresponding author. Tel.: +62-21-7590929; fax: +62-21-7560549.

E-mail address: ajeng.arum.sari@lipi.go.id

1876-6102 © 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of Scientific Committee of ICSEEA 2014
doi:10.1016/j.egypro.2015.03.254
 Ajeng Arum Sari et al. / Energy Procedia 68 (2015) 254 – 262 255

1. Introduction

Lignocellulosic biomass as a renewable energy resource is potent for ethanol production because it does not
interfere food security like other energy resources such as corn and wheat. One of lignocellulosic biomass that
developed in Indonesia for bioethanol production came from oil palm empty fruit bunches (OPEFB) because
Indonesia is the largest producer of palm oil in the world [1]. The oil palm plantation as ca. 8.5 million ha results oil
palm biomass waste as 33 million t year -1[2]. OPEFB have been utilized for bioethanol production in laboratory
and experimental pilot scale [2, 3]. Lignocellulose is composed by cellulose, hemicellulose, lignin, extractive, and
some inorganic materials [4]. Ethanol production from OPEFB consists of three main processes, which are
pretreatment, hydrolysis and fermentation. Pretreatment is needed to produce macroscopic and microscopic biomass
structure used for hydrolysis into fermentable sugar and to break lignin and hemicellulose bonding and cystal
structure to be simple sugar compound [5]. The one method for pretreatment process is usage of alkali chemical
NaOH [3]. During this process, high amounts of wastewater (called black liquor) is produced. Production of
bioethanol in a pilot plant as 76.46 kg resulted 3,000 liters wastewater from pretreatment process that dominantly
obtained from alkaline solution (NaOH). The black color is mainly due to the degradation process of lignocelluloses
and it is an indirect measurement of the amount of lignin compounds present in the effluent stream. Lignin is a
complex heterogeneousphenyl propanoid biopolymer containing a diverse array of stable carbon–carbon bonds with
aryl/alkyl ether linkages and may be cross-linked to hemicelluloses [4].
The poisonous properties of black liquor wastewater is harmful to aquatic ecosystems such as daphnia, fish, and
planktons if discharge directly into waters without any particular treatment system [6, 7, 8]. The characteristics of
black liquor wastewater consist of organic compounds 40%, lignin 25%, hemicellulosic 7.5%, organic acid 7.5%,
and anorganic compounds such as NaOH 20% [9]. Lignin is composed by phenolic, hydroxyl, carbonyl, methoxy,
and aldehyde groups that serve as potential adsorption of pollutants [10]. Black liquor wastewater can be minimized
in many technologies including physicochemical treatments (sedimentation, coagulation-flocculation, adsorption,
chemical oxidation, membrane filtration, and ozonation) and biological treatment (aerobic treatment, anaerobic
treatment, and fungal treatment) [11, 12]. Table 1 shows comparison each method for black liquor wastewater
treatment. However, each of treatment has several advantages and disadvantages. The disadvantages that commonly
obtained are expensive, ineffective in high COD concentration, and producing sludge as new waste. To best our
knowledge, research about wastewater treatment of black liquor in bioethanol process from oil palm empty fruit
bunches has not been conducted yet.
Coagulation-flocculation method using ferric chloride, lime, alum, ferrous sulphate and poly aluminum chlorida
(PAC)is one of the best option to treat black liquor wastewaterbecause it is cheaper than other methods such as
membrane filtration and ozonation [9]. The major advantage of chemical treatment is that color, COD, and TSS are
being reduced during this process therefore it can lead to make it more cost effective before other treatments. Irfan et
al. (2013) reported that by using combination of ferric chloride and aluminum chloride, black liquor wastewater
from pulp and paper mills process was decolorized up to 95%. On the other hand, white-rot fungi (WRF) are often
used to degrade a wide variety of pollutants which resembling lignin structure because these fungi secrete one or
more enzymes such as lignin peroxidase, manganese peroxidase, and laccase [17]. Because the biggest content of
black liquor is lignin, WRFare expected to decolorize black liquor wastewater [14]. Several WRF such as
Phanerochaete chrysosporium, Trametes versicolor, and T. elegansare recognized for their ability to degrade lignin
and black liquor wastewater [18]. In addition, the degradation by using WRF is a promising method for efficient,
does not require high cost, and applicable [19].
This study aimed to determine decolorization of black liquor wastewater by using chemical and biological
methods. Coagulation-flocculation method by using combination of coagulants in different ratio was also
investigated. This study also identified the ability of WRF to decolorize black liquor wastewater on agar and in
liquid medium.
256 Ajeng Arum Sari et al. / Energy Procedia 68 (2015) 254 – 262

Table 1. Comparison removal of black liquor wastewater by using several methods.

Method Initial concentration Removal Time References
Removal of black liquor from Lignin from pine 23.7 g/L pH 2, 70°C 3h 13
papermaking industry by acidification Lignin from bamboo 19.6 g/L Lignin from pine: 92.3%
Lignin from bamboo: 93.7%
Removal of COD, TSS, and color COD: 28270 ppm Alum: COD 11%, TSS 46%, color 52% 33 min 9
of black liquor from pulp and paper mills TSS: 11455 ppm PAC: COD 14%, TSS 50%, color 46%
Color: 1640 pt-co/hazen
Decolorization of black liquor from Color: 4% (v/v) Coriolus antarcticus BAFC 266 88.52% 60 d 14
kraft pulping process by white-rot fungi Phanerochaete sordida BAFC 2122 60.08%
Pycnoporussanguineus BAFC 2126 82.03%
Steccherinum sp. BAFC 1171 91.86%
Trametes elegans 2127 ND
Decolorization of black liquor effluent Color: 1000 (Pt.Co.) 48% 5d 15
based pulp and paper mills by BOD: 300 ppm 33.3%
white-rot fungus Schizophyllum commune COD: 960 ppm 39.5%
Black liquor degradation by 33 % (w/v) 40-50% 15 d 16
white-rot fungus Trametes elegans

2. Materials and methods

2.1. Wastewater condition

Black liquor wastewater was obtained from bioethanol process in pilot plant Research Center for Chemistry,
Indonesian Institute of Sciences. The waste liquor formed in the pretreatment process consists of an alkaline
aqueous solution that contains organic and inorganic solids and has a distinctive dark coloration. pH wastewater is
13 and the color is black. Sample was diluted with distilled water to obtain the concentration of black liquor was
30,000 ppm.

2.2. Decolorization experiment by coagulation-flocculation

The coagulants used in this work for decolorization of black liquor were Poly Aluminium Chloride (PAC) and
alum (Al2(SO4)3). Concentration of each coagulant was taken as 0.1-2%. The method was slight modified method
from Irfan et al. (2013). During investigation, 300 ml of effluent sample was taken in a glass beaker. The sample
was strongly mixed for one minute by using a jar test at 200 rpm and thereafter slowly mixed for two minute at 50
rpm. The effluent sample was left steady for 30 min for sedimentation. pH before and after decolorization and pH
without black liquor were measured by using pH meter. The filtrate was taken for measurement of decolorization. In
this experiment, the combination of PAC-alumin different ratio was conducted.

2.3. Decolorization experiment by white-rot fungi

2.3.1. Decolorization on agar medium

The white-rot fungiTrametes versicolor U80, T. versicolor U97, and T. versicolor YS001 were used in this study.
They were maintained on malt extract agar (MEA) medium at 25°C for 7 days in a stationary culture [17]. An agar
plug of fungi on maintained agar medium in Petri dish with diameter 9 cm was tested for growth on AG medium
containing 30,000 ppm of black liquor [14]. In the first layer, AG medium was made which consist of agar 15 g L-1,
glucose 10 g L-1, KH2PO4 0.5 g L-1, K2HPO4 0.6 g L-1, CuSO4 0.4 mg L-1, MnCl2 0.09 mg L-1, Na2MoO4 0.02 mg L-
1
, FeCl3 1 mg L-1, ZnCl2 3.5 mg L-1, and thiamine hydrochloride 0.1 mg L-1 at pH 4.5. Agar 15 g L-1 mixed with
black liquor 30,000 ppm was added on the second layer. A control plate with no black liquor added was also
inoculated with each strain. The inoculated plates were incubated at 25°C for 7 d and the level of growth was
 Ajeng Arum Sari et al. / Energy Procedia 68 (2015) 254 – 262 257

assessed by measuring radial extension of mycelium. The growth percentage was calculated by measuring the
growth disc diameter of each fungus, according the formula (1):

§ Ic  Is ·
% growth = ¨¨ ¸¸ x100%
© Ic ¹ (1)

where Øs is fungal diameter in treated sample with black liquor (cm), Øc is fungal diameter in control without black
liquor (cm).

2.3.2. Decolorization in liquid medium

Three fungi were used for further experiments in liquid medium. Three agar plugs of each fungus, punched from
isolated MEA, were inoculated in 100 mL of AG liquid medium (without agar) in 300-mL Erlenmeyer flasks.
Inoculated flasks were pre-incubated for 7 daysunder static conditions at 25°C.After pre-incubation, each inoculated
flask was supplemented with 30,000 ppm black liquor and then incubated for 7, 15, 21, and 30 days. After
incubation, samples were filtrated to get supernatant that measured in the next experiment. Different ratio of
combinated PAC-alumwas also used in this study.

2.4. Decolorization measurement

All samples were diluted for 10 times. The change in absorbance of black liquor was determined at 365 nm with
a UV-Vis spectrophotometer (Optizen 2120 UV). AG medium only containing black liquor was used as a control.
The percentage of decolorization was calculated as folows:

§ Cc  Cs ·
Decolorization (%) = ¨ ¸ x100%
© Cc ¹ (2)

where Cc is the initial dye concentration (ppm) and Cs is the final dye concentration (ppm).
All experimental set up si explained in Fig. 1.

Fig. 1. Flow chart for experiment.

258 Ajeng Arum Sari et al. / Energy Procedia 68 (2015) 254 – 262

3. Results and discussion

3.1. Decolorization by using coagulation-flocculation

Poly aluminum chloride (PAC) and alum were used as coagulant to decolorize black liquor wastewater. PAC is a
pre-polymerized Al(III) chemical, containing a range of hydrolysis and polymeric species. Compare with
conventional aluminum chloride, PAC may slow the rate of hydroxide precipitation upon dilution, can be
maintained for a longer time, and the more strongly adsorb at surfaces [20]. Table 2 shows pH of coagulants
solution before adding black liquor wastewater. All results showed that pH coagulants worked in acid condition.
This data is useful to know the effect of pH concentration for decolorization of black liquor wastewater since pH of
wastewater is approximately 12.

Table 2. The initial pH of coagulants.

Coagulant Concentration (%) pH
PAC 0.1 4.28
0.5 4.03
1 3.92
2 3.82
Alum 0.1 3.93
0.5 3.70
1 3.62
2 3.51

Coagulation–flocculation is treatment that commonly using cationic inorganic metal salts for coagulants[21].
Most of the suspendedparticles in wastewater carry negative charge in aqueousmedium so that after addition of
inorganic coagulant, metal saltswill hydrolyse rapidly in wastewater toform cationic species, which are adsorbed by
negativelycharged colloidal particles, resulting in simultaneous surfacecharge reduction and formation of micro-
flocs [22]. Fig. 2 shows the effect of concentration of PAC and pH for decolorization of black liquor wastewater.

100 Decolorization (%) pH 14

12
80
10
Decolorization (%)

60 8
pH

40 6
4
20
2
0 0
0.1% 0.5% 1,00% 2,00%
PAC concentration (v/v)

Fig. 2. Effect of PAC and pH for decolorization of black liquor wastewater.

 Ajeng Arum Sari et al. / Energy Procedia 68 (2015) 254 – 262 259

The determination of PAC variation is needed to obtain the maximum decolorization. This figure shows that
black liquor was decolorized in the range 38-98% where the highest decolorization was obtained in PAC
concentration 1% and at final pH 4.05. The greater PAC concentration was added, the higher decolorization was
obtained. However, 2% PAC was the saturated concentration for coagulation-flocculation. On the other hand,
adding coagulant in line with decreasing pH was may caused by increasing amount of chloride. These observations
showed that this decolorization was highly dependent with pH of PAC. Irfan et al. (2013) reported that PAC is
excellent for color reduction. Sludge obtained from decolorization by using PAC was brownish.
Because of the initial and final pH were below 4.5, it was assumed that the dominant compounds in black liquor
wastewater were carboxylic and phenolic groups which react with metal cations of PAC. Adsorption of aluminium
cation to anion of black liquor functional groups was occured. At low pH, anionic organic molecules interacted with
metal cations and form insoluble metal complexes which interact with one another and form large agregations
precipitating in the forms of flocs [9, 23]. Furthermore, Fig. 3 shows the effect of concentration of alum and pH for
decolorization of black liquor wastewater. Unfortunately, black liquor was only decolorized in range of 0-63%.

100 14

Decolorization (%) 12
80
Decolorization (%)

pH 10
60 8

pH
40 6

4
20
2

0 0
0.1% 0.5% 1,00% 2,00%
Alum concentration

Fig. 3. Effect of alum and pH for decolorization of black liquor wastewater.

The highest decolorization was obtained at pH 3.79 with concentration of alum 2%. Same with PAC,
decolorization by using alum was better at low pH. Optimum coagulation occurs when pH wastewater is between 6
and 8. The higher decolorization by using PAC than by using alum was may caused the addition of preformed
polymers in PAC affect on longer reaction time and strongly adsorbtion on the surface [20]. Alum is a non-
polymeric inorganic coagulants with low molecular weight and difficult to be hydrolyzed. As a result, low colloid
destabilization and flocculation process were not optimal so that the lower decolorization was obtained. By using
coagulation-flocculation process, particles collide to form macro particles called as flocs. Usually coagulants form
hydroxide when dissolved in water where they have amorphous structure as well as large surface area and possess
positive charge [9].
A combination of PAC and alum was used for further experiment to know the effectively this combination for
decolorization of black liquor wastewater. The concentration of coagulants (PAC-alum) for treatment was kept
uniform as 1%. This value was chosen because it was the optimum concentration for combination of PAC-alum with
the decolorization as 95.3% (data was not shown).
A combination of PAC and alum in all ratio showed similarity decolorization. In this system, alum played a role
in coagulation process for the removal of high-molecular-weight fraction. On the other hand, PAC played a role in
adsorption process for the removal of low-molecular-weight fraction[24]. PAC was faster to form floc because
aluminate and polymer chains from polyelectrolyte has bind colloidal so floc became denser clumps. Furthermore,
the addition of alum into this reaction will increase molecular weight, thus increasing the decolorization. However,
260 Ajeng Arum Sari et al. / Energy Procedia 68 (2015) 254 – 262

in the field application, the combination alum and PAC in ratio 3:1 can be a good option for treatment in economical
cost due to low price of alum especially in Indonesia. However, decolorization by using coagulation-flocculation
involves high consumption of coagulants and the production of large amounts of sludge.

3.2. Decolorization by using white-rot fungi

Trametes versicolor U80 andT. versicolor U97 were obtained from laboratory of Plant Chemistry, Faculty of
Agriculture, Ehime University, Japan and T. versicolor YS001 was obtained from Research Center for Chemistry,
Indonesian Institute of Sciences. They were chosen in this experiment because they have ability to decolorize RBBR
on agar medium by using secretion of ligninolytic enzymes [25, 26]. Fungi having ability to decolorize RBBR will
be able to degrade several xenobiotic compounds including black liquor. Both agar medium contaminated black
liquor and without contaminated black liquor were used in this experiment. Three fungi tested in black liquor agar
medium were able to growth at some degree, around 0.9-1.1 cmd-1. The results showed that Trametes versicolor
U97 is able to grow to 70% on an agar medium covered with black liquor waste water on the seventh day. Compare
with control, the growth on black liquor medium was slower means that black liquor was little toxic for fungi (Fig.
4). However the ability of fungi to grow shows that fungi may able to degrade black liquor.

10 U80 No BL
9
Diameter growth (cm/d)

8 U97 No BL
7
6 YS001 No
BL
5 U80
4
3
2
1
0
1 2 3 4 5 6 7
Days

Fig. 4. Diameter growth of white-rot fungi on agar medium.

The decrease in the absorbance of black liquor after decolorization with fungi has been considered as a criterion
for the extent of lignin biodegradation. In liquid medium, black liquor was decolorized above 50% in 15 days after
the black liquor addition. The most degraded fungus was T. versicolor U97. At the end of incubation period, T.
versicolor U97 had decolorized 89% (Fig. 5). The removal of the major visible light absorbance areas observed after
incubation time suggested a degradation of all the chromogenic molecules contained in the black liquor [14]. T.
versicolor U97 cultures grown in liquid medium turned brown on the 10th day means the production of the enzymes
of the lignin degradation system was occured.
 Ajeng Arum Sari et al. / Energy Procedia 68 (2015) 254 – 262 261

100

80

Decolorization (%) 60

40
U80 U97 YS001
20

0
7 15 21 30
Days

Fig. 5. Decolorization of black liquor in liquid medium by several white-rot fungi.

Previous studies showed that T. versicolor U97 was able to degrade RBBR and DDT [17, 25]. However, compare
with chemical treatment such as coagulation-flocculation, this method was not so effective. Biological treatment
method such as decolorization by white-rot fungi (WRF) is not much impressive in reducing colour but reduction in
BOD and COD has been found to be much satisfactory [27].Furthermore, this system needs longer time than
coagulation-flocculation. The advantages of the biological treatment are no sludge and less toxic because enzyme
activity produced by fungi can breakdown the structure of black liquor. Unfortunately, very limited experience is
available on the possibility of direct degradation of highly contaminant black liquor by WRF.

4. Conclusion

It was concluded that black liquor wastewater with initial concentration 30,000 ppm can be decolorized by using
two methods, coagulation-flocculation and decolorization by WRF. The optimum condition to obtain 96%
decolorization was using combination of alum-PAC with concentration 1%, ratio 3:1, and total retention time 33
minutes. On the other hand, T. versicolor U97 decolorized 89% black liquor in liquid medium at 30 d incubation
time. Coagulation-flocculation can be used as primary treatment and decolorization by WRF is used as secondary
treatment to reduce usage of chemicals and toxicity of black liquor.The presented results open the possibility for the
use of combined physco-chemical and biological in the treatment of blackliquor wastewater from bioethanol process.
However, in the field application, utilization of coagulant sludge still needs further consideration.

Acknowledgements

The authors are grateful to Hardianti Ramadhanti Lukman, University of Halu Oleo forhelping the experiment.
This research was supported by National Priority from Indonesian Government of Fiscal Year 2014.

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