Journal Pre-proofs

Valorization of Palm Biomass Wastes for Biodiesel Feedstock and Clean Solid
Biofuel through Non-sterile Repeated Solid-State Fermentation

Rawitsara Intasit, Benjamas Cheirsilp, Yasmi Louhasakul, Piyarat Boonsawang,

Sumate Chaiprapat, Jarucha Yeesang

PII: S0960-8524(19)31781-X
DOI: https://doi.org/10.1016/j.biortech.2019.122551
Reference: BITE 122551

To appear in: Bioresource Technology

Received Date: 7 October 2019

Revised Date: 29 November 2019
Accepted Date: 30 November 2019

Please cite this article as: Intasit, R., Cheirsilp, B., Louhasakul, Y., Boonsawang, P., Chaiprapat, S., Yeesang, J.,
Valorization of Palm Biomass Wastes for Biodiesel Feedstock and Clean Solid Biofuel through Non-sterile Repeated
Solid-State Fermentation, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122551

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Valorization of Palm Biomass Wastes for Biodiesel Feedstock and Clean

Solid Biofuel through Non-sterile Repeated Solid-State Fermentation

Rawitsara Intasita, Benjamas Cheirsilp,a,*, Yasmi Louhasakulb, Piyarat Boonsawanga, Sumate

Chaiprapatc and Jarucha Yeesangd

aBiotechnology for Bioresource Utilization Laboratory,

Department of Industrial Biotechnology, Faculty of Agro-Industry,

Prince of Songkla University, Hat Yai, Songkhla, 90112 Thailand

bBiology Program, Faculty of Science Technology and Agriculture, Yala Rajabhat

University, Sateng, Muang, Yala, 95000 Thailand

cDepartment of Civil and Environmental Engineering, Faculty of Engineering, Prince of

Songkla University, Hat Yai, Songkhla 90110, Thailand

dFaculty of Science and Technology, Nakhon Pathom Rajabhat University, Nakhon Pathom

73000, Thailand

*Corresponding author: Prof. Dr. Benjamas Cheirsilp

Email: benjamas.che@psu.ac.th
 2

Abstract

Palm biomass wastes are currently considered as promising solid biofuels. However,

their high potassium content leads to formation of slag in combustion chambers and causes

frequent power-plant shutdowns for maintenance. Therefore, this study aimed to develop a

low-cost practical biological pretreatment for these wastes. Oleaginous fungi Aspergillus

tubingensis TSIP9, which originates from palm wastes, was used to pretreat biomass wastes

and simultaneously produce oils through non-sterile solid state fermentation (SoSF). The

operating conditions were optimized through response surface methodology. The fungi could

grow and produce oils with good biodiesel fuel properties. After SoSF, potassium content in

biomass wastes was reduced by 90% and cellulose content increased to >57%, making it

suitable as clean solid biofuel. Repeated-SoSF with 90% substrate replacement was highly

effective in continuously pretreating biomass wastes and producing fungal oils. This study

demonstrates the cost-effective and environmentally friendly process for production of clean

renewable energy through zero-waste strategy.

Keywords: Biodiesel; oleaginous fungi; palm biomass wastes; solid biofuel; solid state

fermentation
 3

Introduction

Oil palm is the most important oil crop in Thailand. The area devoted to oil palm

plantations is about 882,389 ha, mainly in southern Thailand which accounts for 767,677 ha

or accounting for 87 % of the total area (Nutongkaew et al., 2019). In general, 100 tons of

fresh palm fruit bunches generates about 50 tons of biomass wastes, especially empty fruit

bunches (EFB) (Tan et al., 2018). EFB are currently considered as a potential solid biofuel.

However, the high content of potassium in EFB (>10,000 mg/kg-dried EFB) leads to the

formation of a potassium crust and slag with a low melting point on the surface of tubes and

the grate floor of combustion chamber. The accumulation of this potassium crust and slag

hinders heat transfer and results in low thermal efficiency. This causes power plants to be

frequently shut down for maintenance (Theis et al., 2006). Therefore, the potassium content

is used as an indicator of the potential ash fusion or ash deposition of a solid biofuel.

A number of chemical and hydrothermal pretreatments have been applied to reduce

the potassium content in biomass prior to its use as a solid biofuel in order to improve its

properties as clean solid biofuel (Novianti et al., 2014; Nurdiawati et al., 2015; Tan et al.,

2018). However, chemical pretreatment entails both handling and environmental problems

and hydrothermal pretreatment can lead to the energy input being higher than the energy

output. As an alternative, biological pretreatment of biomass prior to its combustion as a solid

biofuel not only improves the fuel quality but also aids the production of value-added

products in order to overcome the economic issues associated with other forms of

pretreatment.

Oleaginous fungi capable of lignocellulolytic activity are suitable for the

bioconversion of lignocellulosic biomass into fungal oils (Cheirsilp and Kitcha, 2015). They

may also potentially reduce inorganic materials in the biomass. The fungi are currently
 4

cultivated using either submerged fermentation (SmF) or solid-state fermentation (SoSF)

(Singhania et al., 2010; Farinas et al., 2011). In SoSF, the fungi mostly grow on moist solid

substrates in direct contact with the oxygen in the air (Pérez-Rodríguez et al., 2014). SoSF

has been widely applied for the cultivation of fungi due to its economical and practical

advantages over SmF. These advantages include lower capital investment, lower plant-

operation costs, the simplicity of the cultivation system, higher product concentration, easier

product recovery, and lower wastewater output.

This study aimed to pretreat abundant lignocellulosic palm wastes in the form of EFB

and simultaneously produce fungal oils using the oleaginous fungus Aspergillus tubingensis

TSIP9. The operating parameters for practical non-sterile SoSF were optimized through

response surface methodology (RSM) and the repeated- SoSF strategy was attempted to

improve the efficiency of the process and its economic performance. The fuel properties of

the fungal oils as biodiesel feedstocks and fungal-treated EFB as a clean solid biofuel were

evaluated. Finally, a preliminary mass balance of the consolidated process was performed.

Materials and methods

Microorganism

Aspergillus tubingensis TSIP9 is an oleaginous fungus originating from palm biomass

wastes. A stock of the fungus was obtained from Biotechnology for the Bioresource

Utilization Laboratory, Faculty of Agro-Industry, Prince of Songkla University, Thailand.

The fungal strain was maintained on sterile potato dextrose agar (PDA) slants at 30±2 °C.

Fungal spore suspension prepared with 0.1 % Tween 80 was inoculated in 50 mL potato

dextrose broth (PDB) and incubated for 24 h at 150 rpm and 30±2 °C before use as seed

culture (Cheirsilp and Kitcha, 2015).

 5

EFB preparation

EFB and effluent from an anaerobic digester (AD) were collected from a local palm

oil mill, in southern Thailand. The collected EFB were sun-dried, shredded into 2–5 cm

pieces before use. The EFB contained 45.76±7.96 % cellulose, 29.48±4.41 % hemicellulose

and 19.23±2.53 % lignin. The AD effluent had a chemical oxygen demand (COD) of

1,618±30 mg/L, total nitrogen of 62.2±1.9 mg/L and pH 6.24±0.83.

Process optimization in solid state bioreactor

The SoSF was performed in a tank bioreactor with a volume of 5 L, a 15 cm inner

diameter and a height of 30 cm; it was equipped with a water spray system. A spore

suspension of the fungus was inoculated on 100 g of EFB at 108 spores/g-EFB. The AD

effluent was sprayed on EFB as nutrient source and moisturizing agent for the fungi during

SoSF. The substrate layer height was 3-4 cm. The initial moisture content was in the range of

65-70%. It should be noted that after AD effluent spraying, more than 67% of liquid was

absorbed by the EFB. The experimental design was based on a Box Benhken Design (BBD)

and a quadratic model was employed to evaluate the individual and combined effects of AD

effluent volume (50, 125 and 200 %v/w) and interval time of feeding (1, 2 and 3 days). The

fermented solid and liquid were adequately mixed every day. The cultures were incubated at

room temperature (30±2oC) for 10 days. The fermented solid and liquid were recovered. The

enzyme activities in the fermented liquid were analyzed. The fungal biomass and oils were

extracted from the fermented solid. The fungal oils and fungal-treated EFB were then

evaluated for their properties as biodiesel and solid biofuel, respectively.

The repeated-SoSF was performed using the optimal AD effluent volume and interval

feeding time from the above experiments. The cultures were incubated at room temperature
 6

(30±2oC) for 10 days. The fermented solid were replaced with fresh EFB at 50 % and 90 %

based on the dry weight of initial EFB. The remaining fermented solid, fresh EFB and

fermented liquid were adequately mixed to initialize the next batch of SoSF. The SoSF was

repeated for four cycles. The fungal oil yields and the enzyme activities in the fermented

liquid of each cycle were analyzed. The recovered oils were calculated by multiplying the

fungal oil yields with the recovered fermented EFB from each cycle.

Prospective biodiesel fuel properties of fungal oils

The prospective biodiesel fuel properties of the fungal oils were calculated using

empirical equations based on the fatty acid compositions (Jham et al., 1982). These include

cetane number (CN), iodine value (IV), saponification value (SV), oxidation stability (OS),

higher heating value (HHV) and cold filter plugging point (CFP). Following GC analysis, the

molecular structures of the fatty acid methyl esters (FAME) of the fungal oils were used to

estimate the important fuel properties of biodiesel. The SV, IV and OS were estimated using

the following Eqs. (1-3):

SV = ∑ (560×P)/Mw (1)

IV = ∑ (254×D×P)/Mw (2)

OS = [117.9295/( %wt C18:2+ %wt C18:3)]+2.8905 (3)

where P and Mw are the percentage and the molecular weight of each FAME component,

respectively and D is the number of double bonds in each FAME component.

Eqs. (4) and (5) were used to estimate the CN and HHV properties:

CN = 46.3 + (5458/SV) – (0.255×IV) (4)

HHV = 49.43– (0.041×SV) – (0.015×IV) (5)

The long-chain saturation factor (LCF) and CFP were calculated using Eqs. (6) and (7):
 7

LCF = (0.1×C16:0 + 0.5×C18:0 + 1×C20:0 + 1.5×C22:0 + 2×C24:0)

(6)

CFP = (3.1417×LCS) – 16.477 (7)

Analytical methods

Fungal biomass separation and oil extraction

The fungal biomass were recovered from the treated EFB using 0.1 % Tween 80 at a

solid:liquid ratio of 1:10 and vortex for 5 min. The extraction process was repeated twice.

This method was applied according to the properties of Tween 80 as spore extraction solution

(Da Silva et al., 2011) and dispersing agent for fungal aggregates (Lee et al., 2017). The

recovered fungal biomass were dried at 60oC to constant weight. The fungal oils were

extracted from the fungal biomass using chloroform: methanol method (Folch et al., 1957).

The mixture was sonicated for 30 min before filtering to obtain the solvent phase containing

the fungal oils. The extraction step was repeated twice to ensure complete extraction. The

solvents were separated from the fungal oils using an evaporator. The fungal oil yield was

calculated as the amount of oils in milligrams per gram of dried EFB.

Cellulase activity

The cellulase activity of the fermented liquid was determined according to the method

of Mandels and Weber (1969). Enzyme solution 0.5 mL was added with 0.5 mL 1 %

carboxymethylcellulose (CMC) in 0.05 M citrate buffer (pH 5.0). The reaction mixture was

incubated at 50 °C for 10 min. The reducing sugars released were quantified using glucose as

the standard. One unit of cellulase (IU) was defined as the amount of enzyme releasing 1

μmol of glucose per min under the assay conditions

Xylanase activity
 8

The xylanase activity of the fermented liquid was determined according to the method

of Warzywoda et al. (1983). Enzyme solution 0.5 mL was added with 0.5 mL 1 % xylan

dissolved in 0.05 M citrate buffer (pH 5.0). The reaction mixture was incubated at 50 °C for

10 min. The reducing sugars released were quantified using xylose as the standard. One unit

of xylanase is defined as the amount of enzyme releasing 1 μmol of xylose equivalents per

minute under the assay conditions.

Chemical composition of EFB

The chemical composition of EFB was analyzed according to Goering and Van Soest

(cited in Charnnok et al., 2018). Firstly, neutral detergent fiber (NDF), acid detergent fiber

(ADF), and acid detergent lignin (ADL) were analyzed and used to calculate the contents of

hemicellulose, cellulose, and lignin as shown in Eqs. (8), (9), and (10), respectively.

Hemicellulose (%) = %NDF− %ADF (8)

Cellulose (%) = %ADF− %ADL (9)

Lignin (%) = %ADL− %Ash (10)

The potassium in EFB was analyzed using the Inductively Coupled Plasma Optical

Emission Spectrometry (ICP-OES, Optima 8000, Perkin Elmer, USA).

The statistical significance of the results was evaluated by one-way analysis of

variance (ANOVA) and Duncan’s multiple range tests (P value < 0.05) using the SPSS

version 22.0 software.

Results and Discussion

Non-sterile solid state fermentation of EFB by oleaginous fungi

The oleaginous fungus, A. tubingensis TSIP9, was used to biologically pretreat non-

sterile EFB and simultaneously produce fungal oils through SoSF. The AD effluent was used

as a low-cost nutrient source and moisture adjusting agent. The moisture content of the
 9

substrate is one of the most important parameters affecting the efficiency of SoSF. With too

low moisture content, the solubility of the nutrients is limited and this hinders the effective

nutrient uptake by the fungi. On the other hand, with too high moisture level the void spaces

in the solids are filled with water, resulting in oxygen limitation (Yoon et al., 2014).

The effects of two important operating parameters, volume of AD effluent and

interval time of feeding were determined through RSM. These two operating parameters were

optimized to adequately supply both nutrients and water content. The operating conditions

expressed in terms of uncoded values and the resulting experimental data from SoSF are

shown in Table 1. Multiple regression analysis was performed to evaluate the individual and

interaction effects among independent and dependent variables. Consequently, quadratic

models that well explain the variations of fungal oil and enzyme productions as a function of

volume of AD effluent and interval time of feeding, were established. The second order

regression equations for fungal oil, cellulase activity and xylanase activity as volume of AD

effluent (VE) and interval time of feeding (IT) are given as follows:

Fungal oil (mg/g-EFB) = -24.925+2.869* VE -65.735* IT-0.00984*

VE 2+16.112* IT 2+0.04203* VE* IT (11)

Cellulase (U/g-EFB) = 13.456+0.07859* VE -10.088* IT+0.00009* VE2

+2.019 * IT2-0.0163* VE*IT (12)

Xylanase (U/g-EFB) = 37.932+0.9629* VE-32.268* IT+3.439E-003*

VE2+11.217* IT2-0.519* VE * IT (13)

The F-test for ANOVA was used to evaluate the statistical significance of the models’

equations. The P values for the fungal oil and enzyme production are lower than 0.05

indicating the significance of the models and the models can be used to visualize the effects

of operating parameters on the responses.

 10

A series of experiments showed that the production of fungal oils increased with

increasing the volume of AD effluent. While, interval time did not much affect the fungal oil

production. The optimal values of volume of AD effluent and interval time of feeding were

determined. The maximum fungal oil production was 169±3.2 mg/g-EFB, achieved with the

use of AD effluent at 125 % v/w and fed every 3 days (which resulted in an average moisture

content of 78.9-81.4 %). Meanwhile, the cellulase and xylanase activities increased with

increasing volume of AD effluent up to 200 % v/w and the suitable interval time of feeding

was everyday (in that case the average moisture content was as high as 96.1-96.3 %). The

highest cellulase and xylanase activity were 22.2±2.9 U/g-EFB and 247.5±4.5 U/g-EFB,

respectively. Since the moisture content is the key factor affecting the mass transfer of

nutrients between solid and liquid phases in SoSF, the higher amount of water facilitated

more nutrient solubility and enhanced enzyme production (Lonsane et al., 1992). In addition,

higher moisture content also increases the degree of swelling capacity of substrate and

enhances the fungal growth (Pérez-Rodríguez et al., 2014). However, it was obvious that

lower nutrient supplementation and lower moisture content was favored for fungal oil

production which generally relates to the nutrient imbalance (high C/N ratio) and the

availability of oxygen. The most suitable moisture content also depends on the structure of

the solid substrate. The specific surface area and porosity of the solid particles govern the

efficiency of air diffusion and also the water-holding capacity (Freitas et al., 2013).

Several previous studies have reported the use of oleaginous yeasts for microbial oil

production (Liu et al. 2012; Xue et al. 2015; Wang et al. 2016). However, those studies

performed either a two-step process of separate hydrolysis and fermentation (SHF) or a

simultaneous saccharification and fermentation (SSF), which requires costly commercial

enzymes. Nevertheless, the oil yields were relatively low, in the range of 27-39 mg/g-dried

substrate. Recently, Dai et al. (2019) found that the oleaginous yeast Rhodosporidium
 11

toruloides AS 2.1389 was able to produce yeast oils with a higher yield of 71-80 mg/g-dried

substrate. Peng and Chen (2007) reported the direct conversion of lignocellulosic biomass

into fungal oils using the endophytic fungus Microsphaeropsis sp. through SoSF. This fungus

produced fungal oil directly from the biomass without separated hydrolysis step. However,

the fungal oil yields were only 19-42 mg/g-dried substrate. Later, Lin et al. (2010) studied the

fungal oil production from wheat straw using cellulolytic A. oryzae A-4 through SoSF. They

reported a higher yield of fungal oil at 62.9 mg/g-dried substrate. Qiao et al. (2018) reported

the SoSF of mulberry branches by Mucor circinelloides Q531. This SoSF gave fungal oils at

42.43 mg/g-dried substrate. Our previous studies found that the cellulolytic oleaginous A.

tubingensis TSIP9 was able to produce fungal oils from EFB mixed with palm kernel cake

with a higher yield of 79.9 mg/g-dried substrate. However, all these studies used aseptic

technique which is not practical in industrial scale. This study has shown more practical and

low-cost SoSF using non-sterile EFB. Through this process, not only a much higher yield of

fungal oils was obtained but the solid fuel derived was also cleaner.

Repeated-solid state fermentation of EFB

Repeated-SoSF of EFB was performed in order to increase the effectiveness of the

process and reduce production costs. AD effluent was used to supply the nutrients and

moisture at 125 % v/w every 3 days. The fermented EFB were replaced with fresh EFB every

10 days. The EFB replacement was varied at 50 % and 90 % based on the dry weight of initial

EFB. Each batch of SoSF was incubated at room temperature for four cycles. The fungal oil

yields recovered from each cycle and the enzyme activities detected in the fermented liquid

are shown in Figure 3. It should be noted that the fungal oils and enzymes were able to be

effectively produced during four cycles of repeated-SoSF. This was probably because there

was a sufficient supply of nutrients and the moisture content in the SoSF system was suitable.
 12

The EFB replacement at 50 % gave fungal oil yields during the four cycles of SoSF in the

range of 140-156 mg/g-EFB. The recovered oils were calculated by multiplying the fungal oil

yields with the recovered fermented EFB from each cycle. The recovered oils from each cycle

were in the range of 70-78 g per 1 kg EFB-based SoSF with 0.5 kg of fermented EFB being

recovered from each cycle. The cellulase and xylanase recovered after four cycles were

121±13 and 291±16 U/g-EFB, respectively. With a higher percent replacement of 90 %, the

fungal oil yields were slightly increased to 147-169 mg/g-EFB possibly due to the greater

availability of substrate for the fungi. The recovered oils from each cycle (132-152 g per 1 kg

EFB-based SoSF) were much higher than those at 50 % EFB replacement due to the higher

amount of fermented EFB recovered (0.9 kg of fermented EFB was recovered from each

cycle). The cellulase and xylanase recovered after four cycles were in the range of 210-689

U/g-EFB, respectively. It is therefore reasonable to suggest that the repeated-SoSF process

employed in this study is highly effective for continuously and efficiently producing fungal

oils and cellulolytic enzymes.

Evaluation of fungal oils as biodiesel feedstock

The production of biodiesel, namely fatty acid methyl esters (FAME), from fungal

oils involves hydrolysis of the oils followed by esterification with methanol. Table 2 shows

the FAME profile of fungal oils from A. tubingensis TSIP9. The composition of fungal oils

was mainly long chain fatty acids with C16-C18. These include 60.96±4.2 % palmitic acid

(C16:0), 19.69±0.33 % oleic acid (C18:1), 8.28±1.47 % stearic acid (C18:0), and 4.55±0.34

% linoleic acid (C18:2). The fungal oils differ from palm oils in being quite rich in saturated

fatty acids. Biodiesel quality parameters including cetane number (CN), iodine value (IV),

cold filter plugging point (CFP), oxidative stability (OS) and others, are estimated as shown

in Table 3. These parameters are generally used to evaluate the suitability of the fungal oils as
 13

biodiesel feedstocks. The IV measures the content of unsaturated fatty acids in the biodiesel

sample indicating the possible formation of partially oxidized compounds that will cause

deposits in the injection system of an engine. As shown in Table 3, the IV estimated for

fungal oils derived biodiesel would meet the requirements of the international standards. The

CN value measures the combustion property of the biodiesel which is associated with the

fatty acid compositions. The CN value largely depends on the chain length and the degree of

unsaturated fatty acids. A high CN value indicates a shorter time before ignition. In this

study, the estimated CN value of 64-69 complies with the ASTM D6751 international

standard (a minimum of 47). The CFP value shows the filterability of the biodiesel at low

temperature which depends on the degree of saturated fatty acids. The OS value indicates the

biodiesel resistance to degradation over storage periods. In this study, the OS value estimated

for the fungal oils derived biodiesel was 23-27 h which would comply with the EN14214 and

ASTM D6751 standards which require a minimum 6 h or 3 h, respectively. This result was in

agreement with the high saturation degree of FAME from fungal oils (Table 2) indicating

their high oxidative stability. It can be concluded that most of the parameters estimated for

biodiesel derived from the fungal oils produced in this study would satisfy the international

standards.

Composition of fungal-treated EFB

The compositions of EFB after biological pretreatment by the oleaginous fungi A.

tubingensis TSIP9 were determined as shown in Table 1. In order to describe the effect of

volume of AD effluent and interval time of feeding on chemical composition of fungal-

treated EFB, the multiple regression analysis was performed. The second order regression

equations with the percentages of cellulose, hemicellulose and lignin as dependent variables
 14

and the volume of AD effluent (VE) and interval time of feeding (IT) as the independent

variables are given as follows:

Cellulose (%) = 51.491-0.148 * VE+21.179* IT+0.0004* VE2

-6.3214* IT2+0.0292* VE* IT (14)

Hemicellulose (%) = 33.067+0.266* VB-24.499* IT-0.00095*VB2+6.233 *

IT2-0.0032* VB * IT (15)

Lignin (%) = 6.373-0.0181* VB+6.283* IT- 0.00004* VB2-1.277*

VB20.0083* VB * IT (16)

The statistical significance of the model equations was evaluated by the F-test for

ANOVA. The P values for the three models were lower than 0.05 indicating that the

quadratic models were significant. Eqs. (14) – (16) were then used to visualize the effects of

independent variables on responses. All series of experiments showed that the SoSF by the

fungi was able to reduce lignin content as well as increase cellulose content of the EFB, while

the hemicellulose did not greatly vary. Figure 2 shows the effect of the interactions between

the independent variables on chemical composition of fungal-treated EFB. The highest

cellulose content of 67.3±1.1 % was obtained with the use of AD effluent at 125 %v/w and

the interval feeding time of 2 days. While the lowest amount of lignin (7.01±0.5 %) was

obtained when using AD effluent at 200 %v/w with everyday feeding, which corresponded to

the optimal condition for enzyme production. These results suggest that the oleaginous fungi

A. tubingensis TSIP9 could be used to remove lignin (delignify) and increase cellulose

content of lignocellulosic biomass. The delignification by the fungi involves certain steps of

depolymerization and aromatic ring cleavage by several extracellular enzymes. Firstly, -O-4

linkages of lignin are oxidized to arylglycerol compounds. Secondly, aromatic rings are

cleaved following the -ketoadipate pathway; and finally the cleaved aromatic rings coupled
 15

with -O-4 oxidation leads to the formation of cyclic carbonate structures (Javaid et al.,

2019). It has also been reported that the fungi can degrade lignin via non-enzymatic oxidation

reactions that partially oxidize lignin via aromatic ring demethylation. Many white and

brown-rot fungi have been shown to produce hydrogen peroxide which enters the Fenton

reaction and results in release of -OH. These radicals create a number of cleavages. It should

be also noted that the fungi can cause selective or nonselective delignification. In selective

delignification, lignin is removed without any marked loss of cellulose, and in nonselective

delignification all the major cell wall components are degraded (Priyanga and Kannahi,

2018). The delignification efficiency in this study was as high as 63 % which was much

higher than those reported in previous studies in the range of 25-27% for the fungi T.

versicolor TISTR 3224 (Kamcharoen et al., 2014), Pleurotus floridanus (Ishola et al., 2014)

and Phanerochaete chrysosporium ATCC 32629 (Hamisan et al., 2009).

Evaluation of fungal-treated EFB as solid biofuel

EFB is one of the major lignocellulosic biomass that can be utilized as solid biofuel.

However, the raw EFB contained potassium content as high as 11,164±118 mg/kg

(1.1164±0.0118 %). The high potassium content makes EFB not suitable for combustion due

to the slack formation in the heat exchange surface and grate floor in combustion chamber.

These cause low heat transfer efficiency. After SoSF by the oleaginous fungi, the potassium

content in EFB was reduced to 1,158±25 mg/kg (0.1158±0.0025 %) corresponding to an

approximately 90 % reduction. The analysis of fungal biomass and SoSF effluent indicated

that during SoSF the potassium content in EFB was mainly solubilized into SoSF effluent

(>90%) and partly assimilated by the fungi (<10%). It has been reported that the fungi can

solubilize potassium from solid substrate through the mechanisms of acidolysis, chelation,

exchange reactions, complexolysis and production of organic acid anions mainly citrate,
 16

malate and oxalate (Meena et al., 2014). These results suggest that the SoSF by oleaginous

fungi could be effective biological pretreatment of EFB prior to being used as clean solid

biofuel. Nurdiawati et al. (2015) studied the production of low-potassium solid fuel from

EFB employing a hydrothermal treatment and water washing process. The EFB was

subjected to hydrothermal treatment at the optimal temperature of 180°C with a holding time

of 30 min, followed by washing. Tan et al. (2018) also evaluated hydrothermal treatment for

potassium removal from EFB but using lower temperature of 120oC for 120 min. This

process removed potassium by 90 %. In addition, Charnok et al. (2019) studied hydrothermal

pretreatment with sulfonated bentonite catalyst for potassium removal. The hydrothermal

pretreatment of EFB at 200 °C for 25 min removed potassium by 70 %. Compared to these

physical-chemical pretreatments, the potassium removal by fungal pretreatment investigated

in this study is both more energy efficient and environmentally friendly.

Preliminary mass balance analysis

A preliminary mass balance analysis was carried out for the SoSF of non-sterile EFB

using cellulolytic oleaginous fungi (Figure 4). The major components in the dried EFB were:

cellulose 45.76 %, hemicellulose 29.48 % and lignin 19.23 %. The potassium content in EFB

was 11,164 mg/kg. The SoSF of 1 kg dried EFB requires 0.1 L of fungi inoculum (108

spores/mL) and 1.25 L of AD effluent added three times during 10 days cultivation (3.75 L in

total). Based on the above experimental data, SoSF of 1 kg EFB would yield 169 g fungal

oils and 207 g residual fungal biomass. The SoSF effluent was 2.46 L containing 4,470 U/L

cellulase and 14,227 U/L xylanase which could be further applied in bioconversion of

lignocellulosic biomass into fermentable sugars. Finally, 724 g of fungal-treated EFB would

be obtained. The cellulose and hemicellulose contents in the treated EFB increase up to 57.8

% and 32.6 %, respectively, while the lignin and potassium contents decrease to 9.6 % and
 17

1,158 mg/kg, respectively. This integrated process of biological treatment and solid state

fermentation of non-sterile palm biomass wastes for co-productions of fungal oil and solid

biofuel may greatly increase the competiveness of the palm oil and bioenergy industries.

Conclusions

This study has shown that oleaginous fungi Aspergillus tubingensis TSIP9 could grow

on and pretreat palm biomass wastes through non-sterile solid state fermentation using

anaerobic digester effluent as nutrient and moisturizing sources. The fungi also produced

fungal oils with good biodiesel fuel properties. The fungal-treated biomass wastes contained

much lower amount of potassium and higher content of cellulose suggesting their cleaner

properties as solid fuel. Repeated-solid state fermentation effectively increased the process

performance and contributed to the operating cost reduction. This study may contribute

greatly to the cost-effective and environmental friendly process for bioconversion of biomass

wastes into valuable products.

E-supplementary data for this work can be found in e-version of this paper online.

CRediT author statement

Rawitsara Intasit: Investigation, Data curation, Writing- Original draft preparation. Benjamas

Cheirsilp: Conceptualization, Methodology, Funding acquisition, Writing - review & editing.

Yasmi Louhasakul: Methodology, Writing - review & editing. Piyarat Boonsawang: Writing

- review & editing. Sumate Chaiprapat: Writing - review & editing. Jarucha Yeesang:

Writing - review & editing

 18

Acknowledgements

This research was financially supported by Prince of Songkla University under Grant

No. AGR600494S and partially by Energy Policy and Planning Office (EPPO) (AEPSU-59-

02). The first author was financially supported by the PSU-Ph.D. scholarship, Graduate

School, Prince of Songkla University. The second author was supported by Thailand

Research Fund Grant No. RTA6280014. The authors also would like to acknowledge the

generosity of Chumporn Palm Oil Industry, Thailand for providing palm materials.

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Figure legends

Figure 1 Response surface plots of effects of volume of AD effluent and interval time of

feeding on fungal oil (a), cellulase (b) and xylanase (c) production by oleaginous

fungi.

Figure 2 Response surface plots of effects of volume of AD effluent and interval time of

feeding on cellulose (a), hemicellulose (b) and lignin (c) contents of fungal-treated

EFB.

Figure 3 Fungal oil and enzyme productions by repeated-SoSF with 50 % (a) and 90 % (b)

EFB replacement. Different letters above columns indicate significant differences

between cycles at P < 0.05. Vertical bars represent standard deviations (n = 3).
 23

Figure 4 Preliminary mass balances for non-sterile solid state fermentation (SoSF) of 1 ton

empty fruit bunches (EFB) using cellulolytic oleaginous fungi and anaerobic

digester (AD) effluennt.

Table 1. Operational conditions considered in this study expressed in terms of volume of AD effluent
and interval time of feeding and experimental results

Volume Interval Recovered value added products F

of AD time of Fungal oils Cellulase Xylanase Cellulose
Run effluent feeding (mg/g-EFB) (U/g-EFB) (U/g-EFB) (%)
(%v/w) (days) Actual Predicted Actual Predicted Actual Predicted Actual Predicted
1 50 1 48.80 45.71 8.50 8.74 49.50 47.85 61.20 61.23
2 125 1 129.59 137.03 14.23 14.63 117.67 126.11 57.45 56.56
3 200 1 117.85 113.51 22.22 21.58 247.50 240.71 54.60 55.51
4 50 2 37.26 29.61 4.64 3.89 27.42 22.34 63.01 64.90
5 125 2 118.64 124.08 8.69 8.57 63.11 62.57 61.27 62.42
6 200 2 108.87 103.71 13.29 14.30 133.92 139.12 63.50 63.55
7 50 3 39.48 50.23 2.58 3.09 12.93 19.66 57.81 55.93
8 125 3 168.10 147.85 6.70 6.55 30.16 21.84 55.50 55.64
9 200 3 121.14 130.64 11.41 11.05 58.76 60.35 59.92 58.96
10 125 2 118.70 124.08 9.26 8.57 61.76 62.57 61.24 62.42
11 125 2 125.56 124.08 8.31 8.57 61.75 62.57 67.03 62.42
12 125 2 128.30 124.08 8.50 8.57 64.14 62.57 61.24 62.42

13 125 2 116.40 124.08 8.35 8.57 60.16 62.57 63.26 62.42

Table 2. Fatty acid compositions of fungal oils compared with palm oil

Fatty acid compositions Formula Fungal oils Palm oil*

Lauric acid C12:0 0.33±0.11 0.2

Myristic acid C14:0 1.21±0.02 1.1

Pentadecanoic C15:0 0.25±0.01 nd

 24

Palmitic acid C16:0 60.96±4.2 44.0

Palmitoleic acid C16:1 0.23±0.01 0.1

Heptadecanoic acid C17:0 0.37±0.07 nd

Stearic acid C18:0 8.28±1.47 4.5

Oleic acid C18:1 19.69±0.33 39.2

Linoleic acid C18:2 4.55±0.34 10.1

Linolenic acid C18:3 1.31±0.35 0.4

Arachidic acid C20:0 0.90±0.59 0.1

Behenic acid C22:0 0.42±0.18 nd

Lignoceric acid C24:0 1.50±0.18 nd

Saturated fatty acids - 74.3±0.7 49.9

Unsaturated fatty acids - 25.7±0.7 49.7

* Data from Edem et al. (2002)

nd, not detected.

Table 3. Comparison of the estimated biodiesel properties of fungal oils

Biodiesel property Biodiesel from fungal oils Limits Specification

SV 189-210 ns -

IV 25-29 120, max EN 142514

CN 64-69 47, min ASTM D6751

LCF 14-15 ns -

CFP 27-30 - Thailand’s regulations*

OS 23-27 3, min ASTM D6751

HHV 40-41 Report ASTM D6751

* Standard specification for biodiesel fuel 2009 from Department of Energy Business,

Ministry of Energy, Thailand.

 25

ns, not a specified limit.

SV: Saponification Value, IV: Iodine Value, CN: Cetane number, LCF: Long Chain

Saturated Factor, CFP: Cold Filter Plugging Point, CP: Cloud Point, PP: Pour Point, OS:

Oxidation Stability, HHV: Higher Heating Value.