Ecotoxicology and Environmental Safety 203 (2020) 110991

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Dose-response analysis of toxic effect from palm oil mill effluent (POME)
by-products on biohydrogen producing bacteria – A preliminary study on
microbial density and determination of EC50
Shalini Narayanan Arisht a, Peer Mohamed Abdul a, b, *, Jannatulhawa Jasni a,
Nazlina Haiza Mohd Yasin c, Sheng-Kai Lin d, Shu-Yii Wu d, e, Mohd Sobri Takriff a, b,
Jamaliah Md. Jahim a, b
a
Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia
b
Research Centre for Sustainable Process Technology, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600, UKM, Bangi, Selangor,
Malaysia
c
Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia
d
Department of Chemical Engineering, Feng Chia University, Taichung, 40724, Taiwan
e
Green Energy Development Center, Feng Chia University, Taichung, 40724, Taiwan

A B S T R A C T

The stimulant and toxicity effects of reported organic (acetic acid, propionic acid, butyric acid, formic acid, oil & grease) and inorganic (copper) by-products
presented in palm oil mill effluent on anaerobic bacterial population were examined in this paper. The toxicity test had shown that acetic, propionic and butyric
acids tend to stimulate the bacterial density level (survival rate more than 50%), while formic acid, copper, oil and grease were shown to have suppressed the density
level (survival rate less than 50%). The highest biomass recorded was 1.66 mg/L for the concentration of acetic acid at 216 mg/L and lowest biomass concentration,
0.90 mg/L for copper at 1.40 mg/L. Biohydrogen-producing bacteria have a favourable growth rate around pH 5.5. The comparison of half maximal effective
concentration (EC50) values between two test duration on the effects of organic and inorganic by-products postulate that bacteria had a higher tolerance towards
volatile fatty acids. While acetic, butyric and propionic acids had exhibited higher tolerance EC50 values for bacteria, but the opposite trend was observed for formic
acid, copper and oil & grease.

1. Introduction
It is expected that the global palm oil industry will grow substantially
to meet the growing global demand for edible oil that is widely used in
various industries. By 2035, the palm oil manufacturing sector is fore­
casted to boost its total production upto 25 million tonnes. Palm oil
(Elaeis guineensis) grows largely in Asia, Latin America and Africa.
Indonesia and Malaysia are the world largest producers of palm oil,
followed by Thailand, Colombia and Nigeria. Indonesia and Malaysia
are currently top palm oil exporters, with the highest sustainably
certified palm oil plantation. Global palm oil production in the 2019/
2020 marketing year stood at around 72.27 million metric tonnne, down
from around 74.02 million metric tonne in 2018/2019 (Shahbandeh,
2020). The crude oil palm extraction process is generally involving
thermochemical methods and largely chemical free process. Every tonne
of crude palm oil processed would generate about 3.05 tonne of
wastewater known as palm oil mill effluent (POME) and it is considered
as a significant source of water contamination to the industry (Iwuagwu
and Ugwuanyi, 2014). POME is high strength wastewater with a low pH
(attributable to organic and free fatty acids) resulting through partial
degradation of palm fruits during the oil extraction process, resulting in
high demand for chemical oxygen (COD) and biochemical oxygen (BOD)
between 15,000 and 100,000 mg/L and 10,250–43,750 mg/L, respec­
tively (Elreedy et al., 2016; Maaroff et al., 2019; Zainal et al., 2018).
POME also contains a high degree of nutrients and organic materials that
make it potentially a cheap substrate substitute for biological processes.
An efficient handling system is therefore so critical and has been a major
challenge in tackling the abundant POME (Nasution et al., 2018).
In traditional linear economic model at palm oil mill, POME was
often overlooked as wastewater and frequently subjected to open
ponding treatment system prior to discharge. Several alternative treat­
ment methods have been developed recently for POME before being

* Corresponding author. Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600,
Bangi, Selangor, Malaysia.
E-mail address: peer@ukm.edu.my (P.M. Abdul).

https://doi.org/10.1016/j.ecoenv.2020.110991
Received 16 April 2020; Received in revised form 19 June 2020; Accepted 5 July 2020
Available online 21 July 2020
0147-6513/© 2020 Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S.N. Arisht et al.
discharged from a mill, including coagulation-flocculation (Zinatizadeh
et al., 2007), anaerobic digestion (Mahmod et al., 2020) and phycor­
emediation (Hariz and Takriff, 2017). Anaerobic digestion of POME
using indigenous bacteria is relatively more advantageous among
different sequential treatment methods. This is because anaerobic
digestion using bacteria comes with its higher treatment effectiveness
coupled with the biogas production for energy recovery (Abdeshahian
et al., 2016; Jiang et al., 2019). POME is a good biomass resource and
would play a central role in the advancement of the circular economy in
the oil palm industry. POME possibly required monitoring before can be
fed into the digester as a feedstock because of possible by-products in­
hibition as the bacteria are highly susceptible to the biotic and abiotic
components of the digester. Additionally, it is also important to retain
high bacterial biomass concentration in a given process to have high
treatment capacity (Faudzi et al., 2019; Rajeshwari et al., 2000).
POME was reported to include variety of soluble and insoluble
components ranging from acetate, propionate, butyrate, valerate (Azn­
ury et al., 2012; Choong et al., 2017; Hong et al., 2009; Loh et al., 2017),
oleic acid, glycerol (Fazli and Hertadi, 2019), macronutrients; nitrogen,
phosphorus, potassium, calcium (Madaki and Seng, 2013), and micro­
nutrient such as trace elements (magnesium, iron, manganese, lead)
(Oviasogie and Aghimien, 2002). Hence, it is important to be familiar
with the probable mill processing that could form the contaminant(s)
and the appropriate operational corrective action(s) that should be
taken in minimizing or mitigating the impact of the toxicants on
wastewater treatment plants (Strotmann and Pagga, 1996). Acetic,
butyric, propionic and formic acids are volatile fatty acids and listed as a
volatile organic compound (VOC) that can negatively impact the envi­
ronment (Page et al., 2014) and also identified as anaerobic digestion’s
intermediate metabolites (Jasni et al., 2020). Copper detected in POME
known to have toxic effects on the living organism which hinders and
totally eradicates the growth of bacteria in the water bodies and there­
fore had long been controlled in the discharge system. Oviasogie and
Aghimien (2002) confirmed in their work that the proper use and safe
disposal of POME in the soil environment would lead to improved soil
fertility and contributes to the overall environmental sustainability.
Another results showed that soil enrichment with phosphorus, nitrogen,
calcium, magnesium, sodium and potassium following the application of
the POME whereas copper was said to remain predominantly in its
forms, carcinogenic in soil (Madaki and Seng, 2013). Whereas, excessive
amounts of oil and grease in wastewater hinders sedimentation, which
can result in the loss of biomass and affect the overall efficiency of
treatment stations (Nzila et al., 2016).
A number of microbial toxicity bioassays have been employed to
assess trace metals and organic toxicants using endpoints such as cell
division, inhibition of growth, respiration or other enzyme activity with
varying degrees of success (Stauber and Davies, 2000). Alternatively,
the level of toxicity effectiveness can be expressed as EC50, half the
toxicant’s maximum effective concentration inhibiting 50% of bacterial
growth viability. It is measured by exposing the selected microbial to
various concentration of test substances over a specified period of time,
such as after 24, 48, 72 or 96 h (Anderson and Abdelghani, 1980; Arisht
et al., 2019). By using the biological experiments, a direct and appro­
priate toxicity measure complementing the physico-chemical waste­
water quality measures presented and are used in many countries as a
baseline data for environmental hazard and risk assessment of chem­
icals. Due to the highly dynamic structures of the bacteria, it is however
difficult to determine the actual results of the inhibition test. The results
may vary according to the biotic (type and nature of test organism used),
and abiotic factors of the culture condition (De Filippis and Filippis,
1994). Treatment plants could be subjected to a wide range of organic
and inorganic toxic compounds that may inhibit organic matter
removal. Therefore, due to the existence of a whole range of chemicals, a
selective chemical analysis may be insufficient to be used solely as a
control instrument. In general, while only a fraction of inhibition can be
explained by the compounds identified through a chemical analysis,
2
Ecotoxicology and Environmental Safety 203 (2020) 110991
direct biotoxicity measurements had proven to be the more preferred
method in capturing the wastewater’s complexity (Eilersen et al., 2004).
The objective of this present work is to assess the toxicity effect of
selected organic (oil & grease, acetic, butyric, propionic and formic
acids) and inorganic (copper) constituents found in POME on test or­
ganisms (anaerobic bacterial biomass). Half maximal effective concen­
tration (EC50) for biomass allows the estimation of the complete toxic
effects on bacterial growth concentration. The effect of toxicants on the
pH properties of bacterial growth parameters has also been investigated.
These results serve as a proposed method to determine potential toxic
concentration levels of industrial waste for discharge treatment opera­
tions prior to the implementation of biological treatments.
2. Materials and methods
2.1. Test microorganism species
The anaerobic mixed culture seed sludge was obtained from the
POME wastewater treatment plant at Sime Darby palm oil mill located in
Bestari Jaya, Kuala Selangor, Malaysia. Seed sludge was revived and
undesirable methanogenic bacteria was removed by heating at
approximately 90 � 5 � C for 1 h. The populations were isolated and
identified as spore-forming acidogenic biohydrogen producing bacteria,
Clostridium sp., Lactobacillus sp. and Thermoanaerobacterium sp. (as
described previously) (Maaroff et al., 2019; Roslan et al., 2018a, 2018b).
2.2. Preparation of test medium
The test medium is a complete synthetic culture medium for the test
organism (biohydrogen producing bacteria) to yield a toxic response
when being exposed to the test substance (butyric acid, acetic acid,
propionic acid, oil & grease, copper and formic acid). The test substance
was dissolved in the test medium. Test solutions of certain concentration
levels were prepared by mixing a stock solution of the test substance
with the Endo medium and inoculum culture. The Endo medium that
comprised of (per litre of water) glucose, 20000 mg; urea, 4400 mg;
NaHCO3, 6720 mg; CaCl2, 100 mg; K2HPO4, 125 mg; FeSO4.7H2O, 25
mg; CuSO4.7H2O, 5 mg; CoCl2.6H2O, 0.125 mg; MgCl2. 6H2O 100 mg;
MnSO4.4H2O, 15 mg (Endo et al., 1982) was used as the test medium. All
of the chemical components from the test medium was diluted in
Milli-Ro ultrapure water (Kemflo AICRO-Q Water Filters, USA) and that
the medium presents a neutral pH value of 7.0.
2.3. Preparation of bacteria culture
The inoculum cultures in the test medium were prepared five days
prior to the beginning of the test. The heat-shocked mixed culture bac­
teria were inoculated into a 500 ml Schott bottle containing Endo media
and purged with nitrogen gas for 3 min to provide an anaerobic condi­
tion. Then, the culture was incubated anaerobically for five days in a
shaker bath (Bio-Equip KW-1000DC, China) at 40 � C until the optical
density (OD) has reached 1.0 and above. A correlation standard cali­
bration curve was modelled with different concentrations of the dry
biomass weight of bacteria (mg/L) over five days and was generated by
using an OD wavelength of 530 nm. The cell concentration was
measured at 530 nm with UV/VIS detector in the Varian Cary 50 spec­
trophotometer (Varian Cary, Agilent,USA).
2.4. Standard chemical toxicants
The range of concentrations of chemical toxicants in which reactions
are likely to occur may be assessed from the findings of range experi­
ments. Table 1 shows the range of the respective chemical constituents
of POME, while Table 2 depicts six selected series of concentration that
is in the range of POME content concentration used in this experiment.
S.N. Arisht et al. Ecotoxicology and Environmental Safety 203 (2020) 110991

Table 1 �
Characteristics of major pollutants in POME (Aznury et al., 2012; Choong et al.,
Bsample Bblank
% ​ cell ​ survival ​ vitality ¼ � 100 (1)
2017; Hong et al., 2009; Loh et al., 2017). Bcontrol Bblank
Constituenta Mean (mg/L) Range (mg/L)
where, Bsample is the bacterial biomass concentration at specific toxicant
Acetic acid (AA) 3540 3030–4050 concentration; Bblank is the bacterial biomass concentration in blank
Butyric Acid (BA) 153 16–290 (only Endo medium but without toxicant) and Bcontrol is the biomass
Formic acid (FA) 1000 300–1700
Propionic acid (PA) 389 58–720
concentration of control (contain only toxicant solution but without
Copper (Cu) 0.85 0.80–0.90 cells).
Oil & grease (O&G) 7213 5614–8812
a
All the values are in mg/L. 2.6.2. Half-maximal effective concentration (EC50)
To determine the EC50 value, the half maximal toxicity was calcu­
lated using linear regression line from equation (2) (Anderson and
Table 2 Abdelghani, 1980; Arisht et al., 2019; Sebaugh, 2011) for each test
Test concentration range used in experiment. substances. EC50 value in the mg/L concentration is identified when at
Toxicants (mg/L) Test range which 50% cell survivability from the overall cell viability percentage in
the toxicants achieved.
1 2 3 4 5 6

Organic by-product AA 3000 3200 3400 3600 3800 4100 Y ¼ aX ​ þ ​ c ​ ða ​ is ​ in þ ve ​ or ​ ve ​ depending ​ on ​ the ​ dataÞ (2)
BA 66 116 216 266 316 366
FA 300 600 900 1200 1500 1800 where, Y is the overall maximum cell viability percentage (y-axis); X is
PA 50 200 350 500 650 800 concentration value in the mg/L; values a is positive or negative steep
O&G 4500 5500 6500 7500 8500 9500 slope and c is the intercept on y-axis are generated by the SigmaPlot
Inorganic by-product Cu 0.4 0.6 0.8 1.0 1.2 1.4
Control 0
linear fit curve operation.

3. Results and discussion

2.5. Bacteria culture preparation in the toxicants
Stock solutions were prepared by dissolving the substance in the test
medium, while the initial pH level of the medium was adjusted to 7.0.
Endo media containing these condition toxicants (Test 1 to 6) (Table 2)
inoculated with mixed culture bacteria and were incubated anaerobi­
cally for 120 h in a shaker bath of 120 rpm at 40 � C. The bacterial
conditions were checked regularly using Olympus BX51 fluorescence
microscope (Olympus, USA) to ensure no occurrence of fungal
contamination. The initial biomass content (BC) was standardized to 1.0
� 102 mg/L, which was equivalent to 8.3 � 107 cells (CFU)/mL for each
120 ml serum bottle with 100 ml substrate. A mixed bacterial culture of
2 ml was pipetted into the serum bottle and was thoroughly mixed with
the Endo media. Before incubation, nitrogen gas at a flow rate of 40 ml/
min was passed through the culture medium for 3 min to provide the
anaerobic conditions. All of the batch tests were performed in triplicates.
The counting of the initial and daily cellular concentrations was per­
formed through the use of a spectrophotometer. The concentration-
response curves (dose-response curve) and EC50 values were also esti­
mated for the bacteria. Apart from toxicity, pH reading was also recor­
ded. The pH reading was obtained daily for a period of 120 h (5 days),
using a pH meter (APHA, 1998). The initial pH level was adjusted to the
desired level through the use of sodium hydroxide or hydrochloric acid.
This analysis had experimentally manipulated two variables, namely the
pH level and toxicants concentration as a way of assessing the in­
teractions of organism effects on the toxicity levels (Meador, 1991).
3.1. Standard growth curve of anaerobic bacteria in endo medium
Bacterial growth generally has six different phases in batch culture
(Monod, 1949): lag phase, exponential phase, declining growth phase,
stationary phase, and death phase (Fig. 1) culture. The growth slowed
down in the lag phase of test organisms because of the presence of
un-viable cells or physiological adjustments to new environments. In
this experiment, the batch culture technique was used for test organisms
(anaerobic biohydrogen producing bacteria). It is inferred by the find­
ings of Broglia and Petrazzuolo (2014), that in general, once sugars were
introduced as a carbon source to the cultivating medium, the glycolysis
pathway was the preferred method of metabolism. The volatile fatty
acids were then released by bacteria and stopped growth until the me­
dium had been buffered with high concentrations of phosphate. The
anaerobic biohydrogen producing bacteria had a lag-phase of 6 h. This
was then followed by the exponential phase, which by geometrical
progression the cells divide and grow. Due to the limited anaerobic
environment, cell division slows down during the decreasing growth
phase. Hence, the bacterial biomass accumulates at a steady rate until
the nutrient limit the growth and/or biotransformation potential of

2.6. Measurement and analytical method

2.6.1. Bacteria growth curve analysis in the toxicants

Different concentration levels of toxicants with bacterial culture had
been employed in the toxicity test. The calculated biomass concentra­
tion of each culture in the triplicate samples were then compared against
the biomass concentration of the control culture with no toxicants (only
Endo medium). The average specific biomass concentration rate for a
particular period that was calculated as the survival rate (%) from
equation (1) (Chung-Min and Xian, 2015; Patel et al., 2009) against
concentration (mg/L) (OECD, 2011) were plotted using Sigma Plot
Software 14 (Systat Software Inc., USA).
Fig. 1. Standard growth curve of positively-controlled anaerobic bacteria
cultured in Endo media (positive control), R2: 0.982. *All biomass concentra­
tion x 102 mg/L

3
S.N. Arisht et al.
microbial cells (Bunch, 1994). Into the stationary phase, the aggregation
of toxic metabolic products, changes in ion balance and, in particular,
pH are some of the factors that limit bacterial cell growth. This is the
stage where the cell concentration remains constant at its maximum
value. The rate of growth instead reaches zero throughout the stationary
phase due to the drained nutrients in the culture medium. The
Fig. 2. Experimental data of the bacteria’s growth kinetics from equation (1) at different days for a) acetic acid, b) butyric acid, c) formic acid, d) propionic acid, e)
copper, and f) oil & grease.
4
Ecotoxicology and Environmental Safety 203 (2020) 110991
concentration of bacterial cells then decreases rapidly during the death
phase or “crash phase” as a result of either overheating, pH disturbance,
contamination or depletion of nutrients. (Roszak and Colwell, 1987).
By following the OECD (2001) guidelines, a preliminary standard
growth concentration experiment was performed to allow the adapta­
tion required for the test organism to be at its optimum cell metabolism
S.N. Arisht et al.
condition for the growth experiment. The Endo media was used at test
medium due to its specificity and bacterial adaptability to the nutrients
in the media. According to Grabow and Preez (1979), the Endo media
had yielded the highest average cultured bacterial count from waste­
water samples and is therefore regarded as the best-simulating media for
studying bacterial physiochemical characters. In this experiment, the
anaerobic bacteria that were used at the initial standard concentration
for the subsequent toxicity test was taken during the intermediate log
phase, where the cells had been the most dynamic at 8.3 � 107 cells
(CFU)/mL and were cultured for 120 h under a controlled environment
for bacterial toxicity assessment. Fig. 1 shows the bacterial-glucose
assimilation from a synthetic Endo media at an initial pH level of 7. In
the absence of toxicants, the cellular glucose assimilation had increased
and reached maximum biomass of about 1.5 � 102 mg/L, which was
equivalent to 12.5 � 107 cells (CFU)/mL during the log phase. The
specific rate of growth is usually decreasing as the microbial population
density is reaching the maximum. The mixed bacterial culture had
required a log phase of 4 h and by 44th hours for the population to grow
into the stationary phase. The toxicity assessment for the bacterial
population was conducted on the second day since the bacteria was
predicted to be at its optimum condition at that point of time.
3.2. Bacteria growth response curve in the toxicants
The initial starting concentration of bacteria without the toxicants
was standardized at ~1.0 � 102 mg/L (day 0) before toxicant (Table 2)
was added (test 1 to test 6 toxicant concentration). Following that, the
growth inhibition tests were carried out for 120 h and the EC50 was
calculated. Growth curves for anaerobic bacteria in the presence of each
toxicant are presented in Fig. 2. Initial bacterial response to the presence
of toxicants was reduced ability to grow or multiply. Traditionally this
has formed the basis for bacterial bioassays. Organic acids (acetic acid,
butyric acid and propionic acid) used in this experiment at all six
different levels of concentrations has a biomass stimulant response on
bacteria growth. A fluctuation of biomass (quadratic model curves) can
be observed in acetic acid, butyric acid, formic acid, oil & grease and
copper. Whereas in the presence of propionic acid, the biomass con­
centration increased for the first 24 h and remained stagnant till 120 h.
Among different concentration variation of test substances, acetic acid
(Fig. 2(a)) at the highest concentration, 4100 mg/L identified being the
highest growth stimulant as bacterial biomass peaked up to 1.80 � 102
mg/L within the initial 24 h of incubation. However, the viable bacterial
biomass content declined slightly to 1.46 � 102 mg/L as the incubation
time prolonged till 120 h.
Highest final bacterial biomass concentration was obtained at BA
concentration of 166 mg/L level. The total biomass at 96-h incubation
onwards was 1.62 � 102 mg/L which was more than the initial 1.0 �
102 mg/L concentration. While the lowest biomass growth was observed
at 1.4 mg/L copper toxicant which is about 0.90 � 102 mg/L after 120 h
of incubation. Whereas, suppression on bacteria biomass concentration
as compared to control can be observed in test with formic acid, oil &
grease and copper (Fig. 2). Microbial abundance might be inhibited up
to 9%, 4% and 10% by formic acid, oil & grease and copper, respec­
tively. The growth phase of the cells assesses the susceptibility of the
growth inhibition bioassay, components in the growth medium and the
species of bacteria used (Stauber and Davies, 2000). Previous work has
evaluated the significance of organic acid exposure concentration in
suppressing bacterial growth. Standard analyses of bacterial resistance,
tolerance, and absorption of membranes have shown the dependence of
microorganism metabolic responses on organic acid concentrations
(Va�zquez et al., 2011).
The lipopolysaccharides (LPS) chain molecules together with phos­
pholipids, peptidoglycans and exopolysaccharide layers form the outer
membrane in Gram-negative bacteria, comprising vast quantities of
metal-complexing sites, leading to the metal tolerance and sorption
capability of microorganisms (Li et al., 2011). LPS layer greatly
5
Ecotoxicology and Environmental Safety 203 (2020) 110991
contributes to the bacteria’s structural integrity. It preserves the cell
from a wide range of toxic compounds, such as certain digestive en­
zymes (e.g., lysozyme), antibiotics (e.g., penicillin), heavy metals, de­
tergents and colorants (Ahmed and Basumatary, 2019; Caroff and
Karibian, 2003). Supporting this statement, toxicity test on copper
(Fig. 2(e)) shows lowest test concentration of 0.4 mg/L mildly stimulates
bacterial growth to 0.989 � 102 mg/L. However, while the concentra­
tion increases it has posed as suppresser for biomass concentration.
Bacterial biomass growth shows tolerance towards the lowest test of oil
& grease concentration (4500 mg/L) compared to a higher concentra­
tion. Anaerobic bacteria were reported to have similar metabolism in
biodegradation of the unsaturated fatty acids as observed in aerobic
bacteria. Bacteria were required to convert unsaturated fatty acid to
saturated fatty acid before utilizing it efficiently. However, this con­
version rather slow in anaerobic bacteria. Hence, this substrate (oil
&grease) at higher concentration leads to negative effects on the effi­
ciency of the metabolic flux of anaerobic bacteria (Nzila et al., 2016).
From Fig. 2, the lowest concentration levels of acetic, butyric and
propionic acids had served as a positive stimulant for growth rate. While
acetic and butyric acids had exhibited positive effects on the final
biomass content even at higher concentration, propionic acid, on the
other hand, had shown a downward trend of the final biomass content at
increasing concentration levels. All three acetic, butyric and propionic
acids are intermediate metabolites that were produced during the
acidogenesis phase of anaerobic digestion (Chong et al., 2009). In a
facultative anaerobic condition, some butyric acid could be converted
by acetogens to acetic acid. Acetogens often catabolize almost all of the
products produced through acidogenesis into acetic acid that is used by
methanogens to produce methane (Millati et al., 2018). A lower toler­
ance towards increasing propionic acid concentration might suggest a
lower affinity of acetogens towards it. As for formic acid, oil & grease,
and copper, they had a negative impact on the bacterial growth at all
concentration levels. As shown in Fig. 2, copper was observed to be the
strongest inhibitor among tested toxicants. Copper had caused in a
decrease in the final number of cells and a decrease in the exponential
rate of growth or, in precise words, gradually increase in time for the
start of growth (lag phase). However, none of these tested toxicants had
stimulated a substantial lag response time-course like those described
regarding the short-chain n-alkanols (Herrero et al., 1985). The growth
rate had increased immediately (short lag phase) upon the addition of
the respective toxicants. Overall, it is observed that majority of viable
biomass concentration in all test toxicant and control starting to
decrease at around day 5 marking bacterial cell are only susceptible to
the given standardised condition for only 5 days.
3.3. Effect of toxicants concentration on changes in pH over time
The correlation between peaking of biomass concentration, substrate
affinities of bacteria species in mixed culture and pH for all tested tox­
icants except for oil and grease are shown in Fig. 3(a) to 3(e). Deplor­
ably, the effect of pH in oil and grease was not able to be determined
since the pH electrode was not able to immerse and provide proper
reading due to its viscous characteristic. The pH is responsible for
monitoring most chemical and ecosystem process and therefore has a
major impact on processes such as chemical breakdown and bioavail­
ability. pH 7 was used as an original point of all toxicants analysis, as
was the case with the neutral pH of most freshwater habitats (pH 6 to 8),
where proton acidity is the most important component. In contrast,
mineral or organic acidity is a trivial contributing factor (Desireddy
et al., 2020; Russell, 1992). Initially, two pH-dependent conceptual
models were previously proposed for test organism were previously
discussed based on observed responses to variable pH values. These
include Model-I: reduced toxicity with increased pH (positive correla­
tion), Model-II: increased toxicity with decreased pH (negative corre­
lation) (Wang et al., 2016). Based on Fig. 3(a) to 3(e), increasing toxicity
with decreasing pH was observed, which was in agreement with
S.N. Arisht et al. Ecotoxicology and Environmental Safety 203 (2020) 110991

Fig. 3. Concentration levels (mg/L) of the toxicants as a function of initial pH over time (hr) for mixed bacteria culture, (a) acetic acid, (b) butyric acid, (c) formic
acid, (d) propionic acid (e) copper and (f) control.

Model-II.
pH fluctuation between 4.12 and 7.17 was observed during bacterial
growth rates for all five toxicants. Formic acid showed the large fluc­
tuations of pH values, with the lowest pH about 4.02 while highest pH
7.17 during 120 h of incubation. In general, pH values between 4.5 and
5.5 was expected to provide the most conducive environment for bio­
hydrogen producing bacteria (Arisht et al., 2019). Coherently, the pH
fluctuations over the period of 120 h for growth promoting volatile fatty
acid toxicants (butyric acid, acetic acid and propionic acid) were within
this range. Highest bacterial growth rate (by comparing with Fig. 2(a),

6
S.N. Arisht et al.
Fig. 4. Toxicity of selected toxicants on the mixed anaerobic bacterial population at 24 h and 96 h of exposure at 50% survival rate.
(b), and 2(d)) was consistently observed around pH 5.5. This observa­
tion was in agreement with several previous findings which states that
the optimum pH for bacteria to produce biohydrogen gas was at pH 5.5
(Lin et al., 2008). Control pH shows steady decline from 7 to 5.46 for day
0–3, followed by gradual increase to pH 5.49 at day 5. pH for control
does not fluctuate rapidly as compared to pH for five test toxicants (Fig.3
(a)–(e). The association seen between declining intracellular pH,
decreased bacterial biomass and the ability of bacteria to tolerate high
concentrations of fermentation acids at low pH has also been detected in
multiple ruminal bacteria. In order to sustain the pH gradient across the
cell membrane, the membrane-bound ATPases or electron transport
7
Ecotoxicology and Environmental Safety 203 (2020) 110991
systems can stabilize the proton inflow via uncouple, and this activity
reduces the amount of energy that can be used for growth (Herrero et al.,
1985). At the other hand, the high pH value of the medium culture re­
duces the activity of the hydrogenase enzyme by altering the metabolic
pathway. (Abdeshahian et al., 2014). By maintaining a steady pH level
of 5 � 0.5 (not exceeding pH 6) across the bacterial cell membrane, high
and potentially toxic concentrations of fermentation acid anions do not
accumulate significantly due to low pH. These analyses imply that pH
evaluation over growth rates, using mixed culture microorganism pose
advantage than single culture over the toxicants as increased resistance
and enhanced growth rate can be observed. As for copper toxicant, the
S.N. Arisht et al.
lowest concentration of 0.4 mg/L showed higher pH reduction after 72 h
of incubation, whereas other higher concentrations of copper have a
similar pattern of reduction. Changes in alkalinity and pH have been
reported to adjust the extent of hydroxide and carbonate association of
copper thus its toxic effects (Stauber and Davies, 2000). Reasonably, the
growth rate of bacteria in copper shown earlier that at 0.4 mg/L the
bacteria have a better survival rate compared to other tested copper
concentration.
3.4. Half maximal effective concentration (EC50)
The half maximal effective concentration toxicity assessment with
microorganism as the biotest organism is imperative because of its
dynamicity, rapid response to the environmental changes, diverse and
always available throughout the year, more sensitive and inexpensive
when compared to those using others aquatic organisms (OECD, 2011).
Microorganisms are remarkably suitable for biotoxicity testing because
they have almost all biochemical pathways similar to those of other
higher organisms. APHA, 1998, Only recently, bacterial activity has
been widely recognized as ecologically relevant in environmental
impact studies (Stauber and Davies, 2000). Fig. 4 shows the linear line in
the most test substances at both 24 and 96 h with a negative slope graph
(Fig. 4(d), (e) and (f)) except for acetic acid (Fig. 4(a)), which has a
positive slope. The negative linear fit line shows that increased con­
centration of the drug reduces the rate of survival while the positive line
of fit shows vice versa.
Half maximal toxicity concentration assessments on the test organ­
ism were typically expressed as EC50 value for 24 and 96 h’ incubation.
The 24 h incubation could be used to measure short-term toxicity. On
contrary, long-term toxicity test incubation to 96 h would give an
attestation of the adaptive capacity of the population to the chemical in
the examination (APHA, 1998). Fig. 4 shows the representation of the
tested combinations, where experimental data from each culture were
simultaneously fitted to equation (2). From the results achieved in this
study, the toxicity assessment of bacteria shows the potential half
maximal toxicity level of organic and inorganic toxicants towards bac­
terial populations following 24 h and 96 h of exposure. It was observed
that the survival rate of bacteria had not exceeded 100% (>100%). From
Fig. 4, the anaerobic bacteria tested with acetic acid, butyric acid and
propionic acid had a survival rate of more than 60%. In contrast, the
majority survival rate for the oil & grease, copper and formic acid test
concentrations was less than 60%. Moreover, there was a consistent
response pattern upon toxicity exposure and adaptation duration. The
EC50 value in butyric acid and propionic acid toxicant decreases from 24
h to 96 h, 974.53 mg/L to 143.59 mg/L and 1383.60 mg/L to 761.17
mg/L, respectively. Conversely, EC50 value increases from 50.88 mg/L
to 1730.92 mg/L, 148.01 mg/L to 1936.56 mg/L, 0.48–1.01 mg/L and
5153–8979 mg/L for acetic acid, formic acid, copper and oil & grease,
respectively through 24 h–96 h’ incubation. In previous studies on metal
and organic acid toxicity, Liu and Dutka (1984) and Rajapaksha et al.
(2004) have shown to have similar effects on bacterial survival over time
on changes in inhibitory concentrations due to changes in bacterial ac­
tivity over time and the effect of pH.
Whereas, in this experiment, the three main volatile fatty acids tested
acetic acid, butyric acid and propionic acid were observed to exert
positively on bacterial metabolism. Previous work has indicated the
importance of volatile fatty acid exposure concentration in suppressing
bacterial growth (Lim and Vadivelu, 2019; Siegert and Banks, 2005;
Singhania et al., 2013). Consequently, our modelling dose-response
analysis approach provides a consistent and straightforward tool for
such descriptions. As suggested by Cherrington et al. (1991), due to the
variation in physical characteristics, direct comparison of different
organic acids with respect to their antimicrobial activities is difficult.
Fig. 4 indicates that all organic acids exhibited a decrement in survival
rate from 24 to 96-h incubation. Acetic acid led to the highest survival
rate, and the survival rate was increasing with respect to higher acetic
8
Ecotoxicology and Environmental Safety 203 (2020) 110991
acid concentration. Prokaryotic bacteria can convert acetate easily to
acetyl-CoA, the key intermediate metabolite in the central metabolic
pathway. Similarly, butyrate can be assimilated in the cell membrane
and was converted through β-oxidation into acetyl-CoA in the glyox­
ysome (Chalima et al., 2017; Jasni et al., 2020). This finding had sup­
ported the end result that showed the role of volatile fatty acids in
enhancing survival rates. Conversely, the inhibition rate for oil & grease
was decreasing (higher survival rate) for all six test range upon pro­
longed incubation (96 h). This observation could indicate that the test
microorganism was acclimatising well to the provided toxicant con­
centration. This was supported by Nzila et al. (2016) and Cappello et al.
(2016) which states, the consortium of anaerobic bacteria will degrade
the free fatty acids (FFA) and oil and grease (O&G) that are also esters of
fatty acids bound to glycerol molecules, hence utilized extensively in the
removal of oil from wastewater.
In the case of the 24- hour incubated period of bacterial culture
toxicity adaptation, copper (1.4 mg/L) had shown the lowest survival
rate of 30.06%. Increased in copper concentration and incubation time
had gradually reduced the viable bacterial cell with the lowest per­
centage of 24.05% (96 h) after being exposed to 1.4 mg/L of inorganic
particles. Normally, copper is present as a free metal ion or as weak
disintegrating complexes that can dissociate at the cell membrane and
have a greater reactivity to membranes. Copper transport works within a
copper-sensitive network that is capable of engaging copper ions should
they build up to toxic levels. The absorption of heavy metals is often
influenced by biotic factors such as the thickness and nature of the
biological layer of cell walls and cell size (He et al., 2019; Jacob et al.,
2018). According to Fick’s first law, the heavy metal uptake is linked to
the diffusion coefficient as well as the surface area and the thickness of
the cell membrane. These included chemical factors (pH, the concen­
tration of salinity and phosphate) that affect bioavailability either by
altering the metal’s speciation or by complexing it on the organism
surface.
Essentially, Cu and O&G toxicity had significantly reduced the bac­
teria’s survival rate when compared with volatile fatty acid toxicity. This
experimental finding was in close agreement with (Zhu et al., 2018), as
low-dose copper ions may affect the activity of microorganisms in the
soil, but a higher dose commonly found in the environment poses a huge
potential long-term risk to humans and other organisms. As for volatile
fatty acid toxicity, the bacteria were seen to have a high tolerance level
even for a very high acetic acid concentration. Therefore, it can be
concluded that acetic acid can be used as an anaerobic bacteria
mix-culture promoter. It is suggested, if the bacteria are to be cultivated
in wastewater containing both acetic and butyric acids, the biomass may
be manipulated either by increasing the biomass of the initial mix cul­
ture or by manipulating the initial acetate: butyrate ratio (Ramey and
Yang, 2005). The EC50 analysis had demonstrated the obvious adapt­
ability variation between bacteria. From the results gathered in the
experiment, the bacteria seemed to have better toxicity endurance to­
wards certain organic by-products (acetic, butyric and propionic acids).
Therefore, it can be concluded that microorganism characteristics such
as the prokaryotic cell wall physiology structure, had caused it to be
more sensitive towards the higher toxicants concentration levels.
A previous study on the level of toxicity by V� azquez et al. (2011),
also showed that the use of mixed culture as a test microorganism had
increased toxic resilience of chemical substance as compared to using
single culture towards toxicants. Mixed cultures consisted of community
dynamics, where the competitive advantage of more tolerant cultures
resulted in changes in community composition and survival. To assess
the toxicity of pollutants to the microorganism, attention must be
focused onto the specific abiotic physicochemical factors of the test
environment, which may mediate or enhance the toxicity effect of the
contaminant. Complex contaminant samples can be characterized by
laborious and expensive chemical assay, which can only provide limited
information for evaluation of possible toxic effects on biological pro­
cesses at treatment plants and in recipients. The chemical assay cannot
S.N. Arisht et al.
accurately provide information on the combined inhibitory effect of all
chemicals present, nor can the bio-availability be assessed directly. In
general, only a fraction of an experienced inhibition can be explained by
the compounds identified by a chemical analysis (Eilersen et al., 2004).
Therefore, the approach to modelling dose-response analysis provided a
simple and consistent tool for such descriptions (by analysing each
focused toxicants separately).
4. Conclusion
The study of data on toxic effects of inorganic and organic by-
products from POME had shown butyric, acetic and propionic acids to
be biomass growth enhancers on bacteria at which the survival rate had
exceeded 50%, while formic acid, copper and oil, & grease were shown
to have inhibited a considerable growth level with a survival rate that is
less than 50%. The bacteria had shown higher sensitivity towards high
formic acid, copper and oil and grease concentration. These results had
revealed the proposed potential toxic concentration of industrial waste
for discharge treatment operations before the implementation of bio­
logical treatment. To provide constructive environment for industrial
palm oil effluent treatment using mixed anaerobic bacteria, monitoring
the reaction at pH 5 � 0.5 and toxicants concentration of AA, BA and PA
around 1730.92, 136.98 and 761.17 mg/L respectively will surely
ensure better bacterial survival while FA, Cu and O&G has to be below
1936.96, 1.01 and 8978.61 mg/L respectively in every 5 days’ treatment
cycle. Therefore, a bioassay can facilitate a further study of chemical and
biologic mechanisms on the bioavailability and toxicity to bacteria in
treatment plants.
Author statement
Shalini Narayanan Arisht: conducted laboratory investigation and
analysis, validation, data curation, primary manuscript writer. Peer
Mohamed Abdul: Methodology development, data interpretation, su­
pervision, acquired grant, secondary manuscript writer. Jannatulhawa
Jasni: conducted laboratory investigation and analysis, validation.
Nazlina Haiza Mohd Yasin: data interpretation, assisted in manuscript
preparation. Sheng-Kai Lin: conducted laboratory investigation and
analysis, graphical analysis. Shu-Yii Wu: original idea conceptualiza­
tion, methodology and analysis development. Mohd Sobri Takriff:
original idea conceptualization, resources, data interpretation. Jamaliah
Md Jahim: co-supervision, original idea conceptualization, manuscript
preparation
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We thankfully recognise the technical and financial assistance pro­
vided by the Universiti Kebangsaan Malaysia-Yayasan Sime Darby
(UKM-YSD) Chair for Sustainable Development: Zero Waste Technology.
We would also like to extend our gratitude to the Government of
Malaysia and Universti Kebangsaan Malaysia for funding this work
through Geran Universiti Penyelidikan (GUP-2018-105)) and Dana
Impak Perdana (DIP-2019-008). We also thank the Ministry of Science
and Technology of Taiwan for funding this research paper (grant
numbers: MOST 107-2221-E-035-001; MOST 107-2632-E-035-001;
MOST 108-2218-E-035-002). Thanks are also extended to the Precision
Instrument Support Center of Feng Chia University and Makmal Ber­
pusat i-CRIM of UKM for providing the fabrication and measurement
facilities needed for this research.
9
Ecotoxicology and Environmental Safety 203 (2020) 110991
References
Abdeshahian, P., Al-Shorgani, N.K.N., Salih, N.K.M., Shukor, H., Kadier, A., Hamid, A.A.,
Kalil, M.S., 2014. The production of biohydrogen by a novel strain Clostridium sp.
YM1 in dark fermentation process. Int. J. Hydrogen Energy 39, 12524–12531.
https://doi.org/10.1016/j.ijhydene.2014.05.081.
Abdeshahian, P., Lim, J.S., Ho, W.S., Hashim, H., Lee, C.T., 2016. Potential of biogas
production from farm animal waste in Malaysia. Renew. Sustain. Energy Rev. 60,
714–723. https://doi.org/10.1016/j.rser.2016.01.117.
Ahmed, M., Basumatary, S.K., 2019. Applied Microbiology. MJP Publisher.
Anderson, A.C., Abdelghani, A.A., 1980. Toxicity of selected arsenical compounds in
short term bacterial bioassays. Bull. Environ. Contam. Toxicol. 24, 124–127. https://
doi.org/10.1007/BF01608085.
APHA, 1998. Standard Methods for the Examination of Water and Wastewater. Stand.
Methods 541. ISBN 9780875532356.
Arisht, S.N., Abdul, P.M., Liu, C.-M., Lin, S.-K., Wu, S.-Y., Maaroff, R.M., Jahim, J.M.,
2019. Biotoxicity assessment and lignocellulosic structural changes of phosphoric
acid pre-treated young coconut husk hydrolysate for biohydrogen production. Int. J.
Hydrogen Energy 44, 5830–5843. https://doi.org/10.1016/j.ijhydene.2019.01.116.
Aznury, M., Trianto, A., Pancoro, A., Setiadi, T., 2012. Effect of feeding time of volatile
fatty acids from palm oil mill effluent on production of polyhydroxyalkanoates by
ralstonia eutropha jmp 134 in batch fermentation, 74, 701–720.
Broglia, M., Petrazzuolo, F., 2014. Hydrogen production from Klebsiella oxytoca and
medium induced metabolic switches. Agenzia naz. Per le nuove tecnol. Lʼenergia E lo
svilupp. Econ. Sostenibile Hydrog.
Bunch, A.W., 1994. High cell density growth of micro-organisms. Biotechnol. Genet. Eng.
Rev. 12, 535–561. https://doi.org/10.1080/02648725.1994.10647921.
Cappello, S., Volta, A., Santisi, S., Morici, C., Mancini, G., Quatrini, P., Genovese, M.,
Yakimov, M.M., Torregrossa, M., 2016. Oil-degrading bacteria from a membrane
bioreactor (BF-MBR) system for treatment of saline oily waste: isolation,
identification and characterization of the biotechnological potential. Int. Biodeterior.
Biodegrad. 110, 235–244. https://doi.org/10.1016/j.ibiod.2015.12.028.
Caroff, M., Karibian, D., 2003. Structure of bacterial lipopolysaccharides. Carbohydr.
Res. 338, 2431–2447. https://doi.org/10.1016/j.carres.2003.07.010.
Chalima, A., Oliver, L., Fern� andez de Castro, L., Karnaouri, A., Dietrich, T., Topakas, E.,
2017. Utilization of volatile fatty acids from microalgae for the production of high
added value compounds. Fermentatio 3, 54. https://doi.org/10.3390/
fermentation3040054.
Cherrington, C.A., Hinton, M., Mead, G.C., Chopra, I., 1991. Organic acids: chemistry,
antibacterial activity and pratical applications. Adv. Microb. Physiol. 32, 87–108.
https://doi.org/10.1016/S0065-2911(08)60006-5.
Chong, M., Sabaratnam, V., Shirai, Y., Hassan, M.A., 2009. Biohydrogen production from
biomass and industrial wastes by dark fermentation. Int. J. Hydrogen Energy 34,
3277–3287. https://doi.org/10.1016/j.ijhydene.2009.02.010.
Choong, Y.Y., Chou, K.W., Norli, I., 2017. Strategies for improving biogas production of
palm oil mill effluent (POME) anaerobic digestion: a critical review. Renew. Sustain.
Energy Rev. 82, 2993–3006. https://doi.org/10.1016/j.rser.2017.10.036.
Chung-Min, P., Xian, M., 2015. Chapter eight - use of phosphorodithioate-based
compounds as hydrogen sulfide donors. Hydrogen Sulfide in Redox Biology,
pp. 127–142. Part A.
De Filippis, L.F., Filippis, L.F. De, 1994. Heavy metals: sources and biological effects. In:
Rai, L.C., Gaur, J.P., Soeder, C.J. (Eds.), Advances in Limnology Series: Algae and
Water Pollution. E. Scheizerbartsche Press, Stuttgart, pp. 31–77.
Desireddy, S.P.C.S.M., Maliyekkal, S., 2020. Anoxic ammonia removal using granulated
nanostructured Fe oxyhydroxides and the effect of pH, temperature and potential
inhibitors on the process. J. Water Process Eng. 33 https://doi.org/10.1016/j.
jwpe.2019.101066.
Eilersen, A.M., Arvin, E., Henze, M., 2004. Monitoring toxicity of industrial wastewater
and specific chemicals to a green alga, nitrifying bacteria and an aquatic bacterium.
Water Sci. Technol. 50, 277–283.
Elreedy, A., Tawfik, A., Enitan, A., Kumari, S., Bux, F., 2016. Pathways of 3-biofules
(hydrogen, ethanol and methane) production from petrochemical industry
wastewater via anaerobic packed bed baffled reactor inoculated with mixed culture
bacteria. Energy Convers. Manag. 122, 119–130. https://doi.org/10.1016/j.
enconman.2016.05.067.
Endo, G., Noike, T., Matsumoto, J., 1982. Characteristics of cellulose and glucose
decomposition in acidogenic phase of anaerobic digestion. Proc. Japan Soc. Civ. Eng.
1982, 61–68.
Faudzi, M.H.I.M., Jahim, J.M., Abdul, P.M., Chu, C.Y., Wu, S.Y., Takriff, M.S., Harun, S.,
2019. Kinetic model of thermophilic biohydrogen production from POME. Int. J.
Integr. Eng. 11 (7), 219–232. https://doi.org/10.30880/ijie.2019.11.07.028.
Fazli, R.R., Hertadi, R., 2019. Production and characterization of rhamnolipids from
bioconversion of palm oil mill effluent by the halophilic bacterium Pseudomonas
stutzeri BK-AB12. Environ. Prog. Sustain. Energy 38, 1–6. https://doi.org/10.1002/
ep.13007.
Grabow, W.K., Preez, M.D.U., 1979. Comparison of m-endo LES , MacConkey , and teepol
media for membrane. Filtration Counting of Total Coliform Bacteria in Water 38,
351–358.
Hariz, H.B., Takriff, M.S., 2017. Palm oil mill effluent treatment and CO2 sequestration
by using microalgae—sustainable strategies for environmental protection. Environ.
Sci. Pollut. Res. 24, 20209–20240. https://doi.org/10.1007/s11356-017-9742-6.
He, T., Xie, D., Ni, J., Cai, X., Li, Z., 2019. Investigating the effect of copper and
magnesium ions on nitrogen removal capacity of pure cultures by modified non-
competitive inhibition model. Ecotoxicol. Environ. Saf. 170, 479–487. https://doi.
org/10.1016/j.ecoenv.2018.12.019.
S.N. Arisht et al.
Herrero, A.A., Gomez, R.F., Snedecor, B., Tolman, C.J., Roberts, M.F., 1985. Growth
inhibition of Clostridium thermocellum by carboxylic acids: a mechanism based on
uncoupling by weak acids. Appl. Microbiol. Biotechnol. 22, 53–62. https://doi.org/
10.1007/BF00252157.
Hong, S., Shirai, Y., Nor Aini, A., Hassan, M., 2009. Semi-continuous and continuous
anaerobic treatment of palm oil mill effluent for the production of organic acids and
polyhydroxyalkanoates. Res. J. Environ. al Sci. 3, 552–559.
Iwuagwu, J.O., Ugwuanyi, J.O., 2014. Treatment and valorization of palm oil mill
effluent through production of food grade yeast biomass. J. Waste Manag. 2014, 1–9.
https://doi.org/10.1155/2014/439071.
Jacob, J.M., Karthik, C., Saratale, R.G., Kumar, S.S., Prabakar, D., Kadirvelu, K.,
Pugazhendhi, A., 2018. Biological approaches to tackle heavy metal pollution: a
survey of literature. J. Environ. Manag. 217, 56–70. https://doi.org/10.1016/j.
jenvman.2018.03.077.
Jasni, J., Arisht, S.N., Mohd Yasin, N.H., Abdul, P.M., Lin, S.K., Liu, C.M., Wu, S.Y.,
Jahim, J.M., Takriff, M.S., 2020. Comparative toxicity effect of organic and
inorganic substances in palm oil mill effluent (POME) using native microalgae
species. J. Water Process Eng. 34, 101165. https://doi.org/10.1016/j.
jwpe.2020.101165.
Jiang, Y., McAdam, E., Zhang, Y., Heaven, S., Banks, C., Longhurst, P., 2019. Ammonia
inhibition and toxicity in anaerobic digestion: a critical review. J. Water Process Eng.
32, 100899. https://doi.org/10.1016/j.jwpe.2019.100899.
Li, M., Pokhrel, S., Jin, X., M€adler, L., Damoiseaux, R., Hoek, E.M.V., 2011. Stability,
bioavailability, and bacterial toxicity of Zno and iron-doped Zno nanoparticles in
aquatic media. Environ. Sci. Technol. 45, 755–761. https://doi.org/10.1021/
es102266g.
Lim, J.X., Vadivelu, V.M., 2019. Enhanced volatile fatty acid production in sequencing
batch reactor: microbial population and growth kinetics evaluation. AIP Conf. Proc.
2124 https://doi.org/10.1063/1.5117100.
Lin, C.-Y., Chang, C.-C., Hung, C.-H., 2008. Fermentative hydrogen production from
starch using natural mixed cultures. Int. J. Hydrogen Energy 33, 2445–2453.
Liu, D., Dutka, B.J., 1984. Toxicity Screening Procedures Using Bacterial Systems. CRC
Press. https://doi.org/10.1016/0043-1354(83)90265-8.
Loh, S.K., Nasrin, A.B., Azri, S.M., Adela, B.N., Muzzammil, N., Jay, T.D., Eleanor, R.A.S.,
Lim, W.S., Choo, Y.M., Kaltschmitt, M., 2017. First Report on Malaysia ’ s
experiences and development in biogas capture and utilization from palm oil mill e
ffl uent under the Economic Transformation Programme. Current and future
perspectives 74, 1257–1274. https://doi.org/10.1016/j.rser.2017.02.066.
Maaroff, R.M., Md Jahim, J., Azahar, A.M., Abdul, P.M., Masdar, M.S., Nordin, D., Abd
Nasir, M.A., 2019. Biohydrogen production from palm oil mill effluent (POME) by
two stage anaerobic sequencing batch reactor (ASBR) system for better utilization of
carbon sources in POME. Int. J. Hydrogen Energy 44 (6), 3395–3406. https://doi.
org/10.1016/j.ijhydene.2018.06.013.
Madaki, Y.S., Seng, L., 2013. Palm oil mill effluent ( pome ) from Malaysia palm oil Mills
: waste or resource. Int. J. Sci. Environ. Technol. 2, 1138–1155.
Mahmod, S.S., Azahar, A.M., Indera Lutfi, A.A., Abdul, P.M., Mastar, M.S., Anuar, N.,
Takriff, M.S., Jahim, J.M., 2020. Potential utilisation of dark-fermented palm oil mill
effluent in continuous production of biomethane by self-granulated mixed culture.
Sci. Rep. 10 (9167), 1–12. https://doi.org/10.1038/s41598-020-65702-w.
Meador, J.P., 1991. The interaction of pH, dissolved organic carbon, and total copper in
the determination of ionic copper and toxicity. Aquat. Toxicol. 19, 13–31. https://
doi.org/10.1016/0166-445X(91)90025-5.
Millati, R., Lukitawesa, Permanasari, E.D., Sari, K.W., Cahyanto, M.N., Niklasson, C.,
Taherzadeh, M.J., 2018. Anaerobic digestion of citrus waste using two-stage
membrane bioreactor. IOP Conf. Ser. Mater. Sci. Eng. 316, 1–9. https://doi.org/
10.1088/1757-899X/316/1/012063.
Monod, J., 1949. The growth of bacterial cultures. Annu. Rev. Microbiol. 3, 371–394.
Nasution, M.A., Wibawa, D.S., Ahamed, T., Noguchi, R., 2018. Comparative
environmental impact evaluation of palm oil mill effluent treatment using a life cycle
assessment approach: a case study based on composting and a combination for
biogas technologies in North Sumatera of Indonesia. J. Clean. Prod. 184, 1028–1040.
https://doi.org/10.1016/j.jclepro.2018.02.299.
Nzila, A., Thukair, A., Sankara, S., Razzak, S.A., 2016. Characterization of aerobic oil and
grease-degrading bacteria in wastewater. Environ. Technol. 38, 661–670. https://
doi.org/10.1080/09593330.2016.1207712.
10
Ecotoxicology and Environmental Safety 203 (2020) 110991
OECD, 2011. Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test.
OECD Publishing. Elsevier Ltd. https://doi.org/10.1007/BF01611022.
Oviasogie, P.O., Aghimien, A.E., 2002. Macronutrient status and speciation of Cu, Fe, Zn
and Pb in soil containing palm oil mill efulent. Global J. Pure Appl. Sci. 9, 71–80.
https://doi.org/10.4314/gjpas.v9i1.15982.
Page, L.H., Ni, J.Q., Heber, A.J., Mosier, N.S., Liu, X., Joo, H.S., Ndegwa, P.M.,
Harrison, J.H., 2014. Characteristics of volatile fatty acids in stored dairy manure
before and after anaerobic digestion. Biosyst. Eng. 118, 16–28. https://doi.org/
10.1016/j.biosystemseng.2013.11.004.
Patel, S., Gheewala, N., Suthar, A., Shah, A., 2009. In - vitro cytotoxicity activity of
solanum nigrum extract against hela cell line and vero cell line. Int. J. Pharm.
Pharmaceut. Sci. 1, 38–46.
Rajapaksha, R.M.C.P., Tobor-Kapłon, M.A., Bååth, E., 2004. Metal toxicity affects fungal
and bacterial activities in soil differently metal toxicity affects fungal and bacterial
activities in soil differently. Appl. Environ. Microbiol. 70, 2966–2973. https://doi.
org/10.1128/AEM.70.5.2966.
Rajeshwari, K., Balakrishnan, M., Kansal, A., Lata, K., Kishore, V.V., 2000. State-of-the-
art of anaerobic digestion technology for industrial wastewater treatment. Renew.
Sustain. Energy Rev. 4, 135–156. https://doi.org/10.1016/S1364-0321(99)00014-3.
Ramey, D.E., Yang, S.T., 2005. Production of Butyric Acid and Butanol from Biomass.
Environmental Energy Inc., Blacklick, OH (United States), p. 103.
Roslan, R., Abdul, P.M., Jahim, J.M., 2018. Identification of restriction-modification
systems in Clostridium butyricum using bioinformatic tools. Malays. Appl. Biol. 47,
89–100.
Roslan, R., Abdul, P.M., Jahim, M.J., 2018. Endogenous CRISPR/Cas Systems Prediction:
A Glimpse towards Harnessing CRISPR/ Cas Machineries for Genetic Engineering.
Jurnal Kejuruteraan SI 1 (7), 1–9. https://doi.org/10.17576/jkukm-2018-si1(7)-01.
Roszak, D.B., Colwell, R.R., 1987. Survival strategies of bacteria in the natural
environment. Microbiol. Rev. 51, 365–379.
Russell, J.B., 1992. Another explanation for the toxicity of fermentation acids at low pH:
anion accumulation versus uncoupling. J. Appl. Bacteriol. 73, 363–370. https://doi.
org/10.1111/j.1365-2672.1992.tb04990.x.
Sebaugh, J.L., 2011. Guidelines for accurate EC50/IC50 estimation. Pharmaceut. Stat.
10, 128–134. https://doi.org/10.1002/pst.426.
Shahbandeh, M., 2020. Palm Oil: Global Production Volume 2012/13-2019/20. statista.
Siegert, I., Banks, C., 2005. The effect of volatile fatty acid additions on the anaerobic
digestion of cellulose and glucose in batch reactors. Process Biochem. 40,
3412–3418. https://doi.org/10.1016/j.procbio.2005.01.025.
Singhania, R.R., Patel, A.K., Christophe, G., Fontanille, P., Larroche, C., 2013. Biological
upgrading of volatile fatty acids, key intermediates for the valorization of biowaste
through dark anaerobic fermentation. Bioresour. Technol. 145, 166–174. https://
doi.org/10.1016/j.biortech.2012.12.137.
Stauber, J.L., Davies, C.M., 2000. Use and limitations of microbial bioassays for assessing
copper bioavailability in the aquatic environment. Environ. Rev. 8, 255–301.
https://doi.org/10.1139/er-8-4-255.
Strotmann, U.J., Pagga, U., 1996. A growth inhibition test with sewage bacteria - results
of an international ring test 1995. Chemosphere 32, 921–933. https://doi.org/
10.1016/0045-6535(95)00357-6.
V�
azquez, J.A., Dur� an, A., Rodríguez-Amado, I., Prieto, M.A., Rial, D., Murado, M.A.,
2011. Evaluation of toxic effects of several carboxylic acids on bacterial growth by
toxicodynamic modelling. Microb. Cell Factories 10, 100. https://doi.org/10.1186/
1475-2859-10-100.
Wang, Z., Meador, J.P., Leung, K.M.Y., 2016. Metal toxicity to freshwater organisms as a
function of pH: a meta-analysis. Chemosphere 144, 1544–1552. https://doi.org/
10.1016/j.chemosphere.2015.10.032.
Zainal, N.H., Aziz, A.A., Idris, J., Jalani, N.F., 2018. Reduction of POME final discharge
residual using activated bioadsorbent from oil palm kernel shell. J. Clean. Prod.
https://doi.org/10.1016/j.jclepro.2018.02.110.
Zhu, X., Wu, X., Yao, J., Wang, F., Liu, W., Luo, Y., Jiang, X., 2018. Toxic effects of binary
toxicants of cresol frother and Cu (II) on soil microorganisms. Int. Biodeterior.
Biodegrad. 128, 155–163. https://doi.org/10.1016/j.ibiod.2017.04.012.
Zinatizadeh, A.A.L., Mohamed, A.R., Mashitah, M.D., Abdullah, A.Z., Isa, M.H., 2007.
Optimization of pre-treated palm oil mill effluent digestion in an up-flow anaerobic
sludge fixed film bioreactor: a comparative study. Biochem. Eng. J. 35, 226–237.
https://doi.org/10.1016/j.bej.2007.01.018.