Bioresource Technology Reports 18 (2022) 101017

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Bioresource Technology Reports

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Effect of salinity and surfactant on volatile fatty acids production from

kitchen wastewater fermentation
Raj Shekhar Bose a, b, *, Basem S. Zakaria b, Bipro Ranjan Dhar b, Manoj Kumar Tiwari a
a
School of Water Resources, Indian Institute of Technology Kharagpur, WB, India
b
Civil and Environmental Engineering, University of Alberta, Edmonton, AB, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: Effects of the various concentrations of salt (0.5–12 g/L NaCl), surfactants (0.005–0.12 g/L LAS), and their co-
Anaerobic fermentation presence on fermentative volatile fatty acids (VFAs) production from kitchen wastewater were investigated.
Kitchen wastewater VFAs production declined by 17–84% with increasing salt concentration from 3 to 12 g/L, whereas it was
Salts
enhanced by 10–25% at surfactant concentrations of 0.03–0.12 g/L. The addition of 0.06 g/L surfactants in
Surfactants
Volatile fatty acids (VFAs)
kitchen wastewater marginally improved VFAs production in different salt amended (0.5–12 g/L) reactors.
Acetate and propionate were the major VFAs under all conditions; however, the percentage of propionate
increased by 15–22% with increasing surfactant concentration. Higher alpha diversity indices values of the
bacterial population were noted in the reactors amended with surfactant. Enterobacteriales were the dominant
order of bacteria present in the reactor amended with 6 g/L of salt, whereas Lactobacillales were most prominent
in reactor amended with 0.06 g/L of surfactant.

1. Introduction through chemical processes (Lee et al., 2014).

Wastewater generated from the kitchen sink often contains a sig­
nificant organic load from food residues generated during food pro­
cessing, cooking, and utensil washing; with the main components being
carbohydrates, proteins, oil, and suspended solids (Eriksson et al.,
2002). These organics present a significant component of municipal
wastewater. Source-separation and treatment of kitchen wastewater can
markedly reduce a significant fraction of organic load in municipal
wastewater. Currently, there is a growing interest in converting high
organic load waste and wastewater into sustainable products, such as
volatile fatty acids (abbreviated as VFA) via microbial fermentation,
which could be utilized as an alternative carbon source for biological
nutrients removal, and as precursors for methane and hydrogen pro­
duction using various anaerobic biotechnologies (Lee et al., 2014; Dhar
et al., 2015; Lin et al., 2019). VFAs are mainly comprised of acetate,
propionate, butyrate, isobutyrate, and valerate. They have a wide range
of industrial applications, such as the synthesis of bioplastics, bio­
surfactants, lipids, biofuels, and various other industrially relevant
chemicals (Lee et al., 2014). Upscaling of VFA generation from anaer­
obic fermentation of waste/wastewater can significantly lower the cost
of production of VFAs, which is currently synthesized conventionally
Various types of organic wastes, such as food waste, waste activated
sludge, agricultural waste, and industrial waste, have been explored for
VFAs generation using microbial fermentation (Lee et al., 2014;
Jayakrishnan et al., 2019). Moreover, high-strength organic wastewater,
such as source-separated kitchen wastewater (>40,000 mg/L COD) has
been investigated for VFA generation (Lee et al., 2013). However, the
strength of kitchen wastewater varies greatly across the globe and has
been documented to be as low as ≤1000 mg/L COD (Ghaitidak and
Yadav, 2013). Furthermore, the quality of kitchen wastewater is affected
by varying concentrations of salinity and surfactants (Lee et al., 2013;
Liu et al., 2017).
Salinity in kitchen wastewater is contributed from the salt usage in
cooking and sometimes from a range of salinity present already in the
reclaimed water for the use in dishwashing (Liu et al., 2017). Salinity
varies greatly in water across the world, ranging from 20 mg/L in some
freshwater lakes to 12,000 mg/L in large salt lakes, estuaries, and
groundwaters of coastal regions. However, all potable waters usually
have salinity lower than 500 mg/L, the highest permissibility limit (U.S.
EPA, 2014). However, high saline waters are not used for drinking but
are recently being considered for various washing purposes to combat
water scarcity (Chen et al., 2012). High salinity may induce osmotic

* Corresponding author at: School of Water Resources, Indian Institute of Technology Kharagpur, WB, India
E-mail address: rbose1@ualberta.ca (R.S. Bose).

https://doi.org/10.1016/j.biteb.2022.101017
Received 17 February 2022; Received in revised form 8 March 2022; Accepted 9 March 2022
Available online 15 March 2022
2589-014X/© 2022 Elsevier Ltd. All rights reserved.
R.S. Bose et al. Bioresource Technology Reports 18 (2022) 101017

pressure on microorganisms involved in anaerobic bioprocesses causing Table 1

dehydration of the cell components leading to cell death eventually Characteristics of kitchen wastewater and fermented food waste (inoculum).
reducing the efficiency of the process (Wang et al., 2017). Several Parameters Kitchen Fermented food waste
studies reported various inhibition levels of salinity on anaerobic mi­ wastewater slurry (inoculum)
crobes (Wang et al., 2017). Most authors have reported a strong inhib­ Total suspended solids, TSS (mg/ 310 ± 20 21,355 ± 320
itory effect of 20–50 g/L and ≥5 g/L salt concentrations on acidogenic L)
and methanogenic microbes, respectively (Wang et al., 2017). In addi­ Volatile suspended solids, VSS 280 ± 15 20,222 ± 250
tion to salinity, kitchen wastewater also contains a significant level of (mg/L)
Total chemical oxygen demand, 4325 ± 89 21,710 ± 276
surfactants. Notably, linear alkylbenzene sulfonate (LAS) is the most TCOD (mg/L)
widely used surfactant in dishwashing detergents and is the most found Soluble chemical oxygen 3730 ± 50 2540 ± 50
surfactant in kitchen wastewater. Although high concentrations of LAS demand, SCOD (mg/L)
(≥600 mg/L) have been reported to inhibit both acidogenic and meth­ Total volatile fatty acids, TVFA 90 ± 5 650 ± 45
(mg/L)
anogenic microbes (Lee et al., 2013), low concentration ≤ 200 mg/L
Total ammonia nitrogen, TAN 30 ± 3 197 ± 6
improved solubilization and hydrolysis of high organic load substrates (mg/L)
viz. waste activated sludge and food wastes (Jiang et al., 2007; Lee et al., Total phosphates, TP (mg/L) 13 ± 2 72 ± 5
2013), thereby improving the rate and efficiency of anaerobic digestion pH 5.5 ± 0.1 4.5 ± 0.3
(Guan et al., 2017; Luo et al., 2022). Most studies concerning the effect Sugars (mg/L) 2745 ± 55
Proteins (mg/L) 150 ± 25
of salinity or surfactant on anaerobic digestion have been mostly limited Lipids (mg/L) 80 ± 12
to high-strength organic wastes and wastewater. Thus, limited infor­
mation is available on the effects of salt and surfactant (alone and syn­
ergistic) on the anaerobic fermentation of source-separated low-strength fixed surfactant concentration, which exhibited the highest VFA yield
kitchen wastewater. Further, the behavior of fermentation processes has (0.06 g/L LAS), and varying salt concentrations (0.5, 3, 6, 9, and 12 g/L).
been known to vary depending upon the initial substrate concentration Lastly, wastewater containing natural salinity (0.105 g/L) from the
(Yuan et al., 2019). rinsing action on the food leftovers and devoid of addition of salt and/or
Consequently, the present study systematically explores the indi­ surfactant was set as control. The range of salt dosages was chosen based
vidual and combined effect of salt concentration ranging between on previous literature (Liu et al., 2017; Wang et al., 2017). The range of
potable water limits (≤500 mg/L) to high saline levels (~12 g/L) and surfactant dosage was selected based on the range of LAS measured in
varying surfactant concentrations (obtained from sampling values) on the kitchen wastewater at the IIT staff canteen obtained after a month of
the performance of fermenting microbes and efficiency of VFA produc­ sampling. Sodium chloride (99% pure) and LAS (99% pure) were pur­
tion with low strength kitchen wastewater. In addition to VFAs yields chased from Merck (Bengaluru, India) and were used without further
and kinetics, the effects of salt and surfactant on the microbial com­ purification.
munities were also investigated. All experiments were conducted at room temperature and without
mixing in order to generate a natural and cost-effective solution for
2. Materials and methods managing kitchen wastewater. Experiments were conducted for 8 days
in batch mode using 550 mL capacity wash bottles (Tarson) filled up to
2.1. Substrate and inoculum 500 mL (i.e., working volume) as batch reactors. Edges of the reactor
caps were sealed by parafilm wax. F/M ratio at the beginning of the
Tap water having salinity 0.04 g/L was used to generate wastewater experiment was adjusted at 1.2 based on previous literature. The outlet
by gentle rinsing (without using surfactant) of food leftovers, mainly rice of the reactors was connected by a silicon tube to an inverted measuring
(40–50%), pulses (20–30%), curry (10–20%), and yogurt (10–20%) cylinder supported over a 500 mL beaker filled with 3 M Sodium hy­
residues in the dishes, from the kitchen sink of Institute Staff canteen droxide to trap carbon dioxide and measure only methane (if any) by
inside IIT Kharagpur campus, was used as substrate in this study. The downward displacement of water. For conducting various analyses, 5
range of food leftovers % was determined by random sampling of wet mL of sample were drawn out with the help of a 10 mL capacity syringe
mass % of food leftover on 10 sample dishes. Natural salinity of 0.105 g/ from the top of each reactor by removing the silicon tube every day and
L was generated in the wastewater derived from rinsing the food left­ at the same time measuring the volume of water displaced by the gas (if
overs from the utensils. Fermented food waste slurry made of wasted produced) in the measuring cylinder. All the studies were performed in
rice (50–60%) and pulses (40–50%) was used as an inoculum. Salinity triplicate, and results were tabulated in the form of mean and standard
concentration of kitchen wastewater was noticed to be 0.163 ± 0.05 g/L deviation.
obtained by 30 days of sampling, whereas a surfactant concentration of VFA production data from the study were fitted to the first-order
kitchen wastewater was found to be 0.05 ± 0.03 g/L obtained by 30 kinetic equation and modified Gompertz model to estimate first-order
days of sampling. The substrate to inoculum ratio was adjusted to 1.2 kg rate constant, VFA generation potential, maximum VFA production
CODKitchen wastewater/kg VSInoculum based on literature (Liu et al., 2017). rate, and lag phase. The metabolites production patterns from anaerobic
Before incorporating into the substrate, the slurry was subjected to low- biological processes have been assessed by these two kinetic models in
temperature thermal treatment (80 ± 1 ◦ C) for 20 min to attenuate any the literature (Jayakrishnan et al., 2018; Bose et al., 2021). The rate of
existing methanogen (Rafieenia et al., 2018). Characteristics of substrate production of metabolites from biological processes is not the same
and inoculum are detailed in Table 1. throughout the time frame, i.e., the rate is usually slower in the begin­
ning soon after the acclimatization phase of microorganisms, then it
2.2. Experiments gradually increases, reaching the maximum and finally enters a
declining phase. There is also a phase known as the acclimatization
The study consisted of three schemes: first, fermentation experiments phase (also known as the lag phase) of microorganisms during which no
conducted under various adjusted salt concentrations, i.e., ranging be­ metabolites are generated. VFA generation potential is the simulated
tween potable water to high saline water (0.5, 3, 6, 9, and 12 g/L of value of the total cumulative VFA produced during the overall time
sodium chloride); second, fermentation experiments conducted under period of the study. The modified Gompertz model is described in the
various surfactant concentrations (0.005, 0.03, 0.06, 0.09, and 0.12 g/L equation (Eq. 1) below:
of LAS). Based on the performance of fermentation in the presence of
surfactants, third fermentation tests were conducted in presence of a

2
R.S. Bose et al.
[ ]
Rmax − e
(λ− t)+1
Hmax
H(t) = Hmax e − e
(1)
where, cumulative VFA production (mg TVFA) at time t is represented
by H(t); VFA production potential (mg TVFA/g COD/d) is represented
by Hmax; Maximum VFA production rate (mg TVFA/g COD) by Rmax; and
time of lag phase by λ (d).
On the other hand, the following first-order kinetic equation (Eq. 2)
was used to determine the rate of metabolites production (denoted by
d − 1) (Converti et al., 1998).
H(t) = C e− kt
(2)
where, cumulative VFA production (mg TVFA) at time t is represented
by H(t); initial substrate concentration is represented C (mg TCOD); and
k is a constant denoted by (d− 1) presenting the rate of VFA production in
the overall period of the study.
2.3. Analysis of VFAs
Gas Chromatograph (Thermo Scientific model Trace 1310) equipped
with Flame Ionization Detector, and capillary column (mid-polar)
composed of 14% cyanopropylphenyl/86% dimethyl polysiloxane with
dimension 30 m × id 0.25 mm × film thickness of 0.25 μm (Thermo
Scientific TG-1701MS) was used to quantify the major VFAs (acetic acid,
propionic acid, butyric acid, and isobutyric acid). Nitrogen was used as
the carrier gas with constant a flowrate of 1 mL/min, purge flow 5 mL/
min and split flow 30 mL/min, respectively. Injection temperature was
set at 220 ◦ C, detector temperature was set at 220 ◦ C and carrier pressure
at 140 kPa. Mixture of hydrogen and air was used as fuel gas with flow
rate of hydrogen, air and make-up gas set at 30 mL/min, 450 mL/min
and 40 mL/min, respectively. The temperature of the oven was ramped,
starting from 50 ◦ C (hold time of 2 min), 50–100 ◦ C (rate of 10 ◦ C/min),
100 ◦ C (hold time of 2 min), 100–240 ◦ C (rate of 40 ◦ C/min) and 240 ◦ C
(hold time of 2 min). Sample aliquots drawn out from the fermenting
reactors were centrifuged at 10,000g and filtered through 0.45 μm
membrane filters prior to analysis. For precise detection of VFAs, the pH
of the aliquots was reduced to 4 by drop-wise addition of 85% ortho-
phosphoric acid (Merck). The final solution was incorporated in meth­
anol in a ratio 10% (v/v) before injecting into a Gas chromatograph.
2.4. Other analysis
COD, TAN, TS, and VS of kitchen wastewater and inoculum sludge
were determined according to Standard methods (APHA, 1989). pH and
salinity were measured using a multiparameter device (YSI pro plus).
Total sugar was quantified by phenol – sulfuric acid method with
glucose as standard (Nielsen, 2010), while protein was determined by
Bradford assay using BSA as standard (Kruger, 2002). Viable cell
numbers were measured using a Fluorescence Activated Cell Sorter
(FACS Aria III, Singapore) (see Supporting Information). Total oil con­
tent in wastewater was measured by solvent extraction followed by the
gravimetric method (APHA, 1989). All the analyses were done in trip­
licate. All the reagents and salts used in the analysis were purchased
from Merck and were used directly without further purification.
2.5. Microbial community characterization
At the end of batch experiments, microbial communities were
characterized for biomass samples from the sludge, one from each of the
salt (6 g/L) and surfactant (0.06 g/L) amended reactors using 16S rRNA
sequencing. The reactor was first decanted. Then, sludge biomass
accumulated in the triplicate reactors was mixed thoroughly, and 10 mL
of composite sludge sample was used for DNA extraction. Total meta­
genomic DNA was extracted using PowerSoil® DNA Isolation kit (Mol­
Bio Laboratories, Carlsbad, USA) and stored at − 70 ◦ C till further
3
Bioresource Technology Reports 18 (2022) 101017
analysis. The DNA concentration and purity of samples were determined
using a Nanodrop spectrophotometer (2000C, Thermo Scientific, USA).
Microbial cell numbers in the reactor were quantified using qPCR, ac­
cording to Zakaria et al. (2019). Extracted DNA was outsourced to the
Research and Testing Laboratory (Lubbock, TX, USA) for Illumina Miseq
Sequencing of 16S rRNA gene using universal bacterial and archaeal
primer pairs mentioned elsewhere (Bose et al., 2021). Microbial di­
versity and abundance were assigned using the Quantitative Insights
Into Microbial Ecology (QIIME) pipeline (http://qiime.org, Caporaso
et al., 2010) according to Bose et al. (2021).
3. Results and discussions
3.1. Effect of salt on VFA production
Fig. 1a illustrates VFA concentrations under different salt concen­
trations over 8 days of operation. No methane production was observed.
All reactors had an initial VFA concentration of ~160 mg/L during start-
up. A spurt in VFA concentration was noticed in the control reactor right
from day 1. In contrast, at the same time, reactors amended with
different salts concentrations showed signs of inhibition indicated by lag
phases in VFA production. However, after the initial lag phase, VFA
concentrations increased in all salt amended reactors on day 2.
Compared to the control, a prolonged lag phase resulted in 68–79% and
90–95% lower VFA concentration in reactors SL0.5–SL6 and SL9–SL12.
In all reactors, VFA concentration reached a plateau on day 6, and pH
dropped to 4.5–4.8. Despite 68% lower VFA concentration in the first 2
days of operation, SL0.05 (i.e., 0.5 g/L of sodium chloride) exhibited a
similar final VFA concentration to the control after 8 days. Hence,
salinity in potable water concentration range ≤0.5 g/L showed a mini­
mal effect on VFA production. Overall, 3–6 g/L salt resulted in 17–24%
lower VFA concentration, whereas 9–12 g/L of salt led to 58–84% lower
VFA concentration, respectively. Thus, the salt concentration of ≤6 g/L
led to moderate inhibition (<25% decrease in VFAs) and was ecologi­
cally adaptable. In contrast, 9 and 12 g/L salt concentrations led to se­
vere inhibition of VFA production. Viable cell number declined with
increasing salt concentration (refer to Supporting Information). A pre­
vious study by Liu et al. (2017) also showed similar trends. In their
study, salt concentration > 9 g/L led to severe inhibition of acidogenic
fermentation of food waste (40,000 mg TCOD/L). Notably, higher salt
concentrations can affect the microorganisms in two ways: the osmotic
effect and the specific ion effect. Soluble salts increase the osmotic po­
tential (more negative) of the solution, drawing water out of cells
leading to dehydration and, subsequently, plasmolysis. Specific ion ef­
fect refers to the accumulation of a certain ion (sodium or chloride) to an
unusually high concentration, interfering with various metabolic pro­
cesses (Liu et al., 2017; Feijoo et al., 1995; Yan et al., 2015).
Among various VFAs, acetate and propionate combined constituted
the significant fraction (79–84%), while isobutyrate and butyrate
constituted the minor fraction under different conditions (Fig. 2a).
Increasing salt concentration from 3 to 12 g/L, increased acetate fraction
from 63 ± 6% (3 g/L of salt) to 77 ± 8% (12 g/L of salt). At lower salt
concentrations (0.5–3 g/L), VFAs composition was comparable to the
control, indicating a negligible effect of lower salt concentration on the
composition of fermentation products. In contrast, propionate and iso­
butyrate fractions slightly declined; however, the percentage of butyrate
did not vary significantly (p > 0.05) under different salt concentrations.
Table 2 summarizes the impact of salt concentrations on estimated
kinetic parameters using first-order and modified Gompertz kinetic
equations. All estimated kinetic parameters were affected by different
salt concentrations. In general, VFA production rates decreased with
increased salt concentrations. Notably, the salt concentration of ≥9 g/L
showed 61–88% lower VFA production rates (first-order) and 59–86%
lower maximum VFA production rates (modified Gompertz model)
compared to control. Effect on lag phases was prominent even with the
lowest salt concentration of 0.5 g/L; different salt concentrations
R.S. Bose et al. Bioresource Technology Reports 18 (2022) 101017

Fig. 1. VFA concentrations in 8 days of fermentation under (a) different salts concentrations, (b) different surfactants concentrations, and (c) different salts con­
centrations with a fixed surfactant concentration (0.06 g/L).

Fig. 2. Concentrations of different components of VFA under (a) different salts concentrations, (b) different surfactants concentrations, and (c) different salts
concentrations with a fixed surfactant concentration (0.06 g/L).

increased the duration of lag phases by 283–466% compared to the surfactant heads being hydrophilic position themselves, getting exposed
control. However, an increase in salt concentrations showed a non-linear to water, while the tails being hydrophobic are grouped in the center of
impact on the lag phases. the structure protected from the water. The micelles work to disintegrate
large biopolymers into smaller fractions into the solution. The hydro­
phobic tails are attracted to the carbon chain of the biopolymer, while
3.2. Effect of surfactant on VFA production
the hydrophilic heads pull the electron-rich OH− group exposed to water
leading to the disintegration of biopolymer into smaller units engulfed
Fig. 1b shows the impact of various surfactant concentrations
inside the micelles (Aveyard, 2019).
(0.005–0.12 g/L of LAS) on VFA production. Initially, surfactants
As shown in Fig. 2b, acetate and propionate constituted the signifi­
exhibited a slower increase in VFA concentrations than control (41–49%
cant fractions of (77–82%) of total VFAs in both surfactants amended
lower VFAs on day 1). However, such inhibitory effect of surfactant
and control reactors. However, a surfactant concentration of >0.03 g/L
disappeared within 2 days, and all reactors amended with surfactant
resulted in a slight decline in acetate and an increase in propionate.
showed VFA concentration profiles similar to the control. The VFA
However, surfactant concentration between 0.06 g/L and 0.12 g/L led to
concentrations in all reactors reached a plateau after 6 days. Surfactant
a similar VFAs spectrum (p > 0.05) with a slight rise in propionate
concentration of 0.03 g/L (SF0.03) resulted in a marginal increase (by
fraction than control and lower surfactant concentrations. Thus, higher
10%) in VFA production than the control. In contrast, a 0.06–0.12 g/L
surfactant concentration in kitchen wastewater enhanced VFA genera­
(SF0.06–SF0.12) surfactant concentration provided 23–25% higher VFA
tion primarily in form of propionate.
production than control. Viable cell numbers in surfactant amended
Despite the positive impact on VFA production by most surfactant
reactors did not differ significantly compared to the control (Refer to
concentrations, no significant differences (p > 0.05) in VFA production
Supporting Information). Thus, the present range of surfactant concen­
rates were noticed, as estimated from first-order and Gompertz model
trations is presumed not to affect the microbes; instead, it appears to
kinetic model fitting (Table 2). However, duration of lag phases
affect the substrate solely. As suggested in the literature (Guan et al.,
increased in most cases (except for 0.005 g/L LAS) from 0.29 (0.03 g/L)
2017; Luo et al., 2022), the positive impact of surfactants could be
to 0.97 d (0.12 g/L). Overall, surfactant induced slight prolongation in
attributed to the disintegration of biopolymers (e.g., proteins, carbo­
lag phases but eventually led to higher VFA production rates.
hydrates) via tertiary structure unfolding, facilitating the bioavailability
of organic matters to fermentative bacteria. When a sufficient amount of
surfactant molecules is present in the solution, they combine to form
micelles (Wennerström and Lindman, 1979). In the micelles, the

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R.S. Bose et al. Bioresource Technology Reports 18 (2022) 101017

Table 2 concentration. The surfactant addition resulted in a 7% increase in VFA

VFA yield and kinetic parameters estimated with the first order rate equation yield inside 3 g/L salt amended reactor; 1% increase in 6 g/L reactor,
and modified Gompertz model fitting. followed by 6% increase in the reactors amended with 9–12 g/L of salt.
Test samples First order model Modified Gompertz model Furthermore, VFA production rates also decreased with increasing salt
Rate R2 Max. VFA Max. VFA Lag R2
concentrations under the fixed surfactant concentration (Table 2).
constant yield (mg production phase Viable cell number too declined sequentially with increasing salt con­
TVFA/g rate centration (Refer to Supporting Information). Nonetheless, the impact
(d− 1)
COD)
(mg TVFA/ (d) on lag phases showed a non-linear pattern, consistent with the effects of
g COD/d) different salt concentrations alone. Furthermore, no distinguishable
Control 0.31 ± 0.97 2152.71 657.38 ± 0.12 0.99
changes in VFA composition were induced by 0.06 g/L surfactant
0.05 ± ± 37.06 12.62 ± ± addition into varying salt amended reactors, except SF0.06_SL0.5
0.02 0.00 0.00 showed a slightly higher propionate fraction (Fig. 2c). However, it was
SL0.5 0.38 ± 0.99 2173.76 718.19 ± 0.50 0.99 consistent with the impact of surfactant alone, which indicated
0.02 ± 22.57 13.16
enhancement of fermentation via propionate production. As discussed
± ± ±
0.01 0.02 0.00
SL3 0.35 ± 0.99 1812.81 686.97 ± 0.46 0.99 previously, high osmotic pressure induced by high salt concentration >
0.03 ± ± 19.44 10.32 ± ± 3 g/L might lead to significant non-irreversible changes in the microbial
0.00 0.02 0.00 communities, which eventually alleviated the positive benefit of
SL6 0.32 ± 0.99 1655.39 596.87 ± 0.68 0.99 surfactant.
0.02 ± ± 16.46 9.61 ± ±
0.00 0.01 0.00
SL9 0.16 ± 0.99 885.58 ± 270.43 ± 0.66 0.99 3.4. Microbial community
0.03 ± 15.22 7.75 ± ±
0.01 0.03 0.00 The quantitative analysis of microbial communities was performed
SL12 0.05 ± 0.98 336.50 ± 92.85 ± 0.31 0.99
0.02 ± 9.38 3.89 ± ±
for SL6 (6 g/L salt) and SF0.06 (0.06 g/L surfactant) reactors to attain
0.02 0.05 0.00 insight into the respective impact on microbial communities during
SF0.005 0.32 ± 0.99 2077.66 687.97 ± 0.10 0.99 fermentation. The total microbial cell number in the SL6 reactor was
0.04 ± ± 31.29 10.11 ± ± 5.56 × 1010 cells/g sludge, which was lower than SF0.06 (3.06 × 1011
0.00 0.01 0.00
cells/g sludge). The high osmotic pressure introduced by salt possibly
SF0.03 0.40 ± 0.98 2380.80 700.75 ± 0.29 0.99
0.03 ± ± 17.54 12.03 ± ± caused a decrease in the quantity of microbial population in the SL6
0.02 0.02 0.00 reactor. The alpha diversity indices were calculated using QIIME2 to
SF0.06 0.42 ± 0.97 2816.65 663.23 ± 0.52 0.99 estimate the richness and evenness, and diversity of the microbial
0.02 ± ± 20.93 11.29 ± ± community in salt and surfactant amended reactors. The results of
0.02 0.00 0.00
Chao1 (9 vs. 7) and observed taxonomic units (OTUs) (74 vs. 60) showed
SF0.09 0.41 ± 0.97 2789.25 625.99 ± 0.84 0.99
0.01 ± ± 25.39 9.36 ± ± that the microbial richness in SF0.06 was higher than SL6. Similarly, the
0.02 0.02 0.00 evenness and diversity of the microbial community in SF0.06 were
SF0.12 0.44 ± 0.98 2852.68 671.71 ± 0.97 0.99 higher than SL6 in terms of Pielou evenness (0.89 vs. 0.78) and Shannon
0.02 ± 31.91 14.08
± ± ±
index (2.8 vs. 2.2). The higher richness and diversity could be attributed
0.01 0.02 0.00
SF0.06_SL0.5 0.41 ± 0.98 2331.02 798.44 ± 0.41 0.99
to the higher bacterial population in SF0.06 compared to SL6.
0.02 ± ± 20.82 13.41 ± ± The bacteria in both reactors were dominated by two phyla: Pro­
0.02 0.01 0.00 teobacteria and Firmicutes. The relative abundance of Proteobacteria in
SF0.06_SL3 0.36 ± 0.99 2016.74 638.12 ± 0.53 0.99 SL6 and SF0.06 were 93% and 63%, followed by Firmicutes 7% and
0.02 ± 18.05 15.74
37%, respectively. Proteobacteria and Firmicutes have been previously
± ± ±
0.01 0.01 0.00
SF0.06_SL6 0.34 ± 0.99 1828.60 585.31 ± 0.75 0.99 reported to be major bacterial phylum present in the anaerobic digestion
0.01 ± ± 16.98 11.92 ± ± of food waste for volatile fatty acids production (Wainaina et al., 2020;
0.00 0.03 0.00 Liu et al., 2018). Based on the class level, Gammaproteobacteria (93%)
SF0.06_SL9 0.16 ± 0.99 1014.50 276.21 ± 0.54 0.99
was the most dominant class in the SL6 reactor, followed by Bacilli (4%)
0.01 ± ± 14.79 10.88 ± ±
0.00 0.01 0.00
and Clostridia (3%); whereas nearly the equal population of Bacilli and
SF0.06_SL12 0.05 ± 0.99 356.06 ± 83.84 ± 0.55 0.99 Gammaproteobacteria (35–37%) were noticed in SF0.06 followed by a
0.00 ± 11.63 5.10 ± ± similar population of Alphaproteobacteria and Epsilonproteobacteria
0.00 0.02 0.00 (13–15%) (Fig. 3a).
In the SL6 reactor, the class of bacterial population Gammaproteo­
3.3. Combined effect of salt and surfactant on VFA generation bacteria were classified into orders of Enterobacteriales (68%), Pseu­
domonadales (12%), and Alteromonadales (11%); whereas
Based on the positive impact of 0.06 g/L of surfactant on VFA yield, Lactobacillales (4%) and Clostridiales (3%) were the sole order of bac­
this specific surfactant concentration under different salt concentrations teria under the class Bacilli and Clostridia, respectively (Fig. 3b). Based
(0.5–12 g/L) was further tested (see Fig. 1c). The fermentation pattern in on the genus level, the most abundant genus was Proteus (37%), fol­
salt and surfactant amended reactors, concerning lag phase and time of lowed by Acinetobacter (12%), Lactococcus (4%), and Phascolarctobacte­
reaching a plateau, was similar to salt-only fermentation (discussed rium (3%) (Fig. 3c). Proteus, Acinetobacter, and various genera of the
earlier). However, surfactant addition resulted in a 15% increase in VFA Enterobacteriaceae family are well-known mediators of food spoilage
concentration in 0.5 g/L salt amended reactor compared to the reactor and food waste degradation reported in various studies (Wu et al.,
amended with 0.5 g/L salt alone. Furthermore, compared to the control, 2018;). It is worthy to note that Proteus and Acinetobacter were often
VFA concentration for SF0.06_SL0.5 increased by 15.39% (2264 vs. reported for their ability in resistant to high salt concentrations (Abbas
1962 mg/L) compared to the control. Hence, VFA production under the et al., 2016; Zeidler and Müller, 2019; Ke et al., 2021). Even though the
effect of salinity in potable water concentration range ≤ 0.5 g/L is high abundance of genera of Proteobacteria, the high salinity diminishes
stimulated in the presence of surfactant. However, the benefit of the the activity of Proteobacteria, and also inhibit the population of Firmi­
presence of surfactant mostly declined with increasing salt cutes and thereby reducing overall VFA generation.
On the other hand, in the SF0.06 reactor, different classes of phylum

5
R.S. Bose et al.
Fig. 3. Relative abundance of bacterial communities in SL6 and SF0.06 reactors
at the (a) class, (b) order, and (c) genus levels.
Proteobacteria were classified into orders Aeromonadales (21%), Rick­
ettsiales (15%), Enterobacteriales (13%), Campylobacterales (13%) and
Pseudomonadales (2%); whereas phylum Firmicutes solely consisted of
order Lactobacillales (37%) (Fig. 3b). Low acetate concentrations have
been reported to promote the growth of Rickettsiales (Mosbæk et al.,
2016), which is similarly observed in our study. Based on genus level,
Lactococcus (27%) was the most dominant genus, followed by Aeromonas
(21%), Arcobacter (13%), and Saprolegnia (10%). Other genera were
observed Leuconostoc (6%) and Pseudomonas (2%) (Fig. 3c). Lactococcus
is a member of Lactobacillales order, which has the ability to enhance
volatile fatty acids generation (Wainaina et al., 2020; Jin et al., 2019).
Also, members of Lactobacillales order have been reported to produce
propionic acid from lactic acid (Zhang et al., 2010). Thus, increasing the
propionic acid concentration in surfactant amended reactors can be
attributed to the high abundance of Lactobacillales in the reactors.
Moreover, Aeromonas is known to be facultative acetogens (Rodriguez-
Morales and Castañeda-Hernández, 2019), and has been reported in
previous studies for their ability to degrade surfactants (Centurion et al.,
2018; de Souza Dornelles et al., 2020). It seems that the surfactant
addition increased the diversity of Proteobacteria and Firmicutes,
thereby increasing overall VFA production.
6
Bioresource Technology Reports 18 (2022) 101017
4. Conclusions
Our study suggests that low-strength kitchen wastewater having
salinity in the potable water range (0.105–0.5 g/L) can be successfully
utilized for VFA production through fermentation. Further, the presence
of surfactants in the range 0.005–0.12 g/L has a stimulatory effect on
VFA production in the above salt concentration. However, with the
increasing salinity, the feasibility of VFA production from kitchen
wastewater is noticed to decrease, and the presence of surfactants has a
negligible stimulatory effect on VFA production.
CRediT authorship contribution statement
Raj Shekhar Bose: Conceptualization, Methodology, Investigation,
Data curation, Formal analysis, Visualization, Writing – original draft.
Basem S. Zakaria: Writing – review & editing. Bipro Ranjan Dhar:
Supervision, Funding acquisition, Writing – review & editing. Manoj
Kumar Tiwari: Supervision, Project administration, Funding acquisi­
tion, Writing – review & editing.
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
The project was supported by Ministry of Human Resource Devel­
opment, Government of India (R.S. Bose), Izaak Walton Killam Memo­
rial Scholarship (B.S. Zakaria), Natural Sciences and Engineering
Research Council of Canada (NSERC) Discovery Grant (B.R. Dhar),
Ministry of Human Resource Development, Government of India (M. K.
Tiwari).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biteb.2022.101017.
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