Journal of Environmental Management 237 (2019) 228–234

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Journal of Environmental Management

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Research article

Carbon balance of major volatile fatty acids (VFAs) in recycling algal residue T
via a VFA-platform for reproduction of algal biomass
Donghyun Kima, Sungwhan Kima, Jong-In Hanb, Ji-Won Yanga, Yong Keun Changa,
Byung-Gon Ryuc,∗
a
Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, South Korea
b
Department of Civil and Environmental Engineering, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, South Korea
c
Freshwater Bioresources Utilization Bureau, Nakdonggang National Institute of Biological Resources, 137, Donam 2-gil, Sangju-si, Gyeongsangbuk-do 37242, South Korea

ARTICLE INFO ABSTRACT

Keywords: The feasibility of a carbon recycling system that transforms algal residue to volatile fatty acids (VFAs) for re-
Carbon recycling cultivating microalgae was evaluated based on a carbon balance analysis of major VFAs consisting of acetate
Volatile fatty acid (HAc), propionate (HPr), and butyrate (HBu). This system largely involves two processes: (i) bioconversion of
Anaerobic fermentation algal residue to VFAs by anaerobic fermentation, and (ii) cultivation of microalgae using the produced VFAs. The
Microalgae
carbon balance for each unit process was examined to assess how much carbon in algal residue can be converted
to these major VFAs and then assimilated to microalgae biomass. First, the yield and the profile of VFAs from raw
algae (RA) and lipid-extracted algae (LEA) at psychrophilic (15 °C), mesophilic (35 °C), and thermophilic con-
ditions (55 °C) were compared. When digesting the LEA under the thermophilic condition, the highest conver-
sion yield, 0.36 (g carbon in VFAs/g carbon in biomass), with a compositional ratio of 6:1:3 (HAc: HPr: HBu) was
obtained. Consumption of VFAs for microalgal growth reached a maximum value of 0.66 (g VFAs assimilated to
biomass/g VFAs provided) at the compositional ratio of 6:1:3. Consequently, the maximum total carbon re-
cycling ratio was 23.8% when fermenting LEA at the thermophilic condition. Our findings comprehensively
revealed that establishing conditions that convert LEA to higher content of acetate is a decisive factor. It was
estimated that around 40% of the total carbon from the LEA can be recovered for the production of algal
biomass, when increasing the VFA conversion yield beyond 60% by adopting pretreatment methods.

1. Introduction residual biomass remaining after the processing of microalgae (Ykema

The global market based on microalgae is expanding with its potential
as a feedstock for a wide range of useful products such as biodiesel,
bioethanol, proteins, biohydrogen, etc (Mohan and Devi, 2012a). Micro-
algae are advantageous over the conventional (crop or lignocellulosic)
biomass, as they grow faster and accumulate higher lipid content while
requiring less land usage (Qiao and Wang, 2009). Three distinct methods
are available for cultivating microalgae depending on the type of carbon
source for the synthesis of biomass: photoautotrophic, heterotrophic, and
mixotrophic methods (Moon et al., 2013). The heterotrophic and mixo-
trophic methods generally produce more biomass over the photo-
autotrophic approach, but they have a major disadvantage of involving an
expensive organic carbon source such as glucose, which accounts for over
60% of the total production cost (Li et al., 2007; Fei et al., 2011).
Significant efforts have been devoted to finding inexpensive organic
carbon sources such as wheat straw, whey permeate, sorghum, and
et al., 1988; Economou et al., 2010; Yu et al., 2011). In particular, the
algal residue is a byproduct of the lipid extraction process for algal
biofuels, which is an organic waste saturated with toxic solvents.
(Suresh et al., 2013). This on-site waste, which is produced con-
tinuously with biofuels, can either be a stable carbon source for mi-
croalgae, or a pollution source requiring a high oxygen demand to be
degraded if not properly treated (Fei et al., 2011; Suresh et al., 2013;
Chisti, 2007). Thus, recycling organic carbon in the algal residue for
reproduction of the algal biomass is a sustainable strategy that mini-
mizes both the cost and waste (Suresh et al., 2013; Ehimen et al., 2011).
The necessity of a bioconversion process was previously addressed for
transforming unavailable carbon into consumable carbon for micro-
algae (Mohan and Devi, 2012a; Suresh et al., 2013; Liu et al., 2016;
Chang et al., 2010; Naresh Kumar et al., 2018).
A volatile fatty acids (VFAs) platform based on anaerobic digestion,
which generally involves hydrolysis and acidogenesis steps, converts

∗
Corresponding author.
E-mail address: tesia@nnibr.re.kr (B.-G. Ryu).

https://doi.org/10.1016/j.jenvman.2019.02.040
Received 31 August 2018; Received in revised form 28 December 2018; Accepted 7 February 2019
0301-4797/ © 2019 Elsevier Ltd. All rights reserved.
D. Kim, et al. Journal of Environmental Management 237 (2019) 228–234

the carbohydrates or protein in algal residue to VFAs and biohydrogen Table 1

(Kim and Kang, 2015; Lee et al., 2014). The VFAs mainly consist of Characteristics of raw algae (RA) and lipid extracted algae (LEA).
acetic acid (HAc), propionic acid (HPr), and butyric acid (HBu), which Parameter RA LEA
are efficient carbon sources with short biotransformation pathways for
algal growth and lipid accumulation; these major VFAs can serve as Total COD (mg/g) 969.0 ± 11.0 1147.0 ± 101.0
Carbohydrate (mg/g) 204.1 ± 9.7 286.0 ± 5.7
building blocks of carbon chains without being converted to sugar
Protein (mg/g) 475.0 ± 1.0 568.0 ± 1.0
molecules (Moon et al., 2013; Fei et al., 2011; Perez-Garcia et al., 2011; Fatty acids content (mg/g) 90.6 ± 0.3 4.4 ± 0.1
Ryu et al., 2014). A similar concept was practically examined for the Ash (mg/g) 5.9 ± 0.1 5.5 ± 0.0
production of polyhydroxyalkanoates (PHAs) and removal of nutrients Carbon (%) 49.1 ± 0.1 44.7 ± 0.0
from sewage sludge (Lee et al., 2014). Over the past few decades, Hydrogen (%) 7.1 ± 0.1 6.5 ± 0.1
Nitrogen (%) 7.6 ± 0.0 9.1 ± 0.0
however, most previous research has covered each bioprocess (con-
Sulfur (%) 0.4 ± 0.0 0.1 ± 0.0
version of biomass residue into VFAs or biomass production using VFAs Oxygen (%) 30.5 ± 0.2 34.4 ± 0.5
as organic carbon) in a separated manner (Liu et al., 2016). A com-
prehensive evaluation of the whole carbon recycling process, from
conversion of VFAs to biomass production, is also important, but little (TSS), 11.4 ± 0.4 g/L of volatile suspended solids (VSS), and a pH of
research has been dedicated to this. 6.50 ± 0.04. The sludge was predigested for 24 h at room temperature
The main idea behind this study, therefore, is to evaluate the fea- before the experiment to remove remaining organic substrates.
sibility of an integrated system that consists of converting a lipid-ex- A 250-mL serum bottle with 200-mL working volume, containing
tracted algae (LEA, or also can be referred as de-oiled algal biomass) to RA or LEA with a food/microbe ratio (FM ratio) based on VSS of 1:1,
the VFAs and re-cultivation of algal biomass using the VFAs, based on a was prepared for anaerobic fermentation. Time-dependent productiv-
carbon balance analysis. Bioconversion yield from algal residue to the ities of volatile fatty acids (VFAs) and methane were examined during
VFAs and biogas with their compositional ratios at psychrophilic anaerobic digestion at three different temperature conditions: psy-
(15 °C), mesophilic (35 °C), and thermophilic conditions (55 °C) were chrophilic (15 °C), mesophilic (35 °C), and thermophilic (55 °C) condi-
compared. The effects of the VFA compositional ratio, obtained under tions. The bottom plate holding the serum bottles was rotated at
the tested anaerobic fermentation conditions, on the productivity of 50 rpm, which was sufficient to provide mild mixing but was not so
algal biomass were investigated using synthetic VFA containing vigorous that it would break the anaerobic bacterial flocs. Samples for
medium. The carbon balance in each of these steps was comprehen- measuring both biogas and supernatant were taken every 24 h and all
sively estimated. experiments were carried out in duplicate.

2. Materials and methods

2.3. Microalga cultivation
2.1. Preparation of raw algae (RA) and lipid-extracted algae (LEA) as
To systemically investigate the effects of the VFA compositional ratio
substrate
on the productivity of algal biomass, artificial VFA mixtures, with com-
positional ratios (HAc: HPr: HBu) based on anaerobic fermentation using
A green microalgal strain, Chlorella vulgaris UTEX-265, which is com-
RA and LEA at various temperatures, were prepared. A modified TAP
monly used for biodiesel production, was cultivated in a 30-L outdoor
medium (2.42 g of Tris, 25 mL of TAP salts, 0.375-mL of phosphate so-
photo-bioreactor in a typical N-8 medium containing KNO3 (1000 mg/L),
lution, and 1-mL Hunter's trace elements and 2 g of mixed volatile fatty
K2PO4 (740 mg/L), Na2HPO4·2H2O (260 mg/L), CaCl2·2H2O (13 mg/L),
acids in 1-L volume) consisting of different mass ratios of VFAs (HAc:
Fe EDTA (10 mg/L), MgSO4·7H2O (50 mg/L), and micronutrients (1 mL)
HPr: HBu = 7:1:2, 7:2:1, 7:3:0, 6:1:3, 5:4:1) that were observed in the
(Mandalam and Palsson, 1998) with a 10% CO2 supply in Korea Institute
actual AD effluents was prepared. Since the ratios obtained from the
of Energy Research (KIER, Daejeon, Republic of Korea). The algal biomass
experimental values did not exactly coincide exactly with integers, we set
was obtained after a sequential process of harvesting by using a centrifuge
them to the most similar representative values with reference to the
and drying by using a freeze-dryer, to be utilized as raw algae (RA) feed
existing literature (Chang et al., 2010; Gao et al., 2017; Park et al., 2017).
biomass for anaerobic digestion.
Each medium was sterilized by using an autoclave operated at
For preparing LEA, 250 g of RA biomass was put into a 5-L bottle
120 °C for 20 min. A 250-mL baffled flask with an air filter cap was
filled with 4.5-L of chloroform and ethanol (v/v = 2:1), and stirred for
sterilized in an autoclave at 120 °C for 20 min. The flask was filled with
48 h to extract total fatty acids. After 48 h, the stirring was stopped to
120-mL of the artificial VFA medium. Inoculum concentration was set
induce natural sedimentation of the biomass for 2 h. The supernatant
at an optical density of 0.3 (650 nm of wave length): this was done by
containing the mixture of organic solvents and hydrophilic components
first centrifuging the seed culture and then resuspending the pellet with
such as fatty acids and/or various pigments was poured out carefully,
the medium so as to reach the target cell density. Each algal culture was
and a biomass slurry soaked with the remaining solvents was obtained.
cultivated at 120 rpm and 25 °C for 8 days under fluorescent light in-
The biomass slurry was then centrifuged for 5 min at 3000 rpm to fur-
tensity of 100 μmol/m2∙s. All batch tests were performed in duplicate.
ther separate the biomass and the solvents. The biomass was put in a
hood for 24 h and stored in a dry-oven at 40 °C for an additional 4 h to
evaporate the residual organic solvents. It was confirmed that the fatty 2.4. Carbon balance
acids were extracted from the biomass remaining after the aforemen-
tioned extraction process, and LEA was utilized as feed biomass for the Based on the results from an elemental analysis described in section
anaerobic digestion. Detailed characteristics of the RA and LEA are 2.5.1 below, the carbon contents (w/w biomass) of RA and LEA were
summarized in Table 1. obtained and used for the calculation. The carbon fraction of VFAs and
biogas was calculated by using the sum of the atomic weight of carbon
2.2. Anaerobic fermentation of RA and LEA (12.011) as a proportion of the molecular weight of each VFA, lactate
(90.08 g mol−1), formate (46.03 g mol−1), acetate (59.04 g mol−1),
Microbial seed sludge for the inoculation of anaerobic digestion propionate (74.08 g mol−1), butyrate (87.09 g mol−1), and each biogas,
(AD) was obtained from a mesophilic anaerobic digester (wastewater methane (16.04 g mol−1) and carbon dioxide (44.01 g mol−1). When
treatment plant, Daejeon, Republic of Korea). The anaerobic seed calculating the mole number of the biogas, a standard pressure and
sludge used in this study had 20.2 ± 0.2 g/L of total suspended solids temperature (STP) condition was assumed. The calculation was focused

229
D. Kim, et al.
Fig. 1. Time profile of volatile fatty acids (VFAs) production at psychrophilic, mesophilic, and thermophilic condition during five days of anaerobic fermentation
under a methanogen-inhibitory condition.
on tracking the carbon fraction/mass that was biodegraded from bio-
mass residue to VFAs, or assimilated from VFAs to biomass.
2.5. Analytical methods
2.5.1. Characteristics of RA and LEA
Both RA and LEA as substrates of AD were freeze-dried at −52 °C
for three days and then characterized (Table 1). COD was analyzed by
using a Humas kit (HS-COD-HR, Humas Co., Ltd. Republic of Korea)
and a UV spectrometer (DR 2010; HACH, USA). Total carbohydrate
concentration was determined by using the phenol-sulfuric method
(Dubois et al., 1956). The portions of carbon, hydrogen, nitrogen,
sulfur, and oxygen were analyzed by using an elemental analyzer
(FLASH 2000 series, Thermo Scientific, USA). Protein content was es-
timated based on an elemental analysis (Suresh et al., 2013). Ash
content was measured by calculating the difference in weight between
the biomass before and after digestion in a furnace at 750 °C for 4 h.
2.5.2. Fatty acid methyl esters (FAMEs)
A microalgae sample was centrifuged for 10 min at 3500 rpm to se-
parate it into a pellet and a supernatant, and the pellet was freeze-dried at
−52 °C for three days. The lyophilized biomass (10-mg) was resuspended
with a mixture of chloroform and methanol with a ratio of 2:1 (v/v) to
extract lipids. This extracted oil was converted to fatty acid methyl esters
(FAMEs) with methanol in the presence of sulfuric acid at 100 °C for
20 min. After the addition of deionized water into the mixture, the organic
phase in the bottom layer was collected and analyzed by using a gas
chromatograph (HP5890; Agilent, CA, USA) equipped with an INNOWAX
capillary column (30 m × 0.32 mm × 0.5 μm; Agilent) and a flame io-
nization detector (FID). The temperature of the oven was raised from 50 to
200 °C at 15 °C per min and then to 250 °C at 10 °C per min.
2.5.3. Biogas
Biogas was sampled from 50-mL of a headspace of the serum bottle
and then its CH4 and H2 composition was measured on a daily basis by a
gas chromatograph (Hewlett Packard 6890 Series GC System; city,
country) equipped with Porapak Q packed 80–100 mesh columns (Agilient
230
Journal of Environmental Management 237 (2019) 228–234
Technologies, USA) using nitrogen as a carrier gas. Temperatures of the
column and the oven were set at 250 °C and 50 °C, respectively.
2.5.4. Volatile fatty acids (VFAs)
In the AD-effluent and modified TAP medium, 1-mL of the super-
natant was prepared by centrifugation at 7000 rpm for 10 min and fil-
tration (0.22-μm) for detecting VFAs. The profile and the concentrations
of the VFAs were measured every 24 h during the anaerobic fermen-
tation by means of a high-performance liquid chromatograph
(Younglin, South Korea) equipped with an Aminex HPX-87H column
(Bio-Rad Laboratories, USA) and a refractive index (RI) detector.
Temperatures of the column and the detector were set at 60 °C and
room temperature, respectively. A 5-mM solution of H2SO4 was used as
an eluent.
2.5.5. Growth curve of C. vulgaris
To monitor the growth of microalgae in the modified TAP medium
containing VFAs, 3 mL of culture solution was taken and then analyzed
by using a UV–visible spectrophotometer (UV-1800; Shimadzu, Kyoto,
Japan) at wavelengths of 680 nm and 750 nm. To ensure the optical
density (OD) reading was reliable, the culture sample was diluted to
have an OD between 0.1 and 1.
3. Results and discussion
3.1. Effect of temperature on yield and compositional ratios of major VFAs
from RA and LEA and carbon conversion yield
Fig. 1 presents the time-dependent production of VFAs from RA and
LEA biomass at 15 °C, 35 °C, and 55 °C for five days of anaerobic fer-
mentation. RA was employed as a reference substrate for comparison
with LEA. Total concentrations of VFAs generated from algal biomass
increased with the operating temperature (Fig. 1). At the end of anae-
robic fermentation, the concentrations of VFAs were 0.9 ± 0.07,
1.92 ± 0.01, and 1.97 ± 0.02 g/L at 15 °C, 35 °C, and 55 °C, respec-
tively, when RA was used as a substrate (Fig. 1-a, -c, and -e). The
patterns of VFA production from LEA, which increased with
D. Kim, et al. Journal of Environmental Management 237 (2019) 228–234

Table 2
Carbon conversion yield from algal biomass (RA and LEA) to VFAs. The amount of produced VFAs is presented and the proportion of carbon converted to each VFA
among total converted carbon was calculated.
Conversion of carbon from algal biomass to VFAs

Type of biomass Temperature VFAs ratio Lactatec Formatec Acetatec Propionatec Butyratec Total VFAsc Carbon conversion
(HAc: HPr: HBu) (P1, %) (P1, %)a (P1, %) (P1, %) (P1, %) (YVFAs/S)b
RA 15 7:3:0 0.06 ± 0.06 ND 0.58 ± 0.05 0.24 ± 0.07 0.03 ± 0.01 0.91 ± 0.07 0.14
(6.4 ± 3.2) 0 (60.5 ± 0.4) (29.2 ± 3.1) (4.0 ± 0.5)
35 7:2:1 ND ND 1.31 ± 0.00 0.38 ± 0.00 0.23 ± 0.00 1.92 ± 0.01 0.30
0 0 (62.8 ± 0.0) (22.4 ± 0.1) (14.8 ± 0.1)
55 7:1:2 ND 0.09 ± 0.01 1.23 ± 0.00 0.20 ± 0.00 0.45 ± 0.01 1.97 ± 0.02 0.31
0 (2.8 ± 0.2) (57.5 ± 0.3) (11.2 ± 0.0) (28.5 ± 0.1)
LEA 15 7:3:0 ND 0.05 ± 0.03 0.37 ± 0.07 0.18 ± 0.07 ND 0.60 ± 0.17 0.09
0 (5.3 ± 0.6) (61.2 ± 2.7) (33.5 ± 2.1) 0
35 5:4:1 ND 0.06 ± 0.00 0.88 ± 0.02 0.66 ± 0.00 0.13 ± 0.00 1.71 ± 0.04 0.29
0 (0.9 ± 0.5) (46.6 ± 0.3) (42.7 ± 0.2) (9.7 ± 0.5)
55 6:1:3 ND 0.06 ± 0.01 1.24 ± 0.05 0.27 ± 0.00 0.50 ± 0.01 2.07 ± 0.05 0.36
0 (1.6 ± 0.1) (54.2 ± 0.5) (14.6 ± 0.2) (29.6 ± 0.2)

ND: The element was not detected.

a
P1, %: Carbon proportion (%) of each fatty acid in carbon of total VFAs. Calculation of P1 was performed as follows: P1, % = CFA/CVFAs × 100, where CFA and
CVFAs are the carbon fraction of each fatty acid and total VFAs, respectively.
b
YVFAs/S: Carbon conversion yield; g carbon in total VFAs produced/g algal biomass (substrate).
c
Concentration (g/L) of VFAs produced by anaerobic fermentation from algal biomass.

temperature, are also similar to those observed in RA (Fig. 1-b, -d, and
f); the highest VFAs amounts (2.06 ± 0.05 g/L) were yielded at the
thermophilic condition (55 °C) (Fig. 1-f). These results were consistent
with previous findings that production of organic acids was higher at a
thermophilic condition than that observed in a mesophilic condition
when fermenting algal residue, as relatively sufficient soluble carbo-
hydrate and protein were converted into VFAs efficiently by higher
activities of acidogenic bacterial members (Cho et al., 2015). The
maximum VFA yield at the thermophilic condition (55 °C) was 1.4-fold
higher than that reported by Suresh et al. (2013) who obtained 0.26 g
VFA/g VS with alkali-sonicated algal biomass at a mesophilic condition.
The carbon conversion yield (YVFAs/S value) at this condition reached
the maximum value of 0.36 with high HAc and HBu content (Table 2).
The VFA composition is as important as the VFA conversion yield,
because the VFA composition determines the carbon consumption yield
in microalgae cultivation (Mohan and Devi, 2012a; Moon et al., 2013;
Fei et al., 2015). During the anaerobic fermentation, various VFAs were
produced including lactic acid, formic acid, acetic acid (HAc), propionic
acid (HPr), and n-Butyric acid (HBu). As reported in a previous study
(Chang et al., 2010), three VFAs, HAc, HPr, and HBu, were the major
fatty acids. The portion of HAc was the largest in general; HAc con-
stituted 51.6 ± 0.6% to 68.2 ± 0.1% of total VFAs at all temperatures
(Fig. 1), which corresponded to 46.6–62.8% in regard to the carbon
conversion (P1 value in Table 2). This observation is in line with pre-
vious reports that HAc is a major component during anaerobic VFA
fermentation using microalgal biomass (Suresh et al., 2013; Naresh
Kumar et al., 2018; Cho et al., 2015). The production of HAc increased
with the operating temperature, and thus the total VFAs.
Biogas was also monitored to confirm that the methanogens were suf-
ficiently inhibited; VFA conversion yield can decrease if the methanogen is
not inhibited, as methanogens consume VFAs for producing methane (Wang
et al., 2009). In every temperature condition, only a small amount of me-
thane was detected in RA, and almost none was detected in LEA
(Supplementary Fig. 1). Carbon conversion in the form of methane ac-
counted for less than 3% (RA) and 0.1% (LEA), respectively (Supplementary
Table 1), implying that a negligible amount of carbon was converted to the
methane in both RA and LEA. More inhibition of the methanogenic member
was observed when using LEA, likely due to toxicity of the chloroform and
methanol mixture, which was used to extract the cellular lipids and re-
mained in a small amount. Fortunately, inhibition of microbial members
involved in acidogenesis was negligible at 55 °C, although slight declines in
the VFAs production were observed at 15 °C and 35 °C.
3.2. Effect of compositional ratio of major VFAs on algal growth and carbon
conversion yield
The compositional ratio of VFAs can significantly influence the
cell growth and biomass production when VFAs are utilized as a
carbon source for microalgae (Fei et al., 2011; Ryu et al., 2017).
Biomass (final dried cell weight) production was directly proportional
to the amount of HAc in the medium. As shown in Fig. 2, more than
80% of HAc was consumed by microalgae in all the tested ratios while
HPr and HBr were partially assimilated. Although the differences
were negligible, microalgae tended to consume more HBu than HPr.
This selective preference of microalgae may originate from differ-
ences in the metabolic pathway of utilizing VFAs. HAc could readily
and directly be utilized for the TCA cycle and fatty acid synthesis
metabolism by transforming it into acetyl-coA (Perez-Garcia et al.,
2011). Consumptions of other two VFAs were substantially lower
than HAc, as they need to be transformed through several pathways
to be utilized. HBu can be converted to butyryl-coA and then possibly
degraded to 2 mol of acetyl-CoA via β-oxidation (Mohan and Devi,
2012b). HPr was suggested to be a precursor of oxaloacetate, which is
an intermediate in the TCA cycle and glyoxylate pathway (Callely and
Lloyd, 1964). The more detailed mechanism of HPr consumption is
not fully understood but is assumed to be complex, and HPr con-
sumption is reported to be lower than that of HBu (Mohan and Devi,
2012a). The differences in consumption rate and tolerance of/on in-
dividual VFAs provide useful information for determining desirable
operating conditions for previous anaerobic fermentation; an HAc-
rich condition will be beneficial for providing a readily consumable
carbon source – i.e., a thermophilic condition.
The carbon conversion yield in this step is summarized in Table 3.
The carbon conversion yield (YALG/VFAs value) from VFAs to micro-
algal biomass corresponded to the portion of HAc, and ranged from
0.51 to 0.66 (Table 3). The ratio of 5:4:1 showed the lowest value
(0.51), as the proportion of HAc was the lowest and thus was a readily
available organic carbon source. The consumption (P3 value) of HPr
ranged from 3.2 to 6.3% only, and meanwhile that of HBu ranged
from 6.9 to 16.8%. Even when HBu was absent and HPr accounted for
30% of the total VFAs (7:3:0), the consumption rate was still very low
(3.2%). Among the total carbon converted from VFAs to microalgal
biomass, 79.3–96.8% was in the form of HAc (Table 3). Higher carbon
conversion yield (YALG/VFAs value) was directly correlated with higher
HAc content.

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Fig. 2. Microalgal growth curve displayed with final dried cell weight and initial/final VFAs concentration in the medium. Note that the labeling of the substrate type
and temperature in each sub-figure (i.e. (a) RA, 15 °C) indicates the conditions used for the previous anaerobic fermentation, and the microalgae cultivation was
carried out under a constant temperature condition (25 °C) with an artificial medium reflecting the VFA composition obtained in the previous anaerobic fermen-
tation.

3.3. Total carbon recycling ratio
Total carbon recycling ratios (TCRs) were calculated by multiplying
the carbon conversion yield from biomass to VFAs and that from VFAs
to biomass. The value of the TCR ranged from 6.3 to 23.8% (Table 4).
The maximum carbon conversion yield in the step 1 (VFA platform) and
in step 2 (re-cultivation of microalgae) reached 0.36 and 0.66, respec-
tively. When coupling these two carbon conversion processes, HAc was
a major carbon carrier and accounted more than half of the total carbon
conversion yield in each process. The overall tendency in each process
and in the total process is presented in Fig. 3. The conversion yield of
step 1 (from biomass to VFAs, red line) tended to increase along with
the operating temperature of the anaerobic fermentation. Meanwhile,
that of step 2 (from VFAs to microalgal biomass, green line) appeared to
be strongly related to the portion of HAc in the VFA mixture in the
medium. As the conversion yield of step 2 was almost constant (except
the 5:4:1), the TCR follows the trend of the conversion yield of step 1.
This implies that the TCR can be further improved substantially by
enhancing the anaerobic fermentation.
Under the assumption that the biomass was pretreated sufficiently,
TCR was estimated with hypothetical values of the VFA conversion
yield (YVFAs/S value) ranging from 0.4 to 0.6 (Table 4). The value of 0.6
was based on a previous study that reported two-fold increased acid
yield (0.6 g acid/g VSS) by applying a lime pretreatment on rice straw,
which mainly consisted of lignocellosic biomass (Agbogbo, 2007). This
method can be speculated to be effective on microalgae as well, which

Table 3
Carbon conversion yield from VFAs to microalgal biomass. The amount of consumed VFAs was presented and the proportion of utilized carbon to each VFA among
total consumed carbon was calculated.
Conversion of carbon from VFAs to microalgal biomass

Type of biomass Temperature VFAs ratio HAc (P3, %)a HPr (P3, %) HBu (P3, %) Produced algal biomass by VFAs Carbon conversion (YALG/VFAs)b

RA 15 7:3:0 1.22 ± 0.05 0.03 ± 0.01 ND 1.26 ± 0.06 0.63

(96.8 ± 0.4) (3.2 ± 0.4) 0
35 7:2:1 1.16 ± 0.04 0.06 ± 0.01 0.07 ± 0.01 1.29 ± 0.05 0.65
(87.4 ± 0.6) (5.6 ± 0.2) (7.0 ± 0.4)
55 7:1:2 1.18 ± 0.03 0.03 ± 0.00 0.11 ± 0.00 1.32 ± 0.03 0.66
(87.0 ± 0.1) (2.5 ± 0.0) (10.5 ± 0.1)
LEA 15 7:3:0 1.22 ± 0.05 0.03 ± 0.01 ND 1.26 ± 0.06 0.63
(96.8 ± 0.4) (3.2 ± 0.4) 0
35 5:4:1 0.92 ± 0.01 0.05 ± 0.00 0.05 ± 0.00 1.02 ± 0.01 0.51
(86.8 ± 0.0) (6.3 ± 0.1) (6.9 ± 0.2)
55 6:1:3 1.11 ± 0.02 0.05 ± 0.00 0.17 ± 0.02 1.33 ± 0.03 0.66
(79.3 ± 1.4) (3.9 ± 0.3) (16.8 ± 1.3)

ND: The element was not detected.

c
Concentration (mg/L) of VFAs consumed by microalgae in re-cultivation.
a
P3, %: Carbon proportion (%) of each fatty acid consumed by microalgae in carbon of total VFAs. Calculation of P3 was performed as follows: P3, % = CcFA/
CcVFAs × 100, where CcFA and CcVFAs are the carbon fraction of each fatty acid and total VFAs consumed by microalgae, respectively.
b
YALG/VFAs: Carbon conversion yield; g carbon in algal biomass produced/g VFAs (substrate) where, microalgal growth by other gaseous carbon sources such as
CO2 in ambient air was excluded in this estimation.

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D. Kim, et al. Journal of Environmental Management 237 (2019) 228–234

Table 4
Overall carbon recycling ratio in the integrated system of microalgal cultivation coupled with VFA platform.
Overall recycling rate of carbon in this system

Type of biomass Temperature Step 1. VFA platform Step 2. Re-cultivation of microalgae Total Carbon Recycling ratio (TCR, %)c

YHAc/Sa(g/g) YVFAs/S(g/g) YTGAS/S(g/g) YALG/HAcb(g/g) YALG/VFAs(g/g)

RA 15 0.08 ± 0.01 0.14 ± 0.01 0.00 ± 0.00 0.87 ± 0.04 0.63 ± 0.03 8.8
35 0.19 ± 0.00 0.30 ± 0.00 0.06 ± 0.00 0.83 ± 0.03 0.65 ± 0.03 19.5
55 0.18 ± 0.01 0.31 ± 0.00 0.08 ± 0.00 0.85 ± 0.02 0.66 ± 0.02 20.5
LEA 15 0.06 ± 0.01 0.10 ± 0.03 0.00 ± 0.00 0.87 ± 0.04 0.63 ± 0.03 6.3
35 0.14 ± 0.00 0.30 ± 0.00 0.01 ± 0.00 0.65 ± 0.01 0.51 ± 0.01 15.3
55 0.20 ± 0.00 0.36 ± 0.01 0.07 ± 0.01 0.79 ± 0.01 0.66 ± 0.02 23.8
Theoretical value 0.24g 0.4d 0.66g 26.4d
0.35g 0.5e 0.66g 33.0e
0.42g 0.6f 0.66g 39.6f

ND: The element was not detected.

a
YHAc/S: Carbon conversion yield; g carbon in acetate (HAc)/g algal biomass (substrate).
b
YALG/HAc: Carbon conversion yield; g carbon in produced algal biomass/g HAc (substrate).
c
TCR, %: Overall carbon recycling rate. Estimation of TCR was performed as follows: TCR % = YVFAs/S × YALG/VFAs × 100, where YTGAS/S, is not considered
because it is negligible in this estimation.
d
It is assumed that the value of YVFAs/S is 0.4.
e
It is assumed that the value of YVFAs/S is 0.5.
f
It is assumed that the value of YVFAs/S is 0.6.
g
The values of YHAc/S and YALG/VFAs are such that the compositional ratio of VFAs (HAc: Hbu: HPr) is 6:1:3.

Fig. 3. Carbon conversion yield of each process and whole process in each anaerobic fermentation condition with RA and LEA.

also have strong cell walls mainly composed of hemicellulose and
saccharides (Zheng et al., 2011). When the VFA conversion yield was
more than 0.6, the total carbon recycling ratio was calculated to be
39.6%. The results of the carbon balance analysis in this study suggest
that an integrated process of a VFA platform with a proper pretreatment
technique would be an effective method for carbon recycling of mi-
croalgal biomass.
recycling ratio. The maximum total carbon recycling ratio was found to
be 23.8%. The feasibility of this VFA platform-based carbon recycling
system could be enhanced to recover around 40% of the carbon if the
VFA conversion rate could be increased to more than 60% by em-
ploying pretreatment methods.
Acknowledgements

4. Conclusion
A carbon balance analysis of major VFAs in an integrated system
was performed to evaluate the feasibility of algal residue recycling via a
VFA platform. A thermophilic condition for anaerobic fermentation was
beneficial for increasing both the overall carbon conversion yield and
the proportion of HAc. Microalgae tended to consume significantly
more HAc compared to HPr and HBu. The results indicated that HAc
was a key carbon carrier for both biodegradation of the residual bio-
mass to VFAs and assimilation of VFAs to microalgal biomass. Not only
the total VFA conversion yield, but also the compositional ratio of VFA
with higher HAc content was directly correlated with a higher carbon
This work was supported by the Advanced Biomass R&D Center
(ABC) of the Global Frontier Project of Korea funded by the Ministry of
Science and ICT (ABC-2010-0029728).
This work was supported by a grant from the Nakdonggang National
Institute of Biological Resources (NNIBR), funded by the Ministry of
Environment of the Republic of Korea (NNIBR201901101).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jenvman.2019.02.040.

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