Bioresource Technology 191 (2015) 53–58

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Lactic acid production from acidogenic fermentation of fruit

and vegetable wastes
Yuanyuan Wu, Hailing Ma, Mingyue Zheng, Kaijun Wang ⇑
State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing 100084, China

h i g h l i g h t s

 Lactic acid with amount of 10–20 g/L was stably produced at pH 4.0.
 The fermentation type was finally converted into heterofermentation at pH 4.0.
 Hydrolysis was enhanced and lactic acid fermentation was improved at pH 5.0.
 Bifidobacterium played an important role for lactic acid production at pH 5.0.

a r t i c l e i n f o a b s t r a c t

Article history: This work focused on the lactic acid production from acidogenic fermentation of fruit and vegetable
Received 19 March 2015 wastes treatment. A long term completely stirred tank reactor (CSTR) lasting for 50 days was operated
Received in revised form 23 April 2015 at organic loading rate (OLR) of 11 gVS/(L d) and sludge retention time (SRT) of 3 days with pH controlled
Accepted 25 April 2015
at 4.0 (1–24 day) and 5.0 (25–50 day). The results indicated that high amount of approximately 10–20 g/L
Available online 7 May 2015
lactic acid was produced at pH of 4.0 and the fermentation type converted from coexistence of homofer-
mentation and heterofermentation into heterofermentation. At pH of 5.0, the hydrolysis reaction was
Keywords:
improved and the total concentration of fermentation products increased up to 29.5 gCOD/L. The hetero-
Lactic acid
Heterofermentation
fermentation was maintained, however, bifidus pathway by Bifidobacterium played an important role.
Anaerobic digestion Ó 2015 Elsevier Ltd. All rights reserved.
Acidogenic fermentation
Fruit and vegetable wastes

1. Introduction treatment to keep the stability of whole system at high OLR

In China, fruit and vegetable wastes (FVWs) are greatly gener-
ated approximately 1.3 million tons per day in Chinese cities, how-
ever, less than 20% were properly treated (Shen et al., 2013; Liu
et al., 2012). Anaerobic digestion has been considered to be the
most promising alternative technology for FVWs treatment. With
the characteristics of high volatile solids and easy degradability,
the FVWs could be rapidly hydrolyzed, which might lead to the
acid accumulation and inhibition to methanogenesis if the reactor
is overloaded. Ganesh et al. (2014) summarized the published lit-
eratures and concluded that the maximum OLR for single-phase
anaerobic digestion of FVWs was within 3.6 KgVS/(m3 d).
According to the study of Shen et al. (2013), volatile fatty acids
(VFAs) would accumulate to 2.3 g/L and pH would decrease to
6.8 if the OLR was increased to 3.5 KgVS/(m3 d). And then,
two-phase anaerobic digestion technology was applied for FVWs
⇑ Corresponding author. Tel.: +86 10 62789411; fax: +86 10 62773065.
E-mail address: wkj@tsinghua.edu.cn (K. Wang).
(Mtz-Viturtia et al., 1995; Bouallagui et al., 2004; Shen et al.,
2013). However, the OLR of two-phase anaerobic digestion were
only reported with limited increase to 5.7–7.7 KgVS/(m3 d), which
was summarized by Ganesh et al. (2014). In addition, the construc-
tion and operational cost were also increased for the additional
reactors and alkaline addition.
Recently the concept of VFAs platform has been proposed and
based on that, several end products other than methane such as
polyhydroxyalkanoates (PHAs), medium chain fatty acids, biofuels
or hydrogen gas are being researched (Chang et al., 2010; Reis
et al., 2003; Agler et al., 2012; Steinbusch et al., 2000). There have
been a lot of researches on the VFAs production from different
wastes or wastewaters. Jiang et al. (2013) found that the optimal
condition for VFAs production were pH 6.0, 35 °C, 11gTS/(L d) from
food waste with acetate and butyrate accounting for 60%. Chen
et al. (2007) reported that alkaline pH (8.0–11.0) could signifi-
cantly improve the total VFAs concentration from waste activated
sludge and acetate, propionate and isovaleric acid were the three
main VFAs. Yu and Fang (2002) recommended that productions

http://dx.doi.org/10.1016/j.biortech.2015.04.100
0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
54 Y. Wu et al. / Bioresource Technology 191 (2015) 53–58
of butyrate and acetate were favored at pH of 6.0–6.5 while etha-
nol and propionate at pH 4.0–4.5 from dairy wastewater.
However, most of the studies focused on VFAs production (acet-
ate, propionate, iso-butyrate, butyrate, iso-valerate, valerate), only
a handful of studies paid attention to other fermentation products
such as lactic acid, ethanol or succinate. Lactic acid has been veri-
fied as an available building block for PHAs and it is also widely
used in industries such as the pharmaceutical, biomaterial, deter-
gent, leather, and textile industries (Jiang et al., 2011; Kim et al.,
2012; Mazzoli et al., 2014). Temudo et al. (2007) investigated the
influence of pH on glucose fermentation by mixed culture and indi-
cated that lactic acid with low yield rate of 0.2 mol/mol glucose
was produced at pH of 4.0–5.0. Wang et al. (2014) evaluated the
effect of pH on acidogenic end products from food waste in batch
experiments and found that high concentration of 18 g/L lactic acid
was produced at pH of 4.0. Selective production of lactic acid was
tried with glucose as substrate by Itoh et al. (2012) and results
showed that lactic acid was produced at low pH of 3.5, however,
the lactic fermentation was not stable. Thus, the lactic acid could
be produced from the acidogenic fermentation at low pH, however
further investigation such as the fermentation stability in long
term operation, fermentation mechanism and bacterial community
are still necessary.
Therefore, a long term CSTR reactor was operated to investigate
the possibility and stability of lactic acid production from acido-
genic fermentation of FVWs treatment. The bacterial community
and fermentation mechanism for lactic acid fermentation were
also investigated. In addition, the method of increasing pH was also
tried to improve the lactic acid fermentation.
2. Methods
2.1. Inoculums and substrate
The seed sludge was taken from anaerobic digester in Xiao
Hongmen wastewater treatment plant in Beijing city, China. The
pH, total suspended solids (TSS) and volatile suspended solids
(VSS) concentration of the sludge were 7.8, 8.66 g/L, and 4.86 g/L,
respectively.
Simulated FVWs was used as substrate composing 57% water-
melon, 29% apple and 14% potato by wet weight. After crushed
by a food waste disposer (produced by In Sink erator company,
Model 55, 410w) and fully mixed, the simulated FVWs was stored
at 20 °C in a refrigerator and the frozen FVWs was thawed at
4 °C before use every day. Raw shredded FVWs were analyzed
more than ten times, and the initial total solids (TS) concentration
and volatile solids (VS) were approximately 100 g/kg and 87 g/kg,
respectively. The total COD (tCOD), soluble COD (sCOD) and
NH+4-N were 137.1, 79.3 and 0.07 g/kg, respectively, and the pH
was 4.5–4.8.
2.2. Reactors operation design
A CSTR reactor with a working volume of 1.5 L (total 2 L) was
operated at a greenhouse maintained at 35 °C. The reactor was
fully stirred by magnetically stirring. The SRT of the CSTR reactor
was set at 3 days and OLR was 11 gVS/(L d) by feeding diluted
FVWs (200 g raw FVWs diluted to 500 ml with tap water). The
pH of CSTR reactor was controlled at 4.0 (stage 1: 1–24 day) and
5.0 (stage 2: 25–50 day) by a pH controller with addition of HCl
(2 M) and NaOH (2 M). The reactor was fed and drawn off once a
day and the draw-off effluent was used for analysis. The character-
istics of effluent were determined every day in the first 24 days and
every two days in the 25–50 day.
2.3. Analytical methods
The production of gas was flowed to the gas counters manufac-
tured by Bioprocess AB, Lund, Sweden. Gas samples were taken
through the sampling port located on the top of the reactor while
effluent samples were collected from the draw-off materials every
day.
TS, VS, TSS and VSS were measured according to the standard
methods (APHA, 1998). The tCOD and sCOD were determined by
spectrophotometry (DR6000, HACH Company, Germany) and
sCOD was measured after filtration (0.45 lm). The total COD of fer-
mentation products marked as tCODp was calculated by the sum of
COD of individual fermentation product.
Volatile fatty acids (acetate, propionate, butyrate, iso-butyrate,
valerate, iso-valerate) and other fermentation products including
caproate, lactate, succinic acid and formic acid were determined
by high performance liquid chromatography (SHIMADZU
Company, Japan), using an Aminex HPX-87H column (T = 50 °C)
from Bio Rad coupled to an UV (210 nm) detector, while sulfuric acid
5 mM was used as eluent at a rate of 0.5 ml/min. Ethanol was
determined by gas chromatography (Agilent 7890A, Agilent
Technologies, America) equipped with a capillary column
(DB-FFAP, 30 m  0.53 mm  1 lm) and a flame ionization detec-
tor. The temperature of the injector and detector were both 230 °C.
The column temperature was increased from 70 °C to 180 °C at a rate
of 20 °C/min and kept at 180 °C for an additional 5 min. The nitrogen
gas was used as carrier gas. Gas content (hydrogen gas, carbon
monoxide, carbon dioxide and methane) was determined by gas
chromatograph (Agilent, 7890A) fitted with a thermal conductivity
detector and Agilent Carboxen 1000 column (4.5 m  2 mm; mesh
size 60/80). The argon gas was used as carrier gas with a rate of
30 mL/min. The temperature of injector, detector and column were
150 °C, 250 °C and 150 °C, respectively.
2.4. Bacterial community analysis
2.4.1. Clone library analysis
To analyze the structure of bacterial communities in the CSTR,
the sludge of the 24th day in the CSTR was used for clone library
analysis and high throughput pyrosequencing. In addition, the
sludge of the 50th day was used for high throughput
pyrosequencing.
The deoxyribonucleic acids (DNAs) of different sludge samples
were extracted using the Fast DNA SPIN Kit for Soil (MP
Biomedicals LLC., Califonia, USA). The primers 27F (50 -AGAGTTTGA
TCCTGGCTCAG-30 ) and1522R (50 -AAGGAGGTGATCCAGCCAGCCGC
A-30 ) were used for the amplification of bacterial 16S rRNA genes
(Fuller et al., 2003). The polymerase chain reaction (PCR) was per-
formed in a final volume of 50 lL containing 5 lL of DNA buffer,
4 lL of deoxy-ribonucleoside triphosphate (dNTP), 0.5 lL of each
primer, 1 lL of extracted DNA solutions, 0.5 lL Taq DNA polymerase
and 38.5 lL sterile water. The operations of amplification of bacte-
rial 16S rRNA genes were as follows: 94 °C for 2 min, 30 cycles of
94 °C for 1 min, annealing at 50 °C for 1 min and extension at 72 °C
for 1.5 min and final extension at 72 °C for 5 min. The PCR products
were purified with QIA quick PCR purification kit (Qiagen Company,
Hilden, Germany) and then cloned into PMD18-T vector (Takara
Biotechnology Co., Ltd., Dalian, China). 50 Positive clones were
sequenced (completed by Takara Biotechnology Co., Ltd., Dalian,
China), which were randomly chosen using a 3730XLDNAAnalyzer
(Applied Biosystems Company, USA) and all sequences were sub-
mitted to the BLASTN (NCBI, USA) to obtain the closest relatives.
2.4.2. High throughput pyrosequencing and sequence analysis
The samples were sent to GENEWIZ, Inc. (Jiangsu, China) for
DNA extraction and sequencing on the Illumina MiSeq
 Y. Wu et al. / Bioresource Technology 191 (2015) 53–58 55

pyrosequencing platform. After primers was removed, the raw acidogenic bacteria. Jiang et al. (2013) reported that the VFAs
sequences was demultiplexed, qualified and then clustered to accounted for 6.6% at pH uncontrolled around 3.0 for food waste
operational taxonomical units (OTUs) except that the initial treatment in batch experiment. Doğan and Demirer (2009) found
removal of sequences was aimed at quality score <25. Total of that the ratio of acid produced to sCOD was approximately 19.1%
255980 (the sample in the 24th day) and 85514 (the sample in at pH uncontrolled for municipal solid waste treatment.
the 50th day) high-quality 16S rRNA gene sequences were However, in this study the concentration of tCODp was
obtained with average length larger than 400 bp, but less than 23.5 gCOD/L and then the ratio of tCODp/sCOD was high to
480 bp. 84.1%. Wang et al. (2014) found that the maximum VFAs concen-
trations at pH 4.0 was only 2.9 gCOD/L, however, high concentra-
tion of 18.50 g/L lactic acid was also produced at pH of 4.0. Lactic
3. Results and discussion
acid with concentration of approximately 9.0 g/L was also found
at uncontrolled pH in batch experiment (Kim et al., 2003).
3.1. Lactic acid fermentation
Therefore, lactic acid was an indispensable fermentation product
at pH of 4.0 for FVWs treatment and the acidogenic bacteria still
In stage 1, the pH in the CSTR reactor was controlled between
maintained relatively high activity at low pH of 4.0.
4.0 ± 0.1 by the pH controller lasting for 24 days. As shown in
Fig. 1, the total COD concentration of fermentation products
increased gradually from start-up to 7th day and then maintained 3.1.2. Bacterial community
relatively stable around 23.5 ± 2.5 gCOD/L till the end of stage 1. In To reveal the bacterial community’s structure of the lactic acid
the stable period, lactic acid, acetate and ethanol were the three fermentative CSTR, mixed liquor sample in 24th day was used for
main products. In addition, lactic acid was produced with high high through pyrosequencing and 16S rRNA clone library.
amount of 10–20 g/L. Obviously, the concentration of lactic acid According to the results of the high through pyrosequencing, genus
from the 16th day to 23th day was lower than the concentration Lactobacillus was observed with the largest proportion of 94.2%
from the 7th day to 15th day, however, for ethanol it was exactly (Fig. 3). In addition, according to the results of clone library analy-
on the contrary. Therefore, the whole operational time was sepa- sis, the Lactobacillus bacteria took a proportion of 72% and other
rated into three phases: phase 1 (1–7 day), phase 2 (7–15 day), bacteria uncultured were with a proportion of 28% (Fig. 4). Both
phase 3(16–24 day). the results of pyrosequencing and clone library indicated that the
The results of gas production rate and gas content during the Lactobacillus was the dominant bacteria in the CSTR reactor.
operation of stage 1 were shown in Fig. 2. It is obvious that the According to the literatures, Lactobacillus contains various different
gas production rate was higher in phase 3 than phase 2. The hydro- species (Hove et al., 1999). The main Lactobacillus in this study was
gen gas, carbon monoxide, carbon dioxide and methane were Lactobacillus points (32%), Lactobacillus frumenti (10%), Lactobacillus
determined in this study. As shown in Fig. 2, carbon dioxide took acidophilus (8%) and Lactobacillus amylovorus (6%) (Fig. 4).
the largest proportion of more than 99% and hydrogen gas less than
1% was produced. 3.1.3. Fermentation type
According to literatures, there are two major pathways of lactic
3.1.1. Hydrolysis and acidogenesis acid production occurring within lactic acid bacteria: homofer-
The average concentration of tCOD and sCOD of effluent in stage mentation and heterofermentation. Fermentation products of
1 were 40.7 g/L and 27.9 g/L, while the value of TS, VS, TSS, VSS and homofermentation are exclusively lactic acid, while heterofermen-
NH+4-N were 26.6 g/L, 18.9 g/L, 12.0 g/L, 10.3 g/L and 68.3 mg/L. The tation was with equimolar amounts of CO2, lactate and acetate or
ratio of tCODp to sCOD is important as it indicates the activity of ethanol except bifidobacteria (Kandler, 1983). Bifidobacteria was
total concentation of fermentation products(mgCOD/l)

phase 1 phase 2 phase 3

concentration of fermentation products(mg/l)

32000
propionate
30000 30000
acetate
28000 ethanol
26000 lactic acid
25000
24000 tCODp
22000
20000 20000
18000
16000
15000
14000
12000
10000 10000
8000
6000
5000
4000
2000
0 0
2 4 6 8 10 12 14 16 18 20 22 24
time (d)
Fig. 1. Results of total and individual concentration of fermentation products in stage 1.
56 Y. Wu et al. / Bioresource Technology 191 (2015) 53–58

phase 1 phase 2 phase 3

900
gas volume 100
800 H2
CO2

gas production rate (Nml/d)

700
80
600

gas content(%)
60
500

400
40
300
20
200

100
0
0
2 4 6 8 10 12 14 16 18 20 22 24
time (d)
Fig. 2. Results of gas production rate and gas content in stage 1.

of obligately heterofermentation and obligately homofermentation

proportion of bacterial community in CSTR(%)

took proportion of 69% and 25%, respectively, while the left 6% was
100 facultatively heterofermentation.
other
90 Myroides The average concentrations of lactic acid, ethanol and acetate in
Tissierella_Soehngenia phase 2 were 191.3, 37.0 and 42.3 mM, while in phase 3 were
80 Clostridium
Fructobacillus 114.8, 86.9 and 43.7 mM. The ratio of lactic acid to the sum of etha-
70
Pediococcus nol and acetate in phase 2 was 2.4 while in phase 3 was 0.9.
Stenotrophomonas
Bacillus Therefore, the fermentation type in phase 2 may be the coexistence
60 Chloroplast of heterofermentation and homofermentation, however, heterofer-
Sphingobacterium
50 Acinetobacter mentation was very likely to be the dominant fermentation route
Flavobacterium in phase 3. In addition, the ratio of acetate to ethanol was 0.5,
40 Bacteroides
Leuconostoc meaning that the heterofermentation was mainly by fermentation
30
Dialister with equimolar amounts of CO2, lactate and ethanol.
Gardnerella
Alloscardovia As shown in Fig. 2, the gas production rate was between 140–
20 Aeriscardovia 250 NmL/d in phase 2 and between 300–760 NmL/d in phase 3.
Prevotella
10 Bifidobacterium The gas production rate could be considered as approximately
Lactobacillus the carbon dioxide production rate due to carbon dioxide took
0
the proportion of more than 99%. The carbon dioxide increased
stage 1(pH=4.0) stage 2(pH=5.0)
from phase 2 to phase 3, which was in accordance with the conver-
Fig. 3. The structure of bacterial community in stage 1 and stage 2 based on high sion of fermentation type from coexistence of homofermentation
throughout pyrosequencing. and heterofermentation to heterofermentation.

2% 2% 3.2. Improvement of lactic acid fermentation

4% 2% 2% 4%
6%
8%
In order to improve the lactic acid fermentation, the pH of the
28%
CSTR reactor was adjusted to 5.0 from the 25th day and the con-
centrations of fermentation product were shown in Fig. 5. The total
10%
COD concentration of fermentation products increased gradually
from the 25th to 31th day and then maintained relatively stable
around 32.1 ± 4.8 gCOD/L from the 31th to 49th day. Therefore,
32% the total concentration of fermentation products was improved
Lb. pontis Lb. frumenti Lb. acidophilus compared with stage 1. For individual fermentation product, lactic
Lb. amylovorus Lb. amylolyticus Lb. reuteri acid was produced with high amount of around 15.0 g/L. And the
Lb. secaliphilus Lb. gastricus Lb. coleohominis concentration of ethanol was between 0.8 and 1.5 g/L while acetate
bifidobacterium others
was approximately between 2.5–10.0 g/L. In addition, the gas vol-
Fig. 4. Abundance of bacterial community by clone library analysis in stage 1. ume was increased to 637.5 NmL/d and carbon dioxide was still
with high average proportion of 99.2%.
certified via the ‘bifidus pathway’ to produce 1.5 molecules of acet-
ate and one molecule of lactic acid per molecule of glucose. The fer- 3.2.1. Hydrolysis and acidogenesis
mentation types of lactobacillus involved in this study were The average concentration of tCOD and sCOD of effluent in the
summarized according to Holzapfel and Wood (2014). The bacteria stable period of stage 2 were 43.7 and 35.0 g/L. Therefore, the ratio
 Y. Wu et al. / Bioresource Technology 191 (2015) 53–58 57

total concentration of fementation products(mgCOD/l)

concentration of fementation products(mg/l)
40000 40000

35000 35000

30000 30000

25000 25000

20000 20000

15000 15000

10000 10000

5000 5000

0 0
24 26 28 30 32 34 36 38 40 42 44 46 48 50
time (d)
butyrate propionate acetate ethanol lactic acid tCODp
Fig. 5. Results of total and individual concentration of fermentation products in stage 2.

of sCOD/tCOD was 80.0%, which was obviously higher than 68.6%
when in stage 1 with pH controlled at 4.0. It indicated that the
hydrolysis reaction was obviously improved with the increase of
pH from 4.0 to 5.0. Bouallagui et al. (2004) also reported that the
optimal pH for better hydrolytic bacteria activity was between
5.0 and 6.0. The average concentration of total COD of individual
fermentation product was 29.5 gCOD/L and thus the ratio of
tCODp/sCOD was 84.5%, which was almost same with the ratio of
84.1% in stage 1. Therefore, the improvement of lactic acid fermen-
tation was mainly because of the enhancement of hydrolysis
reaction.
3.2.2. Bacterial community
As shown in Fig. 3, the bacterial community in stage 2 was more
diversity than in stage 1. Lactobacillus was still the most abundant
(45.7%), followed by Bifidobacterium (22.3%), Prevotella (17.4%) and
Aeriscardovia (1.2%). The genus Bifidobacterium is traditionally con-
sidered to be part of the lactic acid bacteria forming acetate and
lactic acid in 3:2 molar ratio (Holzapfel and Wood, 2014). The acid
susceptibility of Bifidobacterium is dependent on the strain, how-
ever, in general, it can be considered that bifidobacteria have a
weak acid tolerance (Maus and Ingham, 2003). Rasic and
Kurmann (1983) reported that the numbers Bifidobacterium rapidly
declined at pH < 4.3. Therefore, the increase of pH to 5.0 benefited
the growth of Bifidobacterium. Prevotella were reported to be able
to grow at pH as low as 5.0–5.5 and could utilize glucose to pro-
duce organic acids such as formic acid, acetate, fumaric acid suc-
cinic acid (Takahashi, 2003). Aeriscardovia as a kind of
Bifidobacteriaceae was a Gram-positive, non-sporeforming bacteria
which degrades various carbohydrates to form acids at pH > 4.2
(Simpson et al., 2004). Therefore, the increase of pH resulted in
the diversity of bacterial community, however, lactic acid bacteria
including Lactobacillus and Bifidobacterium was still the dominant
with the proportion of 68.0%.
3.2.3. Fermentation type
A handful of butyrate was produced from the 35th day which
was very likely due to the appearance of Prevotella and
Aeriscardovia. Lactic acid, acetate and ethanol still took the largest
amount of more than 96% (based on mol concentration). The aver-
age concentration of lactic acid, ethanol and acetate were 172.8,
28.4 and 131.1 mM. Thus, heterofermentation was still the domi-
nant fermentative route when pH was increased to 5.0. In addition,
the ratio of acetate to ethanol changed from 0.5 at phase 3 of stage
1 to 5.0 at stage 2. The high amount of Bifidobacterium with ‘bifidus
pathway’ fermentation in the CSTR may be responsible for the
greatly increase of the ratio of acetate to ethanol.
4. Conclusions
At pH 4.0, lactic acid with amount of 10.0–20.0 g/L was stably
produced from the FVWs treatment. The fermentation type was
converted from coexistence of homofermentation and heterofer-
mentation into heterofermentation. At pH 5.0, the hydrolysis reac-
tion was enhanced and thus the lactic acid fermentation was
improved. The heterofermentation was maintained as dominant
fermentation type, however, bifidus pathway by Bifidobacterium
played an important role.
Acknowledgements
This work was financially supported by National Natural
Science Foundation of China (21206084) and Program for
Changjiang Scholars and Innovative Research Team in University
(No. IRT1152).
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