Science of the Total Environment 598 (2017) 9931000

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Science of the Total Environment

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

Maximizing the production of butyric acid from food waste as a precursor

for ABE-fermentation
Ullrich Heinz Stein a,, B. Wimmer a, M. Ortner b, W. Fuchs a, G. Bochmann a
a
University of Natural Resources and Life Sciences, Institute for Environmental Biotechnology, Vienna, Austria
b
Bioenergy 2020+ GmbH, Graz, Austria

H I G H L I G H T S G R A P H I C A L A B S T R A C T

At mesophilic conditions and pH 9.0 to-

tal volatile fatty acids (TVFA) produc-
tion is increased.
Thermophilic conditions at neutral pH
show the highest butyric acid produc-
tion and share of tVFA.
Volumetric productivity is increased at
HRTs of 2 days.

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

Article history: The current study reports on the maximization of butyric acid production from food waste using a mixed microbial
Received 4 October 2016 fermentation. In semi-continuous fermentations the effect of three different pH values (5.5, 7.0 and 9.0), three dif-
Received in revised form 31 March 2017 ferent temperatures (37 C, 55 C and 70 C) and two levels of hydraulic retention time (HRT, 2 days and 6 days) on
Accepted 19 April 2017
the formation of butyric acid as well as total volatile fatty acid production (tVFA) were investigated.
Available online xxxx
Overall, pH 5.5 provided the lowest butyric acid concentrations regardless of the temperature and the HRT. At
Editor: Chennai Guest Editor mesophilic temperature (37 C) alkaline conditions (pH 9.0) lead to a strong incline of tVFA as well as butyric
acid concentration probably due to a decreased solubilization of the substrate. However, most efcient in
Keywords: terms of butyric acid production was the fermentation conducted at 55 C and pH 7 where a butyric acid concen-
Acidogenesis trations of 10.55 g/L (HRT 2 days) and 13.00 g/L (HRT 6 days) were achieved. Additional experiments at 70 C
Volatile fatty acids showed declining butyric acid production. Increase of the HRT from 2 days to 6 days provided an increment of
Temperature butyric acid concentration throughout almost all experimental settings. However, regarding volumetric produc-
Hydraulic retention time tivity the increase in concentration does not compensate for the bigger reactor volume required to establish a
pH
higher HRT. At pH 7 and 55 C the resulting volumetric production rates were 5.27 g/L d at a HRT 2 days and
only 2.17 g/L d at a HRT of 6 days.
2017 Elsevier B.V. All rights reserved.

1. Introduction

Corresponding author. Sustainable waste management is inevitable for all societies to break
E-mail address: ullrich.stein@boku.ac.at (U.H. Stein). the link between economic growth and waste production. Beside waste

http://dx.doi.org/10.1016/j.scitotenv.2017.04.139
0048-9697/ 2017 Elsevier B.V. All rights reserved.
994 U.H. Stein et al. / Science of the Total Environment 598 (2017) 9931000
minimization, it is necessary to promote the valorization of the waste
derived from a huge number of sources either by conversion into new
products with high added value or generation of energy (Loizidou,
2015).
Moreover, depletion of fossil resources, which are not only used as
energy carriers but also for the production of various chemicals, in-
creases the need to look for alternatives. Food waste is a promising
source material for the production of various compounds that are cur-
rently made from crude oil (Angenent et al., 2004; Jang et al., 2012;
Liu and Wu, 2016) and avoids the food or fuel discussion often associ-
ated with bio-based products (Lin et al., 2013). The massive generation
of food waste is currently a major problem in the EU as it has a direct im-
pact on environmental, economic and social issues. In 2012 for example
87.6 13.7 million tons of food waste were generated in the EU
(Stenmarck et al., 2016) and 37 million tons in the US (United States,
2015). About one third of the worldwide produced food goes to waste
directly (Food and Agriculture Organization of the United Nations,
2013) and the absolute amount is expected to even increase within
the next 40 years (Foley et al., 2011; Tilman et al., 2011). Using food
waste as a raw material on the one hand reduces dependency on
petrol-based resources and on the other hand helps to solve a serious
waste problem.
Food waste has been in the scope of researchers as a resource for var-
ious applications. Examples are biogas production (Agler et al., 2011;
Kim et al., 2006; Lin et al., 2013; Liu and Wu, 2016), hydrogen produc-
tion (Han et al., 2016; Hosseini and Wahid, 2016; Yasin et al., 2013)
and various platform chemicals (Angenent et al., 2004; Liu and Wu,
2016; Uckun Kiran et al., 2015). Another application is the use of food
waste for the production of liquid energy carriers such as butanol via
the acetone-butanol-ethanol (ABE) fermentation using solventogenic
clostridia strains (Patkov et al., 2009). The ABE-fermentation is bi-
phasic: during the lag and exponential growth phase (rst phase,
acidogenesis) volatile fatty acids (VFAs) are produced. During the late
exponential and stationary phase (second phase, solventogenesis) the
VFAs are metabolized and further converted into acetone, butanol and
ethanol. The second phase which is initiated by the formation of organic
acids during acidogenesis and the associated lowering of the pH is close-
ly related to induction of sporulation. This leads to problems during con-
tinuous fermentations as sporulated Clostridia do not proliferate,
hindering an efcient continuous production process (Talukdar et al.,
2015). To avoid or at least minimize this drawback ABE-fermentation
is usually performed in semi-continuous manner. According to the bi-
phasic nature of the process it is split into two separate fermentation
steps (Mutschlechner et al., 2000). Since VFAs aren't solely produced
by Clostridia strains but by a variety of different and ubiquitous species
(Zhang et al., 2009) the rst phase can be conducted as a mixed micro-
bial fermentation.
The VFAs generated in acidogenic fermentations comprise primarily
butyric acid, acetic acid and propionic acid, moreover, to a lower extent,
iso-butyric acid, valeric acid, formic acid and caproic acid. The VFA con-
centrations as well as their distribution pattern are inuenced by the
composition of the mixed microbial consortia and by various process
parameters.
Certain research has been already done on acidogenesis of food
waste, investigating the effect of different temperatures, HRTs and
pH values (Han et al., 2016; Kim et al., 2013; Li et al., 2010; Lim
et al., 2008; Xu et al., 2011). However, an approach with such a
wide scope that includes comparison of 18 different semi-
continuous test runs covering a wide temperature range from
mesophilic over thermophilic to hyperthermophilic conditions has
not been conducted yet. In the current study focus was laid on en-
hancement of butyric acid production. Butyric acid can act as a pre-
cursor and as an enhancer for ABE fermentations as it was shown to
increase butanol production (Chang, 2010), the butanol yield
(Tashiro et al., 2004) and the butanol to acetone ratio (Li et al.,
2014).
Besides ABE fermentation, butyric acid can be used for additional ap-
plications such as precursor for pharmaceutical applications (Heidor
et al., 2012; Kumar et al., 2014), building block in the chemical industry
(Cao et al., 2011; El-Shafee et al., 2001) or avoring agent in cosmetic,
food and beverage industries (Ben Akacha and Gargouri, 2015; Shu
et al., 2011).
2. Materials and methods
2.1. Substrate and inoculum
Food waste derived from the food waste bunker of the biogas plant
Vienna, Austria, operated by the Vienna Municipal Department for
Waste Management (MA48). It was sampled immediately after fresh
waste material was delivered and consists mainly of canteen waste
and street market waste. Depending on the source of collection the
food waste was quite inhomogeneous. To obtain representative materi-
al, samples were taken from different locations within the food waste
bunker.
The waste material was blended and subsequently sieved to remove
tendons, bone particles and large impurities using a 0.25 cm2 mesh.
Samples for characterization of the substrate were taken from the ob-
tained homogenized food waste.
Preliminary fermentation tests revealed severe mixing problems
within the fermenter and build-up of a oating layer. To prevent this,
the food waste was diluted to a total solid (TS) content and volatile
solid (VS) content of 5.89% and 5.01%, respectively.
The characteristics of the food waste after blending and dilution are
presented in Table 1.
The biogas plant Vienna is constructed with a substrate suspension
storage tank to ensure continuous operation. The inoculum used in
the current study was taken from this substrate suspension storage
tank which also acts as a mesophilic pre-acidication ahead of the bio-
gas reactor. Prior to further use, the inoculum was adapted to three dif-
ferent temperatures. As other researchers have demonstrated,
mesophilic cultures can serve as sources of thermophilic and even hy-
perthermophilic microorganisms (De la Rubia et al., 2012). Adaptation
to the different temperatures (37, 55 and 70 C) was performed in
three 6L-glass reactors (constructed in house) by a one-step tempera-
ture raise approach as described by Boukov et al. (2005). The reactors
were operated at a hydraulic retention time (HRT) of 14 days. To bal-
ance organic acid production pH was readjusted to 7.0 every time the
reactor was fed with new substrate. These setups were operated for
6 months until acid concentrations became stable in the reactors before
containing cultures were used as nal inoculum. Prior to use the reac-
tors for adaption of the inoculum were not fed for 10 days to minimize
the entry of VFAs through the inoculum. The corresponding values are
presented in Table 2. When feeding was started again the reactors
reached their former level of acid concentrations within one day (data
not shown). The VFAs incorporated in the inoculum have been consid-
ered for results and they were corrected accordingly.
Table 1
Characteristics of the blended and diluted food waste used in this study.
Homogenized food waste Diluted food waste
TS [%] 18.15 1.40 5.89 1.46
VS [%] 16.46 0.61 5.01 1.63
VS/TS [%] 90.69 2.01 85.06 3.09
COD [g/L] 288.92 0.66 100.84 6.45
NH4-N [g/L] 0.61 0.03 0.22 0.01
pH 4.45 0.12 4.57 0.09
Standard deviations were calculated from triplicates.
 U.H. Stein et al. / Science of the Total Environment 598 (2017) 9931000
Table 2
VFAs in the adaption reactors during stable production and after feed stop for 10 days prior
to use as an inoculum.
Adaption reactors 37 C 55 C
Butyric acid [g/L] 2.55 0.03 9.01 0.07
Acetic acid [g/L] 4.45 0.04 2.05 0.07
Propionic acid [g/L] 1.86 0.02 1.96 0.03
Inoculum 37 C 55 C
Butyric acid [g/L] 0.13 0.12
Acetic acid [g/L] 0.54 0.72
Propionic acid [g/L] 0.21 0.17
Standard deviations for the VFA concentrations of the adaption reactors were calculated
from the stable VFA concentrations, n = 9, 10 and 10 for 37 C, 55 C and 70 C,
respectively.
2.2. Experimental set-up
2.2.1. Fermentation set-up
Reactors were constructed from 1L glass asks equipped with gas
supply and gas discharge lines at the top. Each had a working volume
of 0.8 L and the reactors were operated in a semi-continuous mode. At
the beginning of the experiments, 400 mL of the adapted inoculum
was mixed with 400 mL of diluted food waste and introduced into
each reactor. The reactors were mechanically mixed at 900 rpm at con-
stant speed using magnetic stirrers. Temperature was controlled using
electrical heat jackets. The pH levels were adjusted manually every
day using either 9 M sodium hydroxide (NaOH) or 9 M hydrochloric
acid (HCl). Acid and base of high molarity were used to minimize the di-
lution effects. To ensure anaerobic conditions the reactors were ushed
with oxygen-free nitrogen (purity 5.0) for 5 min in the beginning of
each experiment as well as every time they have been opened for sam-
pling and pH adjustment. Each experiment was started separately and
no temperature, pH or HRT change was made during the experiments.
2.2.2. Effect of pH
To investigate the effect of pH on the production of butyric acid the
reactors were operated at pH 5.5, 7 and 9 at 37 C and at an HRT of 2d,
meaning that every day 400 mL of the fermentation broth was with-
drawn from the reactor and 400 mL of new diluted food waste was
added. The reactors were operated for 10 HRTs each, resulting in total
fermentation periods of 20 days.
2.2.3. Effect of temperature and HRT
Inuence of the temperature was investigated at 37, 55, and 70 C.
Pre-set temperatures were maintained by an electrical heat jacket
with integrated temperature sensor which was connected to a control-
ler ensuring constant temperature levels (0.2 C). Again, three parallel
test runs at pH 5.5, 7.0 and 9.0, respectively, were conducted. All other
conditions were the same as described above.
An additional series of experiments addressed the inuence of a
prolonged HRT on VFA production. To extend the initial HRT of 2 days
to 6 days substrate supply was conducted only every third instead of
each day. In all cases the reactors were operated for 10 HRTs resulting
in total fermentation periods of 20 days and 60 days for reactors run
at HRT 2 days and 6 days, respectively.
Combination of all parameters resulted in 18 single experiments in
total.
2.3. Analytical methods
VFAs were monitored daily in the reactors operated at an HRT of
2 days and every 3 days in the experiments conducted at an HRT of
6 days. Concentrations of acetic acid, propionic acid, iso-butyric acid,
and butyric acid were determined by HPLC analysis as described by
Rachbauer et al. (2016).
995
VFAs are weak acids that do not completely dissociate in aqueous so-
lution. The concentration of the two forms were calculated by the law of
dilution according to Oswald solved for the undissociated Eq. (1) as well
70 C as for the dissociated acids Eq. (2).
0.75 0.03
2.94 0.04 c0  10pH
cHA pH pKa
1
1.44 0.06 10 10
70 C
0.11
c0  10pKa
0.50 cA pH pKa
2
0.57 10 10
The following pKa values (25 C) were used: acetic acid 4.76, butyric
acid 4.82, iso-butyric acid 4.86 and propionic acid 4.87.
3. Results and discussion
3.1. Effect of pH
To investigate the inuence of pH on the production of butyric acid,
semi-continuous reactors (HRT 2d, 37 C) were operated at three differ-
ent pH levels. At the beginning the pH values of the reactors were ad-
justed to pH 5.5, 7 and 9. In the course of the experiments the pH
dropped below the set pH levels due to the production and accumula-
tion of VFAs. Therefore the pH was readjusted daily by manual addition
of NaOH. Fig. 1 shows butyric acid concentration over the course of time.
The fermentations carried out at pH 9 and 5.5 reached stable butyric
acid concentrations within 4 and 5 days, respectively. The fermentation
carried out at pH 7 reached a stable butyric acid concentration even
within the rst day and subsequently only minor variations of the con-
centration were observed. This fast response is probably caused by the
fact that the inoculum derived from a reactor operated also at pH 7
and that no pH adaption was necessary. Generally, results demonstrated
that the mixed microbial consortium can be rapidly adapted to a wide
pH range. In similar experiments also such a fast adaption to other pH
levels has been observed (Jie et al., 2014).
Average butyric acid concentrations reached were 2.06, 2.78 and
8.52 g/L for fermentations carried out at pH 5.5, 7.0 and 9.0, respectively.
In the current experiments raising pH did not only provide higher
butyric acid concentrations but also total VFA production increased sig-
nicantly (Table 3). At stable VFA production the pH decreased within
one day to the following values: 5.3 (pH-0.2) 0.2, 5.0 (pH-
2.0) 0.3 and 5.9 (pH-3.1) 0.2 for fermentations conducted at
pH 5.5, 7.0 and 9.0, respectively. The magnitude of pH decrease correlat-
ed with the amount of produced tVFA.
Results are conrmed by Jie et al. (2014) who also describe a sharp
increase of VFA production at alkaline conditions, however, using excess
sludge as a substrate. Wang et al. (2014) investigated acidication of
food waste. Although they only tested the pH range 4.06.0 they also re-
port higher VFA production with rising pH. Also Dahiya et al. (2015) ob-
served higher VFA production at alkaline conditions (pH 9 and pH 10).
But in contrast to the results gained here, under such conditions the
share of butyric acid decreased and more acetic acid was produced.
The observed impact of pH on tVFA concentration can be linked to
the presence of undissociated VFAs. The lower the pH in the fermenta-
tion broth, the higher the amount of undissociated acids which can per-
meate through the cell membrane and subsequently dissociate within
the cell (Huang et al., 1986). This causes a decrease of the internal pH
and forces the cell either to increase active excretion or re-
metabolization of the acids. In both cases VFA production is impaired
(Grupe and Gottschalk, 1992). To check, the concentrations of undisso-
ciated VFAs in the three reactors were calculated at the measured pH
(Table 4). Contrary to the absolute concentrations, the concentration
of the undissociated acids is the lowest at pH 9. Interestingly, at pH 7.0
the concentration of undissociated acids were by far the highest sug-
gesting that this parameter setting should provide the lowest tVFA con-
centration. This was not the case and indicates that the amount of
996 U.H. Stein et al. / Science of the Total Environment 598 (2017) 9931000

Fig. 1. Butyric acid concentrations during the fermentations of food waste carried out at three different pH values.

undissociated acids is not the only inuential factor. Substrate availabil-
ity may also contribute to the measured strongly enhanced VFA yield at
pH 9. According to various authors, the solubilization of the food waste
increases with rising pH, thus providing the microorganisms with more
utilizable substances (Chen et al., 2007; Dinamarca et al., 2003; Jie et al.,
2014; Kim et al., 2003; Lim et al., 2008). Consequently, this should result
in rising tVFA concentrations from acidic over neutral to alkaline condi-
tions, similar to the observations in the conducted experiments.
Standard deviations were calculated from values during stable pro-
duction of butyric acid (Fig. 1), n = 16, 20 and 17 for pH 5.5, 7.0 and
9.0, respectively.
Table 3 provides also distribution of other VFAs measured during the
fermentations. As shown, higher pH values did not only yield increased
concentrations of butyric acid but also the relative portion of butyric
acid (butyric acid/tVFA) increased. The percent shares were 28.9%,
27.3% and 46.8% at pH 5.5, 7.0 and 9.0, respectively. Besides butyric
acid, acetic acid was produced as the prevalent metabolite. Table 4
lists the ratio of butyric acid to acetic acid to illustrate which type of
acid is favorably produced. At pH 5.5 and pH 7.0 the formation of acetic
acid dominates whereas at pH 9.0 butyric acid is the main product. Jiang
et al. (2013) made similar experiments with food waste, however, only
in the pH range 5.07.0. They conrmed that acetic acid is the prevalent
product towards more acidic conditions. However, differing to the re-
sults gained in the current study, already at pH 6.0 butyric acid exceeded
acetic acid formation.
With view to the other metabolites, the concentrations of propionic
and isobutyric acid were relatively similar in all experiments indepen-
dently of the total VFA amount. Accordingly, their percent share de-
creased towards high pH levels.
Other studies link change in VFA pattern and tVFA concentrations to
the re-metabolization of VFAs in the course of solventogenic activity
(Wang et al., 2014; Zhao et al., 2006). Regestein et al. (2015) obtained
results similar to the current study and assumed that at acidic and neu-
tral conditions consumption of butyric acid is favored over the con-
sumption of acetic acid.
Though at alkaline pH values butyric acid is the main product, acetic
acid is still produced in large amounts.
In addition to solventogenesis, other re-uptake paths may occur as
the mixed microbial consortia is a complex community in which differ-
ent metabolites serve as the substrate for other bacteria, fungi and
archaea.
Total VFA concentrations of 7.19 to 18.19 g/L were reached. In com-
parison, Jiang et al. (2013) report tVFA concentrations of 37.1 g/L at
pH 7.0, which is about four times higher as the result at pH 7 obtained
here. However, they also used a much higher substrate concentration
in terms of VS content. The corresponding yield was 0.36 g tVFA/g VS
fed which is similar to our results providing a yield of 0.41 g tVFA/g VS
Table 4
Concentration of dissociated (diss) and undissociated (undiss) acetic acid, butyric acid and
propionic acid during the acidogenesis of food waste at different temperatures and mea-
sured pH values.

Table 3 Parameter Acetic acid Butyric acid Propionic acid

Results of the effects of different pH values on acidogenesis of food waste. setting
undiss diss undiss diss undiss diss
pH 5.5 7.0 9.0 [g/L] [g/L] [g/L] [g/L] [g/L] [g/L]

tVFAs [g/L] 7.13 0.66 10.17 0.46 18.19 0.72 37 C, pH 5.5 0.5 1.7 0.5 1.5 0.5 1.4
Acetic acid [g/L] 2.48 0.14 4.87 0.10 6.89 0.22 37 C, pH 7.0 1.7 3.2 1.0 1.7 0.8 1.2
Propionic acid [g/L] 1.87 0.13 1.97 0.15 2.24 0.01 37 C, pH 9.0 0.4 6.5 0.6 7.9 0.2 2.1
iso-Butyric ACID [g/L] 0.72 0.10 0.55 0.11 0.55 0.11 55 C, pH 5.5 1.3 0.9 0.4 0.2 1.0 0.5
Butyric acid [g/L] 2.06 0.03 2.78 0.01 8.52 0.10 55 C, pH 7.0 0.3 1.3 2.2 8.4 0.4 1.5
Butyric acid/acetic acid 0.83 0.17 0.57 0.11 1.24 0.32 55 C, pH 9.0 0.3 2.6 0.5 3.3 0.3 1.8
tVFA [g tVFA/g VS fed] 0.285 0.031 0.406 0.018 0.726 0.016 70 C, pH 5.5 1.1 1.0 0.2 0.2 0.9 0.6
Butyric acid [g Butyric acid/g 0.082 0.003 0.111 0.008 0.340 0.011 70 C, pH 7.0 1.3 1.6 0.4 0.5 0.8 0.7
VS fed] 70 C, pH 9.0 0.3 1.8 0.5 2.5 0.3 2.1
 U.H. Stein et al. / Science of the Total Environment 598 (2017) 9931000
fed at pH 7. The optimum pH value for the production of tVFAs as well as
butyric acid, pH 9.0, yielded 0.726 g tVFA/g VS fed, around 80% higher
compared to the yield at pH 7.0. With view to butyric acid production
the improvement is even more remarkable. The yield at pH 9.0,
0.340 g butyric acid/g VS fed, corresponds to a more than 200% increase
compared to pH 7.0.
Generally, results show that pH adjustment is a simple means to
steer complex consortia towards higher butyric acid production. How-
ever, while increase of pH seems to be an effective tool to raise tVFA
and butyric acid yield it needs to be considered that large amounts of
base are required. The associated high costs for chemicals makes this
option economically less favorable.
3.2. Effect of temperature
As demonstrated by other researchers, higher temperatures might
be another means to increase accessibility of the provided substrate
for enhanced conversion into VFAs (Jiang et al., 2013). The impact of
temperature on VFA and in particular butyric acid production was stud-
ied at the same conditions as before. Besides 37 C, two additional tem-
perature levels were tested, 55 C and 70 C, using inocula specically
adapted to the respective temperature. Similar to the previous experi-
ments parallel test runs were conducted at pH 5.5, 7.0 and 9.0. Maxi-
mum butyric acid concentrations of 10.55 g/L 0.17 and 8.52 g/L
0.10 where reached during fermentations at pH 7, 55 C and pH 9,
37 C, respectively.
Fig. 2 shows tVFA concentration and the pattern of VFA distribution
at the different fermentation conditions. Contrary to the expectations,
higher temperatures did not only provide decreasing butyric acid con-
centrations but generally resulted in lower tVFA concentrations.
However, there was one apparent exemption which did not t to the
general pattern. At pH 7.0 and 55 C a remarkable increase of butyric
acid concentration by 280% was achieved compared to the same exper-
iment at mesophilic conditions. The raise in butyric acid was over-
proportional and the parameter setting did not correspond to the fer-
mentation conditions with the highest tVFA concentration (pH 9.0,
37 C).
Fig. 2. VFA distribution during the fermentation of food waste at three different temperatures (37, 55 and 70 C), three different pH values (5.5, 7.0 and 9.0) and an HRT of 2 days.
997
Regarding isobutyric acid it was generally noted that at lower pH
higher concentrations were found. At pH 5.5 and a temperature of
55 C or 70 C, the concentration of the isomeric form even exceeded
the butyric acid concentration. In contrast, propionic acid production
was similar throughout all fermentation conditions.
Some studies report similar observations in terms of increased bu-
tyric acid formation at higher temperature. Yang et al. (2015) examined
a rise of butyric acid concentration at a temperature increase to 55 C
whereas a further elevation of temperature from 55 C to 70 C resulted
in a sharp decline. Qiu et al. (2016) fermented xylose at 37 C and 55 C
with a mixed microbial consortia. They discovered an enrichment of bu-
tyric acid producing microorganisms in the culture as a direct conse-
quence of the temperature increase from mesophilic to thermophilic
conditions. It has been stated that elevated temperatures change the
microbial consortia and typically lowers the diversity of the bacterial
species (Qiu et al., 2016). The lower VFA levels might therefore be ex-
plained by a combination of enhanced re-utilization and the lower pro-
duction due to declining biodiversity of the mixed culture at higher
temperature. The reduced variety of microorganisms at 55 C provides
also an explanation for the low tVFA concentrations at pH 5.5 and 9.0.
Such parameter combinations, an elevated temperature and non-
physiological pH conditions, obviously overstress the adaptive capacity
of the microbial consortium.
As discussed above, undissociated acids might affect VFA production.
Table 4 provides an overview of the amount of dissociated and undisso-
ciated VFAs for all parameter settings examined. Research has been con-
ducted investigating the inhibitory concentrations of undissociated
VFAs. Keke et al. (2016) published that undissociated butyric acid re-
duced the VFA production by 60% at concentrations of 1.3 g/L and undis-
sociated acetic acid by 59% at concentrations of 2.0 g/L, respectively. In
our experiments all concentrations of undissociated acetic acid were
below the value reported. Therefore, in the case of acetic acid, inhibition
effects are assumed to play a minor role. In contrast, butyric acid con-
centrations exceeded the inhibitory concentration in the fermentation
conducted at pH 7 and 55 C.
Taking such inhibitory effects into account, a higher butyric acid con-
centration might be achievable if permanent removal of VFA is applied.
998 U.H. Stein et al. / Science of the Total Environment 598 (2017) 9931000
Extraction of volatile acids through membranes has been proven to be a
suitable method (Tugtas, 2014; Zacharof and Lovitt, 2014) but the in-
vestment and maintenance costs of such bioreactor setups are much
higher.
3.3. Effect of hydraulic retention time
The nal parameter tested was HRT. Several researchers reported
enhanced VFA production from food waste at an extended HRT.
Scoma et al. (2013) observed a 64.9% higher concentration when HRT
was raised from 1 to 5 days whereas a further increase of the HRT
from 5 to 7 days decreased the VFA concentration by 6.6%. Fang and
Yu (2000) observed increased VFA concentrations when they changed
the HRT within a much shorter interval, namely between 4 h and
30 h. Also Han et al. (2016) investigated VFA production using wastewa-
ter from food industry. Under various conditions improvement of VFA
yield with increasing HRT (from 1 to 3 days) was demonstrated. How-
ever, none of these experiments investigated the impact of HRT, pH
and a temperature in their combination. Therefore, additional test
runs using all previous parameter settings but at a longer HRT (6 instead
of 2 days) were conducted. Results are shown in Fig. 3. Generally results
conrmed expectations. In most cases the butyric acid concentrations
were higher at longer HRTs. Exemptions are the fermentations conduct-
ed at 37 C, pH 9.0 and 55 C, pH 9.0. The decrease in concentration of
butyric acid in these experiments were 39% (37 C, pH 9.0) and 4%
(55 C, pH 9.0), respectively. The reduction at 70 C and pH 5.5 is negli-
gible and within the experimental error. The highest ascent in relative
as well as absolute values, from 2.78 g/L to 5.52 g/L, was achieved in
the fermentation conducted at 37 C, pH 7.0. Looking at the conditions
that provided the best results at a HRT of 2 days (pH 7.0, 55 C), an in-
crease of 23.2% was achieved.
The butyric acid concentration raised from 10.55 g/L even further to
13.00 g/L. With rising butyric acid concentration the yield increased as
well from 0.422 g butyric acid/g VS to 0.520 g butyric acid/g VS.
The higher concentration achieved in such a way is obviously prefer-
able with respect to efcient substrate utilization. However, with view
to volumetric productivity the increase in butyric acid concentration
Fig. 3. Effect of the HRT on the butyric acid concentrations during fermentations of food waste at three different temperatures (37, 55 and 70 C), three pH values (5.5, 7.0 and 9.0) and two
hydraulic retention times (2 and 6 days).
does not compensate for the bigger reactor volume required to establish
a higher HRT. The corresponding volumetric production rates were
5.27 g/L d at a HRT 2 days versus 2.17 g/L d at a HRT of 6 days.
4. Conclusions
The conducted experiments aimed to inuence the VFA spectrum
and concentration in a mixed microbial fermentation of food waste.
Mixed microbial fermentations provide the signicant technological ad-
vantage that unsterile conditions can be applied which makes the pro-
cess highly attractive from the economic point of view.
Unfortunately, the wide variety of different microorganisms in-
volved makes impact of different process conditions much less predict-
able and process optimization is cumbersome. The investigations made
in the current study provided certain unexpected effects. While it was
conrmed that alkaline conditions can signicantly boost VFA produc-
tion, the best way to enhance butyric acid was to raise temperature
from mesophilic to thermophilic conditions at pH 7.0. In contrast, such
a benecial effect of higher temperature was not observed at the other
pH levels. Applying longer HRTs resulted in a further increase of the bu-
tyric acid concentration, however, at the cost of lower volumetric pro-
duction rate or in other words the need for more reactor volume.
Generally, it was clearly demonstrated that the right combination of
temperature and pH strongly increases butyric acid yield. The identied
optimum parameter setting has an additional advantage. While adjust-
ment to pH 9 requires high amounts of costly chemicals a raise in tem-
perature can be easily implemented. Moreover, temperature control is
an immanent tool of every fermentation process, and therefore it is a
simple, yet effective way to steer acidogenic fermentations towards en-
hanced butyric acid production.
Author contributions
The Manuscript was written through contributions of all authors. All
authors have given approval to the nal version of the manuscript.
 U.H. Stein et al. / Science of the Total Environment 598 (2017) 9931000
Funding source
This scientic work was funded by the Austrian Climate and Energy
Fund (Energy Mission Austria, 838708).
Acknowledgements
The authors gratefully acknowledge the contribution of our col-
leagues from the biogas group, the water group at IFA-Tulln and all oth-
er involved third parties.
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