See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/271371252

Acidogenic fermentation of food waste for

volatile fatty acid production with co-
generation of biohydrogen

Article in Bioresource Technology · January 2015

DOI: 10.1016/j.biortech.2015.01.007

CITATIONS READS

30 849

4 authors:

Shikha Dahiya Omprakash Sarkar

Indian Institute of Chemical Technology Indian Institute of Chemical Technology
6 PUBLICATIONS 43 CITATIONS 15 PUBLICATIONS 122 CITATIONS

SEE PROFILE SEE PROFILE

Yerramsetti V Swamy S Venkata Mohan

Indian Institute of Chemical Technology Indian Institute of Chemical Technology
56 PUBLICATIONS 523 CITATIONS 355 PUBLICATIONS 8,718 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Waste to Platform Chemicals and Biofuels View project

Hydrogen Production through Biological Routes (Mission Mode Project) View project

All content following this page was uploaded by Omprakash Sarkar on 17 February 2015.

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document
and are linked to publications on ResearchGate, letting you access and read them immediately.
 Bioresource Technology 182 (2015) 103–113

Contents lists available at ScienceDirect

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

Acidogenic fermentation of food waste for volatile fatty acid production

with co-generation of biohydrogen
Shikha Dahiya, Omprakash Sarkar, Y.V. Swamy, S. Venkata Mohan ⇑
Bioengineering and Environmental Sciences (BEES), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India

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

 VFA production from food waste

fermentation at variable redox
conditions.
 Short chain carboxylic acids synthesis
was higher at alkaline pH.
 Higher H2 production was also
observed at alkaline pH.

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

Article history: Fermentation experiments were designed to elucidate the functional role of the redox microenvironment
Received 16 October 2014 on volatile fatty acid (VFA, short chain carboxylic acid) production and co-generation of biohydrogen
Received in revised form 29 December 2014 (H2). Higher VFA productivity was observed at pH 10 operation (6.3 g/l) followed by pH 9, pH 6, pH 5,
Accepted 3 January 2015
pH 7, pH 8 and pH 11 (3.5 g/l). High degree of acidification, good system buffering capacity along with
Available online 20 January 2015
co-generation of higher H2 production from food waste was also noticed at alkaline condition. Experi-
ments illustrated the role of initial pH on carboxylic acids synthesis. Alkaline redox conditions assist sol-
Keywords:
ubilization of carbohydrates, protein and fats and also suppress the growth of methanogens. Among the
Degree of acidification
Short chain carboxylic acids
carboxylic acids, acetate fraction was higher at alkaline condition than corresponding neutral or acidic
Volatile fatty acids (VFA) platform operations. Integrated process of VFA production from waste with co-generation of H2 can be considered
pH as a green and sustainable platform for value-addition.
Buffering capacity Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction hydrolytic microorganisms and thus lead to the formation of H2

Among the biological routes, acidogenic fermentation (hetero-
trophic-dark) process for biohydrogen (H2) production shows the
promise of practical viability due to its feasibility of utilizing differ-
ent types of wastes as feedstock. In acidogenic microenvironment,
monomers are formed from hydrolysis of organic compounds by
⇑ Corresponding author. Tel./fax: +91 40 27168107.
E-mail address: vmohan_s@yahoo.com (S. Venkata Mohan).
along with a mixture of low-molecular-weight organic acids (vola-
tile fatty acids (VFA) or carboxylic acids) and CO2 as major fraction
(Venkata Mohan, 2009). Bacterial hydrogen gas (H2) production is
the consequence of transfer of cellular reduction equivalents, i.e.
electrons (e), onto protons (H+) and hence H2 generation occurs
with minimal energy requirement in the anaerobic process
(Srikanth and Venkata Mohan, 2014). This H2 when extracted from
the system can be used as a fuel but when retained in the system
it acts as an electron donor to produce both acetic acid (homo-
acetogenesis) and methane (hydrogenoclastic methanogenesis)

http://dx.doi.org/10.1016/j.biortech.2015.01.007
0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
104 S. Dahiya et al. / Bioresource Technology 182 (2015) 103–113
(Noori and Saady, 2013) where CO2 is the electron acceptor. Acido-
genic bacteria facilitate the formation of acetic acid (C2), propionic
acid (C3), butyric acid (C4) and valeric acid (C5), etc., and the
higher chain fatty acids (C3 and above) are further oxidized to ace-
tic acid through the action of syntrophic bacteria (H2-producing
acetogenic bacteria). Acetic acid can also be produced by homo-
acetogens utilizing H2 and CO2. H2 and VFA are the two important
value added products of acidogenic fermentation which if har-
vested properly in an integrated approach will make the whole
process environmentally sustainable and economically viable. Aci-
dogenic H2 production was well studied and documented with var-
ious feedstock and at present it is at the stage of upscaling
(Pasupuleti et al., 2014; Chandra and Venkata Mohan, 2014;
Venkata Mohan et al., 2008). These short chain carboxylic acids
can be utilized further as they are building blocks of various
organic compounds including alcohols, aldehydes, ketones, esters,
olefins, etc. (Singhania et al., 2013). By implementing appropriate
methods, VFA can be also converted to alcohols (Uyar et al.,
2009), biohydrogen (Srikanth et al., 2009) bioplastics (Cai et al.,
2009; Venkata Mohan et al., 2010; Reddy et al., 2012; Amulya
et al., 2014), microalgal lipids (Venkata Mohan and Devi, 2012),
bioeletricity (Mohanakrishna et al., 2010), aldehydes (Spirito
et al., 2014; Silva et al., 2013), etc. and are also used as preserva-
tives in food and beverage industry and in the synthesis of pharma-
ceutical/chemicals.
Earlier reports documented anaerobic acidification of waste
materials viz., urban organic waste, sludge, sugar beet processing
waste, vinasse and vegetable waste as primary feed stocks for
VFA production with different degrees of success (Singhania
et al., 2013; Cai et al., 2004; Dinamarca et al., 2003). However,
for the substrate to be viable as feedstock, it should be available
in a reasonably good amount and should have high biodegradabil-
ity and carbon load. Moreover, wastes containing the above condi-
tions if used as a feedstock, shall contribute greatly to the
ecological and economic efficiency of the process. In this realm,
canteen based food waste which fulfils the above criteria can be
thought of as potential feedstock for acidogenic fermentation.
One third of the food produced globally for human consumption
is wasted and lost accounting to around 1.3 billion tonnes. An
attempt was made to study the viability of carboxylic acid synthe-
sis under uncontrolled redox condition through fermentation of
food waste coupled with the H2 production towards hydrogen-car-
boxylate platform development. Open microbiome batch experi-
ments were designed to assess the carboxylic acid synthesis and
associated H2 production at variable redox conditions (pH, 5–11).
The major objective of this study was to establish optimum redox
conditions for higher fatty acid productivity.
2. Experimental methodology
2.1. Anaerobic consortia
Anaerobic consortium collected from full scale anaerobic reac-
tor treating sewage wastewater was used as parent inoculum.
The sludge (3.6 g VSS/l; 40 ml) after removing grit was enriched
with food waste (15 kg COD/m3-day; 48 h; pH, 7) for four cycles
prior to inoculating the bioreactors.
2.2. Waste
Composite food waste was collected from institute canteen. The
collected food waste after removing non-food particles was masti-
cated using electrical blender and then filtered through a stainless
steel sieve to remove coarse materials that may cause clogging
problems. Oil present in the waste was separated using an oil-sep-
arating system that works based on the gravity. Oil interferes in the
biological activities of the inoculum and covers the carbohydrate
content of the food waste hence making it unavailable for further
digestion. The oil free waste was used as a feedstock after adjusting
pH and organic load (OL) by diluting with domestic sewage. Chem-
ical oxygen demand (COD) of non-diluted waste has 4900 kg COD/l
with a reasonably good biodegradable fraction (BOD/COD) of 0.72.
All the experiments were performed at an organic loading rate of
15 kg COD/m3-day.
2.3. Experimental details
Seven identical bench scale anaerobic reactors were fabricated
using borosilicate-glass bottles to have a total/working volume of
0.5/0.4 l and gas holding capacity of 0.1 l. The reactors were operated
in suspended growth configuration in batch mode for 6 cycles. Each
batch was operated with 48 h of retention time comprising of 20 min
of FILL phase, 47 h of REACT (anaerobic) phase, 20 min of SETTLE
phase and 20 min of DECANT phase in sequencing/periodic discon-
tinuous mode. All the reactors were operated at an ambient temper-
ature (28 ± 2 °C) with organic load rate 15 kg COD/m3-day to study
the relative efficiency of VFA production as a function of pH. Prior
to operation, the pH of each reactor was adjusted to 5, 6, 7, 8, 9, 10,
and 11 using 1 N HCl or 1 N NaOH. Nitrogen gas was sparged into
the reactor for 5 min after every feeding and sampling event to main-
tain anaerobic conditions. The reactors were kept in suspension
mode during REACT phase by continuous mixing (100 rpm). Prior
to startup, all the reactors were inoculated with 10% of inoculum.
2.4. Analysis
Carboxylic acid composition was analyzed using high perfor-
mance liquid chromatography (HPLC; Shimadzu LC10A) employing
UV–Vis detector (210 nm) and C18 reverse phase column
(250  4.6 mm diameter; 5 lm particle size, flow rate: 0.6 ml/h;
wave length: 210 nm). Mobile phase of (40% acetonitrile in 1 mN
H2SO4; pH, 2.5–3.0) and 20 ll sample injection was used. Biogas
composition was monitored using gas chromatography (GC;
NUCON 5765) using thermal conductivity detector (TCD) with 1/
800 X 2 m Heysep Q column employing Argon as carrier gas. The
injector and detector were maintained at 60 °C each and the oven
was operated at 40 °C isothermally. Chemical oxygen demand
(COD-closed refluxing titrimetric method), VFA and pH were esti-
mated by the standard methods. (APHA, 1998) Buffering capacity
(b) was estimated based on the acid-base titrations employing
auto-titrator (Mettler Toledo DL50). (Velvizhi and Venkata
Mohan, 2014). The sample was divided into two parts of 3 ml each
prior to the test. The first part was titrated with 0.1 N HCl till the
end point at pH = 1.9 and the second part was titrated against
0.1 N NaOH till the end point of at pH = 12. The buffering capacity
(b) was calculated using the equation, where, C is the concentra-
tion of acid or base (mol), Vs is the volume of sample (ml), m is
the slope of tangent on curve (Eq. (1))
C
b¼ ð1Þ
Vs  m
3. Results and discussion
3.1. Fatty acids
Experiments illustrated variation in total carboxylic acids (VFA)
concentration and composition as a function of initial pH with
respect to operation time (Fig. 1a). VFA production was analyzed
at every 12th h of the operating cycles. It was observed that in

7000
6000
5000
4000
3000
2000
1000
0
0 50
6000
VFA Production (mg/l)
4500
3000
1500
0
0 10
Fig. 1. (a) Change in VFA production at different pH variations with respect to time; (b) Change in VFA pattern at different pH studied from 0th h to 48th h during the
operation.
all the conditions studied VFA production started without much
lag and there was a noticeable increment at 12 h of cycle operation.
System operated at initial pH 10 illustrated comparatively higher
fatty acids (as VFA; 6.3 g VFA/l) production followed by pH 9
(5.17 g VFA/l), pH 6 (4.5 g VFA/l), pH 5 (4.2 g VFA/l), pH 7
(4.1 g VFA/l), pH 8 (3.8 g VFA/l) and pH 11 (3.5 g VFA/l). By observ-
ing the results it can be elucidated that pH plays a major role in
fatty acid synthesis. This can be attributed to the fact that most
of the acidogens cannot survive in extremely acidic (pH 3) or alka-
line (pH 12) environments (Jie et al., 2014; Dinamarca et al., 2003;
Singhania et al., 2013). The optimal pH values for the production of
VFA fall in the range of 5.25–11, but the specific range depends on
the type of substrate used.
Further more based on the VFA accumulation and degradation
pattern in the results, VFA profiles can be divided into three phases
(Fig. 1b). In first phase (12 h) there was a rapid increase in the
VFA concentration in all the experimental variations studied, due
to the activity of acidogenic bacteria (AB). These AB are fast grow-
ing bacteria, with minimum doubling time of around 30 min and
are capable of fermenting the soluble organic fraction of substrate
within a short span of time. In the second phase (between 12 and
36 h), the rate of production was relatively low in comparison to
the first phase. Accumulation of fatty acids alters the system buf-
fering condition leading to retardation of the rate of VFA produc-
tion. Maintaining a constant pH during the operation is thus
expected to retain high VFA production rate for prolonged periods.
On the contrary, the third phase was more distinct, it was toward
S. Dahiya et al. / Bioresource Technology 182 (2015) 103–113 105
pH 5 pH 6 pH 7 pH 8
pH 9 pH 10 pH 11
100 150 200 250 300
Time (h)
pH 5
pH 6
pH 7
pH 8
pH 9
pH 10
pH 11
20 30 40 50
Time (h)
consumption of fatty acids rather than its accumulation. Consump-
tion/accumulation of fatty acids is generally associated with meth-
anogenesis either by acetoclastic (pH, 6–8) or hydrogenoclastic
(pH, 9–10) archea. Acetotrophic methanogenesis utilizes VFA in
the form of acetate which undergoes a dismutation reaction to pro-
duce CH4 and CO2 (Horiuchi et al., 2002). Hydrogenotrophic meth-
anogenesis utilizes CO2 and H2 for the production of CH4.
Fermentation microenvironment particularly in alkaline redox
microenvironment favored higher carboxylic acid production com-
pared to corresponding neutral and acidic conditions.
In, acidogenic fermentation which comprises four phases
(hydrolysis, acidogenesis, acetogenesis and methanogenesis), the
first hydrolytic step plays a crucial role as it makes the substrate
available to the microbial consortia for the further metabolism
(Mohanakrishna and Venkata Mohan, 2013). In this regard the
redox condition can have an impact. The food waste generally
composed of carbohydrates, proteins and fats, and these complex
compounds have to be broken down to simpler molecules to be
metabolized (carbohydrates to mono/disaccharides; proteins to
amino acids; lipids to fatty acids/glycerol). Compared to acidic
pH, an alkaline redox microenvironment enhances the hydrolysis
of carbohydrates and proteins by causing the ionization of the
charged groups (e.g. carboxylic groups) and therefore, facilitating
the availability of soluble and easily assimilable for fermentation,
which generally occurs at the initial hour of the fermentation. At
high alkaline pH, most of the carbohydrates are anaerobically bio-
degradable (Noike et al., 1985).
106 S. Dahiya et al. / Bioresource Technology 182 (2015) 103–113

Butyric acid (mg/l)

Acetic acid (mg/l)

Valeric acid (mg/l)

Propionic acid (mg/l)

b
Cycle 1 Cycle 2 Cycle 3
Cycle 4 Cycle 5 Cycle 6
6000
VFA Production (mg/l)

5000

4000

3000

0
5 6 7 8 9 10 11
pH

Fig. 2. (a) Production profile of individual VFA produced at different pH with respect to different time interval (i) 4 h (ii) 12 h (iii) 24 h (iv) 48 h. (b) Maximum VFA production
in different cycle with different pH.

The degradation efficiency of soluble proteins under alkaline to less VFA generation at the acidic pH. Neutral pH operation
and neutral conditions was higher than that under acidic condi- showed relatively lower VFA production due to the dominance of
tions. The alkaline pH is beneficial for the solubilization of proteins methanogenic activity. Neutral or nearly neutral conditions (6.8–
and also the degradation of soluble proteins. Protein gets hydro- 7.2) are generally optimum for methanogensis (Yan et al., 2010;
lyzed at alkaline condition due to the availability of OH ion which Venkata Mohan, 2009). Methanogens consume fatty acids along
acts as a nucleophile on the peptide bond resulting in its break- with CO2 and H2 towards CH4 production. Anaerobic metabolism
down and generation of free amino acids. The fats are more soluble shifts to fatty acids consumption in the pH range 5.0–8.0.
if pH value is alkaline (pH 8) compared to the pH of acidifying reac-
tors (5.5–6.0) where the fat is mostly present in insoluble form
3.1.1. Fatty acid composition
manifesting low hydrolysis. Also, strong alkaline redox condition
Carboxylic acids (short-chain volatile fatty acids; C2 to C5) viz.,
prevents the growth of both acetoclastic and hydrogenoclastic
acetic acid (HAc), butyric acid (HBu), propionic acid (HPr) and iso-
methanogens and hence retains fatty acids without converting it
valeric acid (HVa) were produced (Fig. 2a). Among the carboxylic
to terminal methanogenic end products such as CO2 and CH4.
acids, HAc was detected at a relatively higher fraction (4.2 g/l; pH
Operation at pH 11 showed not so stable performance due to the
10) followed by HBu (1.8 g/l; pH 5), HPr (1.4 g/l; pH 9) and HVa
prevailing higher alkaline redox microenvironment. Next to alka-
(0.04 g/l; pH 6) (Fig. 2b). System pH and operation time showed
line, acidophilic conditions showed good fatty acids production.
marked influence on the rate of production as well as its composi-
Low availability of soluble substrate to acidogenic bacteria leads
tion. Acetic acid production was found to be relatively higher in
 S. Dahiya et al. / Bioresource Technology 182 (2015) 103–113 107

alkaline pH operations. Alkaline pH improves the acetic acid accu-
mulation/production and its percentage in the total VFA. With
increment in pH from 5 to 10, a consistent increment in production
of acetic acid was observed followed by an acute drop at pH 11. Its
production was observed until 36 h and thereafter, proceeding
with fermentation resulted in its consumption in all the experi-
ment variations studied. However, butyric acid showed a contrary
trend. Production and consumption of butyric acid was inconsis-
tent with time and pH. Higher production was observed at acidic
pH (5; 1.8 g/l). With rise in pH, a decrement in production rate
was noticed until pH 8 which, stabilized until pH 10. However, at
pH 11 a sharp rise in its production was noticed. On the contrary,
propionic acid showed more or less uniform production at all the
redox conditions studied. Consumption was not noticed at pH 6,
7, 8 and 9 operations. Iso-valeric acids synthesis was relatively
low compared to other carboxylic acids. However, the trend was
quite distinctive, wherein, consumption was observed specifically
at 12 h followed by production at 24 h in all the experimental vari-
ations studied. At 48 h of operation, valeric acid was not detected
indicating its complete consumption in the fermentation process.
3.1.2. Acidification
Degree of acidification (DOA) represents the extent of acid for-
mation achieved due to the production of carboxylic acids in rela-
tion to substrate (as COD) degradation using the relation (Alkaya
and Demirer, 2011; Amulya et al., 2014) (Eq. (2)):
Degree of acidification ðDOA;%Þ ¼ ðSf =Si Þ  100 ð2Þ

b
50
Biohydrogen (%)

pH 5 pH 6 pH 7 pH 8
40 pH 9 pH 10 pH 11

30

20

10

0
30
Biomethane (%)

20

10

0
12 h 24 h 48 h
Time (h)
Fig. 3. (a) Contribution of individual acid and total degree of acidification in terms of acetic acid, butyric acid and propionic acid at different pH. (b) Biogas composition
evolved from the reactor at different pH conditions studied (i) H2 production and (ii) CH4 production.
108 S. Dahiya et al. / Bioresource Technology 182 (2015) 103–113
where, Si represent initial substrate concentration measured in COD
as mg/l and Sf is net VFA concentration (final-initial) expressed as
theoretical equivalents of COD concentration (COD equivalents of
individual VFA viz., acetic acid, 1.066; propionic acid, 1.512; butyric
acid, 1.816) as mg/l. Degree of acidification enumerates the capabil-
ity of system to produce the carboxylic acids. Degree of acidification
was calculated in terms of individual acetic acid, butyric acid and
propionic acid concentrations and also with the mixture of three
fatty acids (Fig. 3a). As valeric acid was produced in very low con-
centrations, it was not included in the acidification calculation.
VFA composition, fermentation time and initial pH showed a
marked influence on the acidification. The degree of acidification
was higher with pH 10 operation followed by pH 9, pH 6, pH 5,
pH 7, pH 11 and pH 8. It is interesting to note that higher acetic acid
concentration contributed to higher acidification (pH 10, 25.29%;
pH 9, 21.26%). On the contrary, higher propionic acid and butyric
acid concentrations documented relatively lower acidification val-
ues (pH 11, 16.81%; pH 7, 18.89%). When acidification was calcu-
lated based on individual fatty acids, acetic acid showed highest
value at pH 10 (14.45%) followed by pH 9 (10.70%), pH 8 (7.55%),
pH 7 (4.74%), pH 5 (3.11), pH 11(2.68%). In the case of butyric acid,
pH 5 (10.85%) showed highest acidification values followed by pH 6
(9.71%), pH 7 (7.82%), pH 11(6.76%), pH 9 (5.07%). Conversion of
propionic acid was less compared to acetic and butyric acid and
highest acidification was recorded at pH 11 (7.38%), followed by
pH 5 (6.83%), pH 10 (6.50%), pH 7 (6.33%), pH 9 (5.49%), pH 6
(4.93%) and pH 8 (4.33%). Degree of acidification is largely influ-
enced by the type of the VFA produced in the system.
3.2. Biogas
Anaerobic fermentation of wastes generates H2 and CH4 which
are two important energy carriers along with CO2. Co-generated
biogas showed the presence of H2 and CH4 along with CO2
(Fig. 3b). The composition and quantity of biogas varied with
respect to the experimental condition adopted. Highest H2 pro-
duced was recorded at 12th h of cycle operation at pH 10 operation
(30%) followed by pH 9 (25%), pH 7 (15%), pH 11(10%), pH 6 (9%),
pH 5 (4%) and pH 8 (3.5%). Subsequent analysis at 24 h and 48 h
showed marked changes. At 24th h, H2 production was found high-
est at pH 11 operation (21.3%) for one cycle followed by pH 10
(10%) and least for pH 6 (1.6%). At the end of the cycle, traces of
H2 were observed at pH 11 operation (11.707%) and least at pH 5
(1%). The H2 production was dependent on the hydrolysis of the
organic substrate into simpler compounds. In the absence of exter-
nal electron acceptors organic compounds get catabolized to
energy rich intermediates by substrate level phosphorylation along
with hydrolysis. With H2 production a redox balance will be
achieved which is one of the means of disposing of excess elec-
trons. Interestingly, higher H2 production was observed at alkaline
pH than at acidic/neutral conditions. It should be noted that the H2
production at pH 11.0 was inconsistent even though higher at 24 h,
suggesting that strong alkaline pH impaired acidogens. Cai et al.
(2004) indicated that the alkaline pH improved the H2 production
from solid waste, such as sewage sludge or food waste.
Maximum CH4 production was observed at pH 7.0 (21%) fol-
lowed by pH 8 (13%), pH 9 (4.6%) and pH 10 (2.5%). No traces of
CH4 were found at pH 5, 6 and 11 during the initial hour of the
operation. A marginal methanogenic activity was observed at the
end of the cycle operation for pH 6 and pH 11 operations. Decre-
ment in CH4 was observed in the later hours as the pH approached
acidic conditions which inhibited CH4 production to large extent.
During acetogenesis VFA are converted to acetate and H2 followed
by methanogenesis where acetate, H2 and CO2 are converted to
methane and water. It is well known that the optimum pH range
for the growth of methanogens is between 6.8 and 7.2, where
CH4 yields are consistent with this range. Methanogenesis removes
the semi-final products of anaerobic digestion: H2, small organic
compounds, and CO2 without which, a great deal of carbon (in
the form of fermentation products) would accumulate in anaerobic
environments (Adegunloye et al., 2013; Venkata Mohan, 2009).
The VFA consumed for CH4 production only accounts for a very
small amount of COD. Otherwise, acidogens will be more active
converting organic substrates to VFA and the resulting fatty acids
accumulation will then further drop in pH to inhibitory levels.
CO2 production was high compared to two other two gases. At
12 h, maximum production was observed at pH 5.0 followed by
pH 8.0, pH 6.0, pH 11.0, pH 10.0 and pH 7.0. At 24 h of the cycle
operation, the CO2 production was low for above neutral pH. At
the end of the cycle CO2 production was found to be similar to
24 h with a slight decrement. Low CO2 concentration in the alka-
line pH may be attributed to the presence of the hydrogenotrophic
methanogenesis while high due to presence of acetoclastic metha-
nogenesis in below neutral. By forming bicarbonate/carbondioxide
buffer system, CO2 positively contributes to system buffering
capacity by giving resistance to a pH-change mainly in the digester
liquid.
3.3. System redox condition
VFA production is influenced by many factors other than sub-
strate degradation making initial pH a critical parameter. The accu-
mulation of short chain fatty acids leads to a pH drop, and their
toxicities are higher when the pH is below 7.0 (Hwang et al.,
2004). Drop in system pH indicated fatty acid accumulation in
the anaerobic system. Change in pH during process operation
may influence the enzyme activities and in turn metabolic activi-
ties of the biocatalyst. Prior to start-up, pH of the systems were ini-
tially adjusted to 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0. Initial pH
greatly influenced the fatty acids synthesis and profile. Decrement
in pH was observed towards acidic condition in all the experimen-
tal variations studied (Fig. 4a). A rapid drop in pH was noticed until
12th h of operation as the easily digestible fraction of organic mat-
ter was hydrolyzed and converted to fatty acids along with CO2 fol-
lowed by a slight decrease and again increment at the end of cycle
period. Increment in system pH at end might be attributed to the
degradation of the fatty acids by obligate proton reducing bacte-
ria/methanogens/heterotrophic acetogens. System operating with
initial pH 11 was unstable for the initial 3 cycles. Fig. 4b shows
the pH change (DpH) as the function experimental conditions
and time to enumerate specific redox change in that particular per-
iod of time interval. With increase in initial pH from acidic to basic
condition, the drop in pH continued up to 24 h of cycle operation,
which confirmed the accumulation of fatty acids. However, at 36 h
and 48 h, consumption of fatty acids was more evident, indicating
shift in pH towards neutral redox condition.
3.3.1. Buffering capacity
Fatty acids production also has a significant influence on the
system’s buffering capacity (Venkata Mohan et al., 2009). System’s
internal buffering capacity was measured for all the pH conditions
at different time intervals (Fig. 4c). Buffers in aqueous systems
tend to resist changes in pH when small amounts of acid (H+) or
base (OH) is added. Initially buffering capacity in all the reactors
increased until 24th h and decreased later except for the pH 10
operation. At 12th h, maximum BC (0.222 bmol) was observed at
pH 8 while for other pH conditions it was in the range of 0.0058
to 0.0183 bmol. At 24th h, highest BC was found at pH 10
(0.030 bmol) followed by pH 11 (0.029 bmol), pH 6 (0.024 bmol),
pH 9 (0.022 bmol), pH 8 (0.021 bmol), pH 5 (0.018 bmol) and pH
7 (0.014 bmol). System’s BC correlated well with the total VFA pro-
duction. More the fatty acids production, higher the system BC,
 S. Dahiya et al. / Bioresource Technology 182 (2015) 103–113 109

which was specifically observed with pH 10 operations. CO2 is also
liberated along with fatty acid and H2 during fermentation by aci-
dogenic as well as acetogenic microbes. The system showed
improved buffering until 24th h of cycle operation, which can be
attributed to CO2 production which helps in system buffering
through carbonate formation. Accumulation and/or consumption
of fatty acids directly influence the system BC. By the end of the
cycle period, highest BC was observed at pH 10 operation
(0.025 bmol) followed by pH 11 (b 0.015), pH 7 (b 0.012), pH 5 (b
0.010), pH 6 (b 0.0096), pH 9 (b 0.009) and pH 8 (b 0.0074). Con-
sumption of accumulated VFA by the homoacetogens and metha-
nogens resulted in improved BC at the later hours (pH 6,
0.0096 bmol; pH 9, 0.009 bmol; pH 8, 0.0074 bmol). With initial
pH 7 and pH 10 operation, buffering increased with time and drop
was observed at the end of the cycle. The buffering capacity of the
biological systems is also associated with in situ produced volatile
buffers in the anaerobic system. The pH is kept stable by the buffer
effect of the protein residues and other macromolecules
(Dinamarca et al., 2003). Fermentation of proteins leads to produc-
tion of ammonia which facilitates formation of ammonium bicar-
bonate (pKa 6.35/9.35) in the presence of CO2, which
supplements additional in situ buffer to the operating system. At

a 12
pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 pH 11
11

10
9

pH 8
7

6
5

4
0 50 100 150 200 250 300
Time (h)

pH
b 1
5 6 7 8 9 10 11

-1
ΔpH

-2

-3

Δ pH 12 (12-0)
Δ p H 24 (24-0)
-4 Δ pH 36 (24-36)
Δ p H 48 (36-48)

-5

c
β

Fig. 4. (a) Change in pH pattern during the fermentation of food waste of individual reactor when operated at uncontrolled pH. (b) Influence of initial pH on DpH calculated at
different time interval to compare the change in pH at different pH studied. (c) Buffering capacity of the system operated at different pH (calculated based on the slope of
tangent on curve at different time interval).
110 S. Dahiya et al. / Bioresource Technology 182 (2015) 103–113

alkaline pH conditions, free ammonia present in the system, which
in association with bicarbonate (pKa 6.35/9.35), carbonate (pKa
6.35/9.25) and fatty acids viz., formic acid (pKa 9.25) and acetic
acid (pKa 4.75/9.25) plays a great role in buffering these biological
systems. The proper balance between the ammonia and carbonate/
bicarbonates/fatty acid leads to enhanced buffering of the system.
Low concentration of acids at acidic pH range attributes to low BC
compared to the alkaline conditions.
3.4. Yields and conversion efficiency
Carboxylic acid production and consumption pattern as a func-
tion of fermentation time helps to understand the dynamics of aci-
dogenic process occurring within the system. The interactions
between the acidogenic bacteria, acetogens, homoacetogens and
methanogens manifest VFA accumulation and/or consumption.
The production/consumption rates of carboxylic acids are calcu-
lated based on the following equations proposed (Eqs. (3) and (4)).
Production rate of carboxylic acids ðPRCa Þ
¼ ðVFAmax  VFAint Þ=T Prod
Consumption rate of carboxylic acids ðCRCa Þ
¼ ðVFADrop  VFAmax Þ=T Drop
where, VFAmax represents maximum VFA concentration (g/l), VFAint
is initial VFA concentration (g/l), VFADrop denotes drop/consump-
tion in VFA concentration due to its consumption (g/l), TProd is pro-
duction time in hours and TDrop represents concentration dropping/
consumption time (hours). Table 1 depicts the production and con-
sumption rates of individual carboxylic acids as a function of fer-
mentation time. Production was calculated from the initial hour
to the time of highest fatty acids accumulation while the consump-
tion was calculated from the point of degradation to end of the
cycle. The positive and negative values explain the rate of produc-
tion of carboxylic acids and consumption respectively (Table 1).
Analysis of the data showed some interesting observations. Increas-
ing trend in PRCa was observed with HAc from pH 5 to pH 10 oper-
ations, which dropped in pH 11 operation. Rate of HAc production
was lower at acidic and neutral pH compared to alkaline redox con-
dition (pH 10 and pH 9 operations). Highest production rate was
registered at pH 10 operation (+0.121 g/h). Higher production rate
with HBu was observed between pH 5 and 7 operations, indicating
feasible condition for its synthesis. In the case of HPr, the production
rate was inconsistent in the pH spectrum studied. In the overall
study HVa had least PRCa at all pH conditions studied. CRCa had dis-
tinct variations with all the carboxylic acids observed during fer-
mentation. CRCa with HAc was found to be moderate in operations
with initial pH 5 (0.026 g/h), pH 6 (0.025 g/h), pH 8 (0.021 g/
h), pH 10 (0.033 g/h) and pH 11 (0.022 g/h) when compared to
those with pH 7 (0.060 g/h) and pH 9 (0.086 g/h). CRCa of HPr
was negligible at pH 6, pH 7 and pH 8 operations which influenced
the total fatty acid production and degree of acidification. Higher
CRCa with HPr was observed at pH 5 (0.038 g/h), pH 9 (0.038 g/
h), pH 10 (0.014 g/h), and pH 11 (0.037 g/h) operations. CRCa of
HVa was observed in all the operating systems. Comparing the pH
spectrum studied, the consumption of HAc was high followed by
valeric acid. On the contrary, HPr and HBu showed production only
at pH 6, 7 and 8 and pH 7 operations, respectively illustrating that
these conditions are not so feasible for their consumption (Fig. 5).
At acidic initial pH operations, pH favored the production of
longer chain fatty acids, since more reducing equivalents are avail-
able that can be incorporated into the fatty acid chains
(Zoetemeyer et al., 1982). Also at low pH, fatty acids in their undis-
sociated form have the ability to diffuse freely across lipid bilayers
and liberate protons in the cytoplasm lowering the cytoplasmic pH
(Booth, 1985). At low cytoplasmic pH anions accumulate inside the
cell (Russell and Diez-Gonzalez, 1998) and undissociated acid
intercalates within the lipid bilayer (Stratford and Anslow, 1998)
disturbing the metabolic activities of the microbes. At external
acidic pH, the acetic acid that is prevalent (pKa = 4.76) in its non-
ionic form passes through the membrane resulting in an overload
ð3Þ of acetic acid inside the cell. Accumulation of acetate inside in the
cell causes inhibition of the phosphoroclastic pathway leading to
the production of butyric acid. Initially two acetyl CoA molecules
condense to form acetoacetyl CoA which in with few further steps
ð4Þ
reduce to butyric acid, leading to high butyric acid concentration at
acidic pH. This phenomenon was evident in this study particularly
below neutral pH operation. The pH strongly influences the rela-
tive amount of VFA. pH range between 4.0 and 5.0 is favorable
for propionate production (H2 sink reaction) while at pH 6.0–7.0,
acetate and butyrate are major formation favored at a transition
zone between pH 5.0 and 6.0.
On the contrary, at alkaline conditions (pH > 7) fatty acids in
ionic form are not able to pass through lipid bilayer which leads
to their accumulation outside the cell (Lier et al., 1993). The phos-
phoroclastic pathway dominates at alkaline conditions resulting in
increased acetate as well as H2 production. The experimental data
correlated with the acetate production (comparatively higher)
observed at pH 10.0. Propionate and butyrate get degraded ther-
modynamically only when acetate and especially H2 are effectively
eliminated by the MB (Stams et al., 1992). Propionate gets catabo-
lized anaerobically to acetate and CO2. However, catabolism may
get inhibited in the presence of other VFAs and the extent of inhi-
bition depends on the fatty acid concentration and the system pH
(Siegert and Banks, 2005). The catabolism of propionate is ender-
gonic in nature, while methanogenic conversion of acetate is exer-
gonic. In general methanogenic oxidation of acetate stimulates
propionate degradation thermodynamically but when concentra-
tions of butyrate and acetate are increased, removal of propionate
decreases. Acetogenic bacteria keeps symbiotic relation and take
profit from both hydrogenotrophic methanogens (reducing H2 par-
tial pressure) and acetoclastic methanogens. Acetate removal has
an influence on the energetics of VFA oxidizing reactions, espe-

Table 1
VFA Production (0–36th h) and consumption rate (36–48th h) during acidogenic fermentation of food waste.

Initial pH PRCa (g/h) CRCa (g/h) VFA (max, g/l) Acid Deg (%)
HAc HPr HBu HVa HAc HPr HBu HVa
5 +0.021 +0.040 +0.055 +0.0001 0.026 0.038 0.073 0.0016 4.2 20.79
6 +0.047 +0.028 +0.049 +0.0005 0.025 +0.042 0.039 0.0028 4.5 20.84
7 +0.036 +0.036 +0.039 +0.0004 0.060 +0.023 +0.034 0.0024 4.1 18.89
8 +0.061 +0.025 +0.017 +0.00003 0.021 +0.003 0.001 0.0016 3.8 15.72
9 +0.088 +0.031 +0.024 +0.00009 0.086 0.038 0.004 0.0019 5.1 21.26
10 +0.121 +0.038 +0.021 +0.0003 0.033 0.014 0.009 0.0022 6.3 25.29
11 +0.018 +0.043 +0.033 +0.00006 0.022 0.037 0.046 0.0015 3.5 16.81

‘+’ represents production of fatty acids; ‘’ represents consumption of fatty acids.
 S. Dahiya et al. / Bioresource Technology 182 (2015) 103–113 111

a 0.15
0.1
Production rate

production/consumption rate (g/h)

Consumption rate
production/consumption rate

0.1

0.05
0.05
Acetic acid

Butyric acid
(g/h)

0
0
5 6 7 8 9 10 11
5 6 7 8 9 10 11
-0.05

-0.05
-0.1

-0.15 -0.1
pH pH
0.0009
0.09

production/consumption rate
acidproduction/consumption

0
0.06
5 6 7 8 9 10 11
Valeric acid
0.03 -0.0009

(g/h)
rate (g/l)
Propioic

0 -0.0018
5 6 7 8 9 10 11

-0.03 -0.0027

-0.06 -0.0036
pH pH

Fig. 5. (a) Profiles showing the production and consumption rate of individual fatty acids with the function of pH and time operation. (b) Role of microorganisms at variable
redox microenvironment for acidogenic fermentation of food waste to VFA along with co-generation of biohydrogen: an overview.
112 S. Dahiya et al. / Bioresource Technology 182 (2015) 103–113
cially during iso-valerate degradation, where three molecules of
acetate and only one molecule of H2 are formed. This might explain
why iso-valerate degradation was found in all the systems.
Higher H2 production was observed at 24th h of the cycle after
which, it decreased at alkaline pH operation. When H2 concentra-
tion was high, proper proton and electron flow is essential where
MB play an important and regulatory role. H2 gets consumed by
hydrogenotrophic methanogenesis as low H2 and CO2 concentra-
tion which was specifically observed with pH 9 and 10 operations
at the later hours. Hydrogenotrophic methanogenesis creates an
electrochemical gradient across cell membrane, which, is used to
generate ATP through chemiosmosis (Goyal et al., 2014). The inhi-
bition of hydrogenotrophic methanogenesis leads to H2 accumula-
tion which in turn inhibits acetogenesis (Zaher et al., 2004). The
consumption of H2 by MB provides a low partial H2 pressure,
which enables the obligate proton reducing bacteria to degrade
the organic acids, which are thermodynamically very unfavorable
reactions and hence at alkaline pH along with hydrogenotrophic
methanogesis, acid consumption was also observed. Along with
this homoacetogenesis may also produce acetate, which consumes
H2 and CO2 counter balancing the acetate accumulation and degra-
dation. The conversion of H2 and CO2 into acetate is energetically
more favorable at higher H2 partial pressures.
At pH 7, higher CO2 and CH4 composition leads to the presence
of acetoclastic methanogenesis, which utilizes fatty acids in the
form of acetate which undergoes a dismutation (simultaneous oxi-
dation and reduction) reaction to produce CH4 and CO2. Aceto-
trophic methanogenesis is enzymatically catalyzed electron
transfer from the carbonyl group (e donor) of the carboxylic group
to the methyl group (e acceptor) of acetic acid to produce CO2 and
CH4 gas. At acidic pH conditions presence of high CO2 and traces of
H2 are attributed to the presence of hydrolytic activity of the acet-
ogens, acidogens and the basic metabolic products of the mixed
consortium.
3.5. Waste remediation
Substrate degradation (based on the COD removal) showed dis-
tinct variation in the treatment efficiencies as a function of initial
pH (Sfig. 1). Substrate degradation efficiency was higher at neutral
pH operation (pH 7, 66%) followed by pH 8 (62%), pH 9 (61%), pH 10
(55%), pH 6 (51%), pH 5 (44%) and pH 11 (30%) operations. Due to
the prevalence of acetoclastic methanogenic activity, highest sub-
strate degradation was observed at pH 7 and 8 operations. At pH
10 operations, due to presence of the hydrogenoclastic methano-
genic activity there was less removal and more accumulation of
acids in the form of COD. Less methanogenic activity and less avail-
ability of substrate may be the reason for less COD removal
observed at pH 5, 6 and 11 operations.
4. Conclusions
Experiments documented the possibility of VFA production at
alkaline pH condition associated with co-generation of H2 during
fermentation of foodwaste. Data illustrated the role of initial pH
on both carboxylic and H2 production. At different initial pH oper-
ations, the interactions between the acetogens, homoacetogens
and methanogens manifest VFA accumulation and/or consump-
tion. To enhance acidification further, the consequence of the drop
in pH at the later hours of operation need to be addressed. Acido-
genic fermentation of waste towards hydrogen-carboxylic plat-
form documents a new dimension for economical production of
value added products from waste in the framework of biorefinery.
Acknowledgements
We are grateful to the Director, CSIR-IICT for her kind support in
carrying out this work. This research was funded by CSIR in the
form of XII five year plan project on ‘Sustainable Waste Manage-
ment Technologies for Chemical and Allied Industries’ (SETCA;
CSC 0113).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2015.01.
007.
References
Adegunloye, D.V., Olosunde, S.Y., Omokanju, A.B., 2013. Evaluation of ratio variation
of water hyacinth (Eichhornia Crassipes) on the production of pig dung biogas.
Int. J. Biol. Sci. 2 (3), 44–48.
Alkaya, E., Demirer, G.N., 2011. Anaerobic acidification of sugar-beet processing
wastes: effect of operational parameters. Biomass Bioenergy 35, 32–39.
Amulya, K., Reddy, M.V., Venkata Mohan, S., 2014. Acidogenic spent wash
valorization through polyhydroxyalkanoate (PHA) synthesis coupled with
fermentative biohydrogen production. Bioresour. Technol. 158, 336–342.
APHA, 1998. Standard Methods for the examination of Water and Wastewater, 20th
ed. American public health association American water works association/
water environment federation, Washington DC, USA.
Booth, I.R., 1985. Regulation of cytoplasmic pH in bacteria. Microbiol. Rev. 49, 359–
378.
Cai, M., Liu, J., Wei, Y., 2004. Enhanced biohydrogen production from sewage sludge
with alkaline pretreatment. Environ. Sci. Technol. 38 (11), 3195–3202.
Cai, M., Chua, H., Zhao, Q., Sin, N.S., Ren, J., 2009. Optimal production of
polyhydroxyalkanoates (PHA) in activated sludge fed by volatile fatty acids
(VFAs) generated from alkaline excess sludge fermentation. Bioresour. Technol.
100, 1399–1405.
Chandra, R., Venkata Mohan, S., 2014. Enhanced bio-hydrogenesis by co-culturing
photosynthetic bacteria with acidogenic process: augmented dark-photo
fermentative hybrid system to regulate volatile fatty acid inhibition. Int. J.
Hydrogen Energy 39 (2014), 7604–7615.
Dinamarca, S., Aroca, G., Chamy, R., Guerrero, L., 2003. The influence of pH in the
hydrolytic stage of anaerobic digestion of the organic fraction of urban solid
waste. Water Sci. Technol. 48 (6), 249–254.
Goyal, N., Widiastuti, H., Karimi, I.A., Zhou, Z., 2014. A genome-scale metabolic
model of Methanococcus maripaludis S2 for CO2 capture and conversion to
methane. Mol. BioSyst. 10, 1043–1054 (RSC).
Horiuchi, J.-I., Shimizu, T., Tada, K., Kanno, T., Kobayashi, M., 2002. Selective
production of organic acids in anaerobic acid reactor by pH control. Bioresour.
Technol. 82, 209–213.
Hwang, M.H., Jang, N.J., Hyun, S.H., Kim, I.S., 2004. Anaerobic biohydrogen
production from ethanol fermentation: the role of pH. J. Biotechnol. 111,
297–309.
Jie, W., Peng, Y., Ren, N., Li, B., 2014. Volatile fatty acids (VFAs) accumulation and
microbial community structure of excess sludge (ES) at different pHs. Bioresour.
Technol. 152, 124–129.
Lier, J.V.B., Grolle, K.C., Frijters, C.T., Stams, A.J., Lettinga, G., 1993. Effects of acetate,
propionate, and butyrate on the thermophilic anaerobic degradation of
propionate by methanogenic sludge and defined cultures. Appl. Environ.
Microbiol. 59 (4), 1003.
Mohanakrishna, G., Venkata Mohan, S., 2013. Multiple process integrations for
broad perspective analysis of fermentative H2 production from wastewater
treatment: Technical and environmental considerations. Appl. Energy. 107,
244–254.
Mohanakrishna, G., Venkata Mohan, S., Sarma, P.N., 2010. Utilizing acid-rich
effluents of fermentative hydrogen production process as substrate for
harnessing bioelectricity: an integrative approach. Int. J. Hydrogen Energy 35
(8), 3440–3449.
Noike, T., Endo, G., Chang, J.-En., Jun-Ichi, Yaguchi, Jun-Ichiro, Matsumoto, 1985.
Characteristics of carbohydrate degradation and the rate-limiting step in
anaerobic digestion. Biotechnol. Bioeng. 27 (10), 1482–1489.
Noori, M., Saady, C., 2013. Homoacetogenesis during hydrogen production by mixed
cultures dark fermentation: unresolved challenge. Int. J. Hydrogen Energy 38
(30), 13172–13191.
Pasupuleti, S.B., Sarkar, O., Venkata, Mohan S., 2014. Upscaling of biohydrogen
production process in semi-pilot scale biofilm reactor: evaluation with food
waste at variable organic loads. Int. J. Hydrogen Energy 39 (14), 7587–7596.
Reddy, Venkateswar M., Nikhil, G.N., Venkata Mohan, S., Swamy, Y.V., 2012.
Pseudomonas otitidis as a potential biocatalyst for polyhydroxyalkanoates
(PHA) synthesis using synthetic wastewater and acidogenic effluents. Bioresour.
Technol. 123, 471–479.
Russell, J.B., Diez-Gonzalez, F., 1998. The effects of fermentation acids on bacterial
growth. Adv. Microb. Physiol. 39, 205–234.
 S. Dahiya et al. / Bioresource Technology 182 (2015) 103–113
Siegert, I., Banks, C., 2005. The effect of volatile fatty acid additions on the anaerobic
digestion of cellulose and glucose in batch reactors. Process Biochem. 40 (11),
3412–3418.
Silva, F.C., Serafim, L.S., Nadais, H., Arroja, L., Capela, I., 2013. Acidogenic
fermentation towards valorisation of organic waste streams into volatile fatty
acids. Chem. Biochem. Eng. Q. 27 (4), 467–476.
Singhania, R.R., Patel, A.K., Christophe, G., Fontanille, P., Larroche, C., 2013. Biological
upgrading of volatile fatty acids, key intermediates for the valorization of
biowaste through dark anaerobic fermentation. Bioresour. Technol. 145, 166–
174.
Spirito, C.M., Richter, H., Rabaey, K., Stams, A.J.M., Angenent, L.T., 2014. Chain
elongation in anaerobic reactor microbiomes to recover resources from waste.
Curr. Opin. Chem. Biol. 27, 115–122.
Srikanth, S., Venkata Mohan, S., 2014. Regulating feedback inhibition caused by the
accumulated acid intermediates during acidogenic hydrogen production
through feed replacement. Int. J. Hydrogen Energy 39, 10028–10040.
Srikanth, S., Venkata Mohan, S., Devi, M.P., Peri, D., Sarma, P.N., 2009. Acetate and
butyrate as substrates for hydrogen production through photo-fermentation:
process optimization and combined performance evaluation. Int. J. Hydrogen
Energy. 34 (17), 7513–7522.
Stams, A.J.M., Grolle, K.C.F., Frijters, C.T.M.J., Van Lier, J.B., 1992. Enrichment of a
thermophilic propionateoxidizing acetogenic bacterium in coculture with
Methanobacterium thermoautotrophicum or Methanobacterium
thermoformicicum. Appl. Environ. Microbiol. 58, 346–352.
Stratford, M., Anslow, P.A., 1998. Evidence that sorbic acid does not inhibit yeast as
a classic ‘weak acid preservative’. Lett. Appl. Microbiol. 27, 203–206.
Uyar, B., Eroglu, I., Yücel, M., Gündüz, U., 2009. Photofermentative hydrogen
production from volatile fatty acids present in dark fermentation effluents. Int.
J. Hydrogen Energy 34, 4517–4523.
View publication stats
113
Velvizhi, G., Venkata Mohan, S., 2014. In situ system buffering capacity dynamics on
bioelectrogenic activity during the remediation of wastewater in microbial fuel
cell. Environ. Prog. Sustain. Energy 33 (2), 454–462.
Venkata Mohan, S., 2009. Harnessing of biohydrogen from wastewater treatment
using mixed fermentative consortia: process evaluation towards optimization.
Int. J. Hydrogen Energy 34, 7460–7474.
Venkata Mohan, S., Devi, M.P., 2012. Fatty acid rich effluents from acidogenic
biohydrogen reactor as substrates for lipid accumulation in heterotrophic
microalgae with simultaneous treatment. Bioresour. Technol. 123, 471–479.
Venkata Mohan, S., Mohanakrishna, G., Sarma, P.N., 2008. Integration of acidogenic
and methanogenic processes for simultaneous production of biohydrogen and
methane from wastewater treatment. Int. J. Hydrogen Energy. 33 (9), 2156–
2166.
Venkata Mohan, S., Mohanakrishna, G., Goud, R.K., Sarma, P.N., 2009. Acidogenic
fermentation of vegetable based market waste to harness biohydrogen with
simultaneous stabilization. Bioresour. Technol. 100 (12), 3061–3068.
Venkata Mohan, S., Venkateswar Reddy, M., Subhash, G.V., Sarma, P.N., 2010.
Fermentative effluents from hydrogen producing bioreactor as substrate for
poly (b-OH) butyrate production with simultaneous treatment: an integrated
approach. Bioresour. Technol. 101 (23), 9382–9386.
Yan, Y.Y., Feng, L.Y., Zhang, C.J., Wisniewski, C., Zhou, Q., 2010. Ultrasonic
enhancement of waste activated sludge hydrolysis and volatile fatty acids
accumulation at pH 10.0. Water Res. 44 (11), 3329–3336.
Zaher, U., Bouvier, J., Steyer, J.P., Vanrolleghem, P.A., 2004. Titrimetric monitoring of
anaerobic digestion: VFA, alkalinities and more. In: Proceedings 10th World
Congress on Anaerobic Digestion (AD10). Montreal, Canada, Aug 29–Sep 2.
Zoetemeyer, R.J., Van den Heuvel, J.C., Cohen, A., 1982. PH influence on acidogenic
dissimilation of glucose in an anaerobic digestor. Water Res. 16 (3), 303–311,
0043–1354.