Accepted Manuscript

Title: Anaerobic treatment of wastewater using a two-stage

packed-bed reactor containing polyvinyl alcohol gel beads as
biofilm carrier

Authors: Siddhartha Pandey, Sudipta Sarkar

PII: S2213-3437(17)30064-7
DOI: http://dx.doi.org/doi:10.1016/j.jece.2017.02.013
Reference: JECE 1479

To appear in:

Received date: 25-11-2016

Revised date: 26-1-2017
Accepted date: 7-2-2017

Please cite this article as: Siddhartha Pandey, Sudipta Sarkar, Anaerobic treatment
of wastewater using a two-stage packed-bed reactor containing polyvinyl alcohol
gel beads as biofilm carrier, Journal of Environmental Chemical Engineering
http://dx.doi.org/10.1016/j.jece.2017.02.013

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Anaerobic treatment of wastewater using a two-stage packed-bed reactor containing

polyvinyl alcohol gel beads as biofilm carrier

Siddhartha Pandey and Sudipta Sarkar*

Department of Civil Engineering, Indian Institute of Technology Roorkee, Roorkee,

Uttarakhand, India, 247667

*Corresponding Author: Tel.: +911332284756. Email: srkarfce@iitr.ac.in

1
GRAPHICAL ABSTRACT

2
 Highlights

Porous PVA-gel beads were used as bio-carrier in a two-stage anaerobic reactor

For 1500 mg/L COD and 96 hr HRT, COD removal efficiency was 89% at steady-state
VFA profiles at low and medium temperatures have been studied
Propionic : acetic acid ratio in acidogenic reactor increased at low temperature

3
Abstract

Anaerobic treatment of wastewater is a viable alternative to aerobic treatment with the

advantage of low net energy requirement and less sludge production. Successful
establishment of a two-stage anaerobic treatment process resulted in better performance and
improved tolerance to perturbations in operating conditions such as high organic loading rate
and higher methane production. In this study we have reported process development,
microbial enrichment and validation of performance of a two-stage anaerobic packed-bed
reactor comprising of an acidogenic reactor followed by a methanogenic reactor, each
containing porous polyvinyl alcohol (PVA)-gel beads as biofilm carrier, harboring different
consortia of microorganisms for treating wastewater at mesophilic temperature. The stage-
separated reactor system demonstrated chemical oxygen demand (COD) removal efficiency
up to 89% with overall methane productivity of 0.08 L methane/(L reactor.d). During steady-
state operation, the acidogenic reactor produced an effluent with concentration ratio of
propionic acid to acetic acid as low as 0.2, which indicated the stability of the reactor system
against environmental shock-related failure. During an event of deliberately imposed low-
temperature shock period of 60 days, the COD removal efficiency decreased from 89% at
steady-state to as low as 33% with concomitant decline in methanogenic productivity. The
predominance in microbial community changed from steady-state characteristics of Gram
positive to Gram negative during the temperature shock. After the shock was withdrawn, the
reactor system regained its original performance within 28 days; the Gram positive bacteria
also switched back to the predominance.

4
1.0 Introduction

Anaerobic treatment of sewage and medium strength industrial wastewaters is a viable

alternative to aerobic treatment processes with the advantages of reduced energy requirement
and sludge generation. To date, various types of possible reactor configurations for anaerobic
treatment have been extensively studied [1,2,3]. Depending on the approaches of biomass
retention, anaerobic treatment processes can be largely grouped into two major categories,
namely suspended growth reactors and surface- or biofilm carrier attached growth reactors
[4]. Examples of the first group are upflow anaerobic sludge blanket (UASB) reactor and
expanded granular sludge bed (EGSB) reactors whereas anaerobic filter (AF) and anaerobic
fluidized-bed reactors (AFBR) belong to the second group [5,6,7,8]. Among the suspended
growth anaerobic reactors, UASB has been extensively investigated for the application of
wastewater treatment [9]. Anaerobic degradation of biodegradable material has two
significantly different and distinguishable consecutive stages, namely acid-forming or
acidogenesis process followed by methane-forming or methanogenesis process [10,11]. In the
acidogenesis process the complex organic substrates undergo bacteria-mediated hydrolysis to
form different volatile fatty acids (VFA) such as valeric acid (C5), n-butyric acid (C4),
propionic acid (C3) and ultimately culminate in the formation of acetic acid (C2), thereby
lowering the pH [12,13,14]. Acetic acid is a key intermediate metabolite and its utilization in
methanogenic process is known as the rate limiting step of overall anaerobic process dealing
with soluble organic species only [15,16]. In the methanogenic process, the VFAs, more
specifically acetic acid, degrade to form methane and carbon dioxide through a series of
complex microbial reactions. This process is susceptible to low pH, and in particular, the
process becomes ineffective when the pH goes below 4.7 [17] Quite naturally,
microorganisms responsible for acidogenesis and methanogenesis processes differ widely in
terms of physical characteristics, growth rates, substrate utilization kinetics, nutrient
requirements and resilience for environmental conditions [18,19,20,21,22].

In conventional systems, both groups of these microorganisms are made to coexist in a single
reactor system. Such an arrangement is irrational, from a process point of view, as these two
sequential processes have widely different operational characteristics [18,23,24,25,26]. In
order to ensure greater overall stability and control, it is more rational to physically separate
these two processes in two separate reactors in series, where optimum environmental
conditions for each group of microorganisms can be provided separately [27]. In the last two
decades, various research studies have been carried out to effectively separate the overall

5
anaerobic process into two stages [28]. This separation not only ensures an improved
performance but also enhance the tolerance of the system against the perturbations in
operating conditions such as temperature and organic loading rate to yield higher methane
production. Further, the process optimization of the individual stages, specifically the
acidogenesis has been studied under different operating conditions with varying nature of
substrates, including both simple and complex. Whereas, methanogenic reactors have been
mostly studied in terms of microbiological diversity, particularly in view of its susceptibility
under low pH condition [29,30,31].

In the conventional anaerobic degradation process, the sludge granulation, bio-film formation
or immobilization of biomass are often observed, which are considered to be associated with
the successful operation of anaerobic wastewater treatment systems. The underlying
fundamental reason is attributed to the increase in SRT (sludge residence time) due to such
immobilization of biomass, irrespective of the HRT (hydraulic retention time) of the reactor
[32,33,34]. In this respect, attached growth processes are attractive but they face several
problems, such as difficulty in controlling bio-film thickness, higher thickness causing bio-
film breakage and washout, among others [35,36,37]. As a consequence, even if a higher
biomass inventory is achieved, the bio-film breakage and washout phenomena may ultimately
result in lower treatment efficiencies. An alternative solution to this problem is
immobilization of bacteria inside biofilm carriers, which has attracted recent interests.
Compared to suspended biomass and biomass attached to bio-films or granulated sludge,
immobilization of bacteria inside a biofilm carrier is known to increase the reaction rates as
well as to offer a better system control so that the operation becomes easier. To date,
synthetic polymers, natural biopolymers, granulated charcoal or inorganic porous host
materials have been used as hosts for enhancing the immobilization of active biomass
[38,39,40]. Polyvinyl alcohol (PVA)-gel beads belong to synthetic polymers, which have
been successfully used in many wastewater applications such as moving bed biofilm reactor
(MBBR), anaerobic wastewater treatment, etc [41,42,43,44]. Its superior performance stems
from its ability to offer shelter for bacteria in its pore for encapsulation and cultivation. The
adhesion to the PVA-gel beads also reduces sloughing of biomass [45]. In anaerobic
wastewater treatment, PVA-gel bead biofilm carrier has been used in UASB, Anaerobic
fluidized bed reactor, Anaerobic fixed bed reactor etc [46,47,48]. However, there has been
only a single instance of study on application of PVA-gel beads in a two-stage anaerobic
reactor has been published so far. It has reported the use of PVA-gel beads in a hybrid two-

6
stage anaerobic digestion followed by membrane bioreactor, where the anaerobic treatment
was performed in thermophilic range [49,50]. To the best of our knowledge, no study has
been reported so far for the use of two-stage anaerobic treatment system incorporating PVA-
gel beads being operated in the mesophilic temperature range.

Bearing all these considerations in mind, present research study was undertaken to establish a
two-stage anaerobic process using packed-bed reactors filled up with PVA-gel beads and
operated under mesophilic temperature conditions. The study included start-up, optimization
of HRTs of acidogenic and methanogenic reactors and steady-state operations over a period
of time including one occasional drop in temperature. The process as well as reactor
performances were assessed through COD (Chemical oxygen demand) removal efficiency,
volatile fatty acid concentration profiles and methane generation rates.

2.0 Materials and Methods

2.1 Experimental Set-up of a Two-Stage Reactor System

The study was conducted on a two packed-bed reactors each containing PVA hydrogel beads
as biofilm carrier and arranged in series according to the schematic provided in Figure 1. The
two anaerobic packed-bed reactors (AnPBR-1 and AnPBR-2) made of acrylic glass were of
different sizes as indicated in Figure 1. The total packed volume was 13L. Both the reactors
were operated under anaerobic condition at a constant temperature of 35 3 C which falls
in the mesophilic temperature range. During operation, the reactors were covered with
plastic-laminated dark black paper in order to avoid the formation of aerobic conditions due
to algal growth. Random grab samples from each reactor were plated on bold basal media
(BBM) which is a specific enrichment media for algal growth, spiked with antibiotics to
prevent bacterial growth. Absence of any colony on the growth plate confirmed that algal
species were absent in the reactor. Apart from the inlet and outlet ports, each of the reactors
had equally spaced lateral sample collection ports along its entire height. During regular
operation, all the lateral ports were closed and joints were sealed airtight. The reactors had
jackets for insulation and maintenance of constant temperature. The reactors were run in up-
flow mode and were fed from temperature-regulated and air-insulated feed tanks through
peristaltic pumps. Biogas production rate from the reactors was measured using displacement
of water or KOH solution, following the procedure described by Sharma et al., 2015 [51].
The free space at the top of the liquid level acted as gas holder and the measurements were

7
based on amount of water or KOH solution displaced by the produced biogas. The hydraulic
retention times (HRT) of the reactors were regulated by changing the flow rate of the feed
pumps. All the experiments were performed in environmental engineering laboratory of
Indian Institute of Technology Roorkee located in the Northern part of India.

2.2 Biofilm carrier

Polyvinyl alcohol (PVA) hydrogel beads were used as biofilm carrier for the immobilization
of microorganisms in both AnPBR-1 and AnPBR-2, which were later optimized to act as
acidogenic and methanogenic reactors, respectively. PVA-gel beads were procured from the
Indian branch of Kuraray Corporation (Tokyo, Japan). The beads were of spherical shape,
with a diameter of 3 to 4 mm, with a specific gravity of 1.025. The effective specific surface
area was reported to be up to 2500 m2/m3. The beads were hydrophilic in nature and a
scanning electron microscope (SEM) image of its porous structure revealed that the porous
interior offered interconnected pores of size 10-30 m suited for immobilization of
microorganisms.

2.3 Enrichment of Microorganisms within the Reactors

Mesophilic anaerobic seed sludge was collected from UASB-based sewage treatment plant
located in Saharanpur, Uttar Pradesh in India and was used as inoculum for reactors. To
enrich the microorganisms, a homogeneous mixture of seed sludge and PVA-Gel beads was
packed in both the reactor columns AnPBR-1 and AnPBR-2, each of which was separately
run in batch mode by continuously re-circulating a properly balanced synthetic wastewater
with quality characteristics as indicated in Table -1. Molasses was the sole carbon source in
the synthetic wastewater, while NH4HCO3 and KH2PO4 were added as nitrogen and
phosphorus source in order to maintain COD:N:P ratio of 100:5:1 [52,53,54]. Both the
columns were run simultaneously in upflow mode at a constant temperature of 35 C. COD,
N and P concentrations of the fed synthetic wastewater were analyzed on every alternate day
and whenever the values of any of these parameters were observed to fall below 50% of its
initial value, the synthetic wastewater was replaced by a new batch of solution. The COD
concentrations were gradually increased from 250 mg/L to 1500 mg/L in steps over a period
of three months. The PVA-gel beads were physically observed for its appearance during this
time period. As the time progressed, the beads which were originally white in appearance,
turned black due to the growth of attached anaerobic bacteria on and inside the pores of the
beads. When all the beads turned black, the operation of the reactors was changed from batch

8
mode to continuous mode with the synthetic wastewater being fed into AnPBR-1. The
effluent from AnPBR-1 was collected in an intermediate tank where from it was fed into
AnPBR-2. During the rest of the enrichment period the reactors were kept running in
continuous mode, each with HRT of 72 h, so that the treated effluent reaches a steady-state in
terms of quality characteristics.

2.4 Effect of HRT on COD removal efficiency and Performance Evaluation at Steady-State

In order to find out the optimum operating conditions under any particular influent COD
concentration, the performances of overall system as well as individual reactors were
evaluated by applying different combinations of HRTs for the reactors. Whenever a new
combination of HRTs was imposed on the reactors, the system was operated until it achieved
a steady-state in terms of COD removal efficiency. The system was allowed to run for some
duration of time in steady-state and samples were collected at that condition before applying
a new combination of HRTs. The COD removal efficiencies at different HRTs were observed
and optimum operating condition was determined. The volatile fatty acid (VFA)
concentrations of the influent and effluent samples were also determined. Once an optimum
running condition with respect to HRTs of each reactor was established, the reactors were
then allowed to run continuously at the optimum HRT condition for rest of the experimental
period. During such steady-state run, influent, effluent and intermediate samples from each of
the reactors were collected for analysis in terms of pH, COD and VFA profiles.

2.5 Analytical Methods

Concentrations of different VFAs, namely acetic acid, propionic acid, and n-butyric acid
were measured using high performance liquid chromatography (HPLC; Waters, model 515)
fitted with Agilent Zorbax Eclipse Plus C18 analytical column (4.6 250mm 5m)
attached with an Eclipse Plus C18 guard column (4.6 2.5mm 5 m). The HPLC had in
line degasser unit, water pump controller unit, connected in series with a photodiode array
detector which was set at 210 nm wave length [55]. Samples for the measurement of soluble
COD and VFA analysis were filtered through a 0.45 m polyvinylidene fluoride (PVDF)
filter (Whatman, UK) prior to analysis. For VFA analysis, the filtered samples were acidified
with 10% H3PO4 (volumetric basis) to lower down the pH <2 and to ensure that acids were
unionized and were able to volatilize. pH of the samples were measured using Cyberscan 510
digital pH meter. All other routine parameters such as total solids (TS), volatile solids (VS),
pH, COD, biological oxygen demand (BOD5) and alkalinity were determined according to

9
the procedure prescribed in Standard Methods [56]. All spectrophotometric measurements
were carried out using a double-beam UVVIS Spectrophotometer (Hach, model DR-6000).
All the chemical analyses were carried out in triplicate and average values are reported.
Biogas production from the reactors was measured using water displacement method by
following the procedure described by Sharma et al., 2015 [51] The measurement was based
on counting the number of displacements of constant KOH volume by the produced methane
over time. The free space at the top of the liquid level acted as gas holder. Gaseous products
were also collected periodically in Tedlar bags during the experiments and analyzed by gas
chromatograph (NEWCHROME 6800 gas chromatograph) with a thermal conductivity
detector (TCD). A stainless steel column (2.0m x 1/8inch i.d.) packed with Porapack-Q (80-
100 mesh) was used for this purpose. The carrier gas was argon at a flow rate of 30 mL/min
and at 75 psi pressure. The injected volume of the sample was 60 l and other operational
conditions were as follows: injector temperature: 80oC, oven temperature: 50oC, detector
temperature: 90oC. The standard gas mixture of H2, CO, CO2 and CH4 was used as the
internal standard for quantification. At the end of the experimental run, the sludge and PVA-
gel beads were withdrawn from both the AnPBR-1 and AnPBR-2 for identification of sludge
morphology using a scanning electron microscope (SEM). For this analysis, sample was first
fixed for 1h at 4C with 2.5%(w/v) gluteraldehyde in phosphate buffer solution and then
dehydrated using a graded series of acetone-water mixtures (10, 25, 50, 75, 90, and 100%).
Each mixture was then brought to equilibrium for 10 min and finally dried off by following
the critical-point drying method before being sputter-coated with gold particles [57]. Finally,
the samples were examined using SEM (LEO 435 VP).

2.6 Microbial characterization of biomass

The following procedure was used for the quantification of attached biomass density on
PVA-gel beads. First, 25 numbers of PVA-gel beads were randomly isolated from the desired
sampling location within the concerned reactor. The beads were then thoroughly washed with
distilled water three times and dried in a hot air oven at a constant temperature of 45C and
weighed. Similarly, 25 numbers of randomly collected pure beads were thoroughly soaked in
distilled water for an hour before drying them in the oven at the same temperature and
weighed. The difference between these two weights was considered to be the dry weight of
the bacterial mass attached with the beads. The attached biomass concentration was
expressed as the dry weight of attached bacteria per unit weight of the dry PVA-gel beads.
Mixed liquor volatile suspended solids (MLVSS) inside the reactor was measured using

10
standard protocol [56]. The PVA-gel beads collected from any specific location(s) within the
reactors were quickly placed inside a glove box kept under nitrogen atmosphere. The beads
were first crushed to expose the interiors from where the bacteria were collected and then
were spread on petri-plates containing mineral salt agar growth medium to purify different
classes of bacteria present in the reactor. Out of the different colonies that grew on the plate,
the predominant ones were sub-cultured on another set of plates in order to grow pure culture
of the dominant species. The bacteria from the suspended phase were also plated and cultured
in similar fashion. Further, the pure cultures were Gram-stained to find out the Grams
dominancy of bacteria.

3.0. Results and Discussion

3.1. Enrichment of the Treatment System
The microorganisms were enriched simultaneously in AnPBR-1 and AnPBR-2 using
mesophilic anaerobic seed sludge collected from UASB-based sewage treatment plant
following a process mentioned in section 2.3 of Materials and Methods. During the
enrichment period, the PVA-gel beads were physically observed for its appearance which
turned from milky white to black as the mass of anaerobic bacteria grew over time. SEM
images of the interior of the PVA-gel beads were also taken before and after enrichment.
Figures 2a to 2C show the photographs and SEM images of the pure and enriched PVA-gel
beads. Upon enrichment, the white PVA-gel beads turned from white to black in appearance.
The comparison of SEM images of interior of the bead before and after enrichment revealed
the growth of a separate phase on the PVA network. When cultured following the procedure
as discussed in section 2.6 in Materials and Methods, this separate phase scraped from the
interior of the enriched beads, grew into large number of bacterial colonies on the culture-
plate. Therefore, it can be inferred that the new separate phase as shown in Figure 2c is
merely comprised of bacterial colonies. In a similar way, the suspended solids separated from
a solution collected from the reactor and grew into large number of colonies on the culture-
plates, thereby confirming that the suspended bacterial mass constitute the suspended solids
in the solution within the reactor. Figure 2d shows SEM image of dried biomass separated
after drying of the solution collected from AnPBR-1. Figure 3 shows the influent and
effluent COD profiles of the reactor system during the enrichment period. The reactor system
was considered to be fully enriched when the effluent concentration reached a steady-state
under a constant influent COD concentration.

3.2 Process Optimization by controlling HRT of each reactor

11
During the enrichment stage the steady-state COD removal efficiency was around 89% for an
applied overall organic loading rate (OLR) of 0.38 kg COD m-3 d-1 at inlet COD
concentration of 1500 mg/L. The reactor system had two reactors working in series, each one
having HRT of 48 h, to maintain an overall HRT of the system was 96 h. In order to find out
the optimum operating conditions under the target influent COD concentration, the reactors
were run at different combinations of HRT following the protocol mentioned in section 2.4 of
Materials and Methods. The COD removal efficiencies at different HRTs are shown in Table
2. It may be observed from the Table that the maximum COD removal efficiency of the
system was 89% at an overall HRT of 4 days, with AnPBR-1 and AnPBR-2 being operated at
HRT of 36 h and 60 h, respectively. The pH of the effluent from the reactors did not undergo
significant variation during optimization of the HRTs. The pH of the effluent from AnPBR-1
was recorded in the range of 5.5-5.9 while the pH of the effluent from the AnPBR-2 remained
in the range of 6.8-7.2.

Concentration of acetic acid in the influent and effluent of each of the component reactors,
AnPBR-1 and AnPBR-2, was also measured at different HRTs. Figure 4 shows the
percentage change in the acetic acid concentrations in the effluent of the individual reactors
as compared to that in the corresponding influent. A positive change indicates production of
acetic acid whereas negative change shows the conversion of acetic acid. It may be observed
from the Figure that the first one of the two reactors working in series, AnPBR-1,
demonstrated a positive change confirming the generation of acetic acid within the reactor.
The maximum percentage change in the AnPBR-1 occurred at an HRT of 36 h and thereafter,
for higher values of HRT, it showed a declining trend in the acetic acid concentration. The
effluent from AnPBR-1, which mostly contained acetic acid, was fed into the next reactor in
series, AnPBR-2. A negative change in AnPBR-2 as observed in Figure 4 indicated that there
was disappearance of acetic acid in this reactor.
Out of the two distinct stages of anaerobic digestion, acidogenesis process culminates into
formation of acetic acid through hydrolysis of complex organic molecules. The other stage,
methanogenesis, involves further conversion of this acetic acid into biogas which is
essentially a mixture of carbon dioxide and methane, and to a little extent hydrogen gas. Both
the reactors used in this study were packed-bed reactors. AnPBR-1 and AnPBR-2 essentially
resemble plug flow reactors in which, due to the absence of mixing in axial direction, the
concentrations of the reactive components and their end products vary along the axial length
of the reactor. It may be observed from Figure 4 that for AnPBR-1, an HRT below 36 h

12
could not completely convert the organic components present in the feed into acetic acid;
whereas a peak at 36 h HRT and subsequent dip in acetic acid concentration for HRT greater
than 36 h meant that the hydrolytic reactions culminating in acetic acid production was
complete within the reactor at HRT of around 36 h. For a residence time greater than 36 h,
the subsequent reaction, methanogenesis starts within AnPBR-1, thereby reducing the
concentration of acetic acid in its effluent. Therefore, it can be inferred that for an HRT of 36
h AnPBR-1 only allows for acidogenesis reaction to near-completion. Under this operating
condition, AnPBR-1 can be termed as acidogenic reactor. When fed by the effluent of
acidogenic reactor running at HRT of 36 h, AnPBR-2 should now act as a methanogenic
reactor. The bottom graph in Figure 4 shows the percentage change in the lag reactor at
different HRTs, when it was fed with the effluent from lead reactor running at optimized
HRT of 36 h. According to the Figure 4, the conversion of acetic acid was found to be
increasing with the increase in HRT, which reached to a maximum at HRT of 60 h. No
further increase in the concentration of acetic acid was observed at higher HRTs. Thus, it can
be concluded that at optimized operating condition i.e. with 36 and 60 h HRT, AnPBR-1 and
AnPBR-2 were effectively converted into acidogenic and methanogenic packed-bed reactors,
respectively. Therefore, it can be concluded that anaerobic degradation running in packed-
beds with attached microorganisms reached to its maximum efficiency when it is stage-
separated.

3.3 Performance evaluation of the acidogenic and methanogenic reactor

Figure 5a shows the total and individual VFAs, and pH profile of the effluent from the
acidogenic reactor over a period of 270 days when the reactor was allowed to run in steady-
state at an HRT of 36 h with influent COD concentration of 1500 mg/L. Throughout the run,
the temperature was maintained at 35C except for a time period between 82nd day to 137th
day when the temperature of the reactor was allowed to fall to an average value of 10C.
Thereafter, the temperature was restored back to 35 C. The methanogenic reactor was also
run at steady-state under operating conditions same as the acidogenic reactor but with HRT of
60 h. The methanogenic reactor was fed with the effluent from the acidogenic reactor.
Methanogenic productivity is defined as the methane production rate per unit volume of the
reactor. Figure 5b shows the time history profile for the rate of generation of total biogas and
methanogenic productivity of the methanogenic reactor. The average overall methanogenic
productivity of the total reactor system at steady state was calculated to be 0.08 L methane/(L
reactor.d). The experimental results showed that at steady-state there was 45-50% methane

13
content in the biogas generated from the methanogenic reactor. The temperature shock was
also applied to the methanogenic reactor with the same degree and duration as that of
acidogenic reactor. From Figures 5a and 5b, it was observed that during a steady-state
condition, a sudden drop in the temperature acted as a temperature shock that caused
destabilization in the activity of the microorganisms within both the reactors. Such
destabilization caused a decline in the total gas production rates as well as methanogenic
activity of the methanogenic reactor, as is evident from Figure 5b. In acidogenic reactor too,
the destabilization caused by the temperature shock manifested in the form of a decrease in
the acetic acid concentration and increase in the propionic acid concentration.

Figure 6 shows the ratio of concentrations of propionic acid to acetic acid in the effluent of
acidogenic reactor during the steady-state run. It was observed that during the steady-state
run, effluent from the acidogenic reactor had nearly constant ratio of the concentrations of
propionic to acetic acid, except for the time period of temperature-shock related
destabilization and restabilization. Propionic acid is considered to be toxic in anaerobic
process. Suitable reactor configurations may help to avoid generation of high proportion of
propionic acid [58]. It is known that propionic acid to acetic acid ratio can be used as an
indicator of digester imbalance and a value greater than 1.4 indicates impending reactor
failure [48,59,60]. The steady-state ratio of propionic acid to acetic acid concentration in the
acidogenic reactor in this study was recorded in the range of 0.2-0.35 which was significantly
lower than the above limiting value. However, the value increased to as high as 3.5 during the
temperature stress before the temperature was brought back to 35C. The reactor, although
seemed to fail as per the above limiting value, quickly restored back to the original state
within 28 days without any further seeding or enrichment. These results revealed that the
microbial community responsible for completion of hydrolysis to acetic acid did not
disappear and just remained dormant during the time of temperature shock so that when the
shock was withdrawn they grew quickly to re-stabilize the reactor back to the original state
that existed prior to the temperature-shock. Therefore, it may be inferred that the acidogenic
reactor in plug flow like configuration having biofilm carrier supported attached
microorganism provided a rather highly suitable and resilient reactor configuration for the
anaerobic degradation process.

Figure 7 shows the overall COD removal efficiency of the whole reactor system during the
steady-state run. It may be observed that during the temperature stress, overall COD removal
efficiency of the treatment process dropped gradually over the period of time. However, as

14
soon as the temperature shock was withdrawn there was no further drop and the performance
for COD removal started improving as may be observed in Figure 7. The sharp ascending
gradient of the COD removal efficiency curve with respect to time indicates that the reactor
system was able to recover quickly after the withdrawal of the temperature-stress. This
indicates that the anaerobic reactor system in the stage-separated plug-flow type
configuration with biofilm carrier attached microorganisms has enough resilience to quickly
recover against long-duration temperature stresses which are not uncommon in cold climates
[61,62,63,64,65].

Figure 8 shows the average concentration profiles of different VFA species at the inlet of
acidogenic reactor and at the effluents of acidogenic and methanogenic reactor during steady
state operation with overall OLR of 0.38 kg COD m-3 d-1 and at optimum HRT condition. In
this regard, following points are important to mention here that: a) acetic acid and butyric
acid were major VFA species while propionic acid was low in proportion and b) The
methanogenic reactor could not convert all the VFA into biogas; a small portion of VFA still
remained in the final effluent.

3.4 Characteristics of microbial community

The average MLVSS concentration in the acidogenic and methanogenic reactor during
steady- state operation at optimized condition under an influent concentration of 1500 mg/L
COD being fed into acidogenic reactor was found to be 3.8 and 5.2 g/L, respectively.
Average attached biomass concentration within the PVA-gel beads was found to be 43.4 and
58.4 mg dry biomass/ g dried PVA-gel beads for acidogenic and methanogenic reactor,
respectively. If the average water contents of attached biomass cells and the PVA-gel beads
are assumed to be the same, then on wet mass basis, the MLVSS concentration is negligible
compared to that of attached biomass within the PVA-gel beads. Such high abundance of
attached biomass is expected in the biofilm carrier based plug-flow type reactor system.

The Grams dominancy study of the reactor system revealed that the Gram positive bacteria
were dominant during the stable performance of the reactor. However, the dominant bacterial
population reversed from Gram positive to Gram negative during the temperature shock.
When the temperature shock was ceased, the system brought back to previous state of
predomination by Gram positive consortia. An anaerobic reactor system is known to have a
diverse range of different classes of bacteria [1,66,67]. According to the prevailing
environmental conditions the bacterial population is known to shifts its dominancy to a

15
population in which consortia can thrive [68,69]. The characteristics of microbial consortia
indicated that gram negative bacteria have better ability to thrive during low temperatures
compared to the Gram positive ones.

4.0 Conclusion

Anaerobic wastewater treatment systems are preferred over aerobic systems on account of
low energy requirements and low volume sludge generation, but often suffer from operational
drawbacks such as process vulnerability under environmental shocks. A stage-separated
anaerobic reactor system consisting of two packed-bed plug-flow type reactors operated in
series and each filled with porous PVA-gel beads used for immobilization and attachment of
active biomass, showed optimistic performance scenario during the study with molasses-
based medium strength synthetic wastewater. The highest COD removal efficiency of 89%
was achieved when the constituent reactors were stage-separated; the first reactor supported
only acidogenesis process while the next reactor supported methanogenesis process. Analysis
of the effluent of the acidogenic reactor at steady-state revealed that acetic acid and butyric
acid were predominant ones among the VFAs produced. The propionic acid to acetic acid
ratio at steady-state was found in the range of 0.2-0.35, which indicated the stability of the
acidogenic reactor against environmental shock. When the reactor system was exposed to
lower temperature, the propionic to acetic acid ratio increased to a value of 3.5 along with
concomitant decrease in the COD removal efficiency and reduced gas production rate, but
bounced back to steady-state condition soon after the temperature shock was withdrawn. The
methanogenic reactor showed an appreciable biogas generation potential with average
methane content of 48% at steady-state. Overall, the anaerobic reactor system in the stage-
separated plug-flow like configuration with biofilm carrier attached microorganisms showed
optimistic performance with demonstrated resilience against low-temperature shock. Such
fall in temperature is quite common in wastewater treatment plants in cold climates. A
preliminary study on the microbial diversity showed that during the shock, the predominance
in microbial community changed from Gram-positive to Gram-negative bacteria. Further
studies need to be performed to demonstrate its resilience against other shock scenario such
as high organic loading rates, and nutrient stress. Further studies on the behavior and
composition of attached microbial consortia on PVA-gel beads during steady-state and shock
operation regimes shall help in better understanding of the mechanism and advantage of
using PVA-gel as a biofilm carrier.

16
Acknowledgement

The authors are thankful to Mr. Vipin rose, Head of operations, Kuraray India Private Ltd for
making the PVA-gel beads available for the study. Authors would also like to extend their
sincere thanks to Ms. Ruchi Panwar, for her helping hands in analytical studies.

References

1 Stuckey, D. C. (2012). Recent developments in anaerobic membrane reactors.

Bioresource Technology, 122, 13748.
2 Chen, Y., Cheng, J. J., & Creamer, K. S. (2008). Inhibition of anaerobic digestion
process: a review. Bioresource Technology, 99(10), 404464.
3 Alkarimiah, R., Mahat, S. B., Yuzir, A., & Din, M. F. (2011). Operational Start-
Up Performance of an Innovative Anaerobic Stage Reactor ( ASR ) using
Synthetic Wastewater. 2011 International Conference on Environment and
Industrial Innovation, 12, 133137.
4 Zekker, I., Rikmann, E., Tenno, T., Loorits, L., Kroon, K., Fritze, H., & DC.
Rubin, S. S. (2015). Nitric oxide for anammox recovery in a nitrite-inhibited
deammonification system. Environmental technology, 36(19), 2477-2487.
5 DiStefano, T. D., & Palomar, A. (2010). Effect of anaerobic reactor
processconfiguration on useful energy production. Water Research, 44(8), 2583
91.
6 Nunez, L. A., & Martinez, B. (1999). Anaerobic treatment of slaughterhouse
wastewater in an expanded granular sludge bed (EGSB) reactor. Water Science
and Technology, 40(8), 99-106.
7 Omil, F., Garrido, J. M., Arrojo, B., & Mndez, R. (2003). Anaerobic filter reactor
performance for the treatment of complex dairy wastewater at industrial scale.
Water Research, 37(17), 4099-4108.
8 Wu, B., Li, Y., Lim, W., Lee, S. L., Guo, Q., Fane, A. G., & Liu, Y. (2017).
Single-stage versus two-stage anaerobic fluidized bed bioreactors in treating
municipal wastewater: Performance, foulant characteristics, and microbial
community. Chemosphere, 171, 158-167.

17
9 Tenno, T., Rikmann, E., Zekker, I., Tenno, T., Daija, L., & Mashirin, A. (2016).
Modelling equilibrium distribution of carbonaceous ions and molecules in a
heterogeneous system of CaCO3watergas. Proceedings of the Estonian
Academy of Sciences, 65(1), 68-77.
10 Salomoni, C., Caputo, a, Bonoli, M., Francioso, O., Rodriguez-Estrada, M. T., &
Palenzona, D. (2011). Enhanced methane production in a two-phase anaerobic
digestion plant, after CO2 capture and addition to organic wastes. Bioresource
Technology, 102(11), 64438.
11 Song, Y., Kwon, S., & Woo, J. (2004). Mesophilic and thermophilic temperature
co-phase anaerobic digestion compared with single-stage mesophilic- and
thermophilic digestion of sewage sludge, 38, 16531662.
12 Madsen, M., Holm-Nielsen, J. B., & Esbensen, K. H. (2011). Monitoring of
anaerobic digestion processes: A review perspective. Renewable and Sustainable
Energy Reviews, 15(6), 31413155.
13 Francioso, O., Rodriguez-Estrada, M. T., Montecchio, D., Salomoni, C., Caputo,
A., & Palenzona, D. (2010). Chemical characterization of municipal wastewater
sludges produced by two-phase anaerobic digestion for biogas production. Journal
of Hazardous Materials, 175(13), 7406.
14 Li, W.-W., & Yu, H.Q. (2011). From wastewater to bioenergy and biochemicals
via two-stage bioconversion processes: a future paradigm. Biotechnology
Advances, 29(6), 97282.
15 Bareither, C. a, Wolfe, G. L., McMahon, K. D., & Benson, C. H. (2013).
Microbial diversity and dynamics during methane production from municipal
solid waste. Waste Management (New York, N.Y.), 33(10), 198292.
16 Angenent, L. T., Banik, G. C., & Sung, S. (1998). Anaerobic migrating blanket
reactor treatment of low-strength wastewater at low temperatures. Water
Environment Research: A Research Publication of the Water Environment
Federation, 73(5), 56774.
17 Grootscholten, T. I. M., Kinsky dal Borgo, F., Hamelers, H. V. M., & Buisman, C.
J. N. (2013). Promoting chain elongation in mixed culture acidification reactors by
addition of ethanol. Biomass and Bioenergy, 48, 1016.
18 Xiao, K. K., Guo, C. H., Zhou, Y., Maspolim, Y., Wang, J. Y., & Ng, W. J.
(2013). Acetic acid inhibition on methanogens in a two-phase anaerobic process.
Biochemical Engineering Journal, 75, 17.

18
19 Ghanimeh, S. a, Saikaly, P. E., Li, D., & El-Fadel, M. (2013). Population
dynamics during startup of thermophilic anaerobic digesters: the mixing factor.
Waste Management (New York, N.Y.), 33(11), 22118.
20 Blume, F., Bergmann, I., Nettmann, E., Schelle, H., Rehde, G., Mundt, K., &
Klocke, M. (2010). Methanogenic population dynamics during semi-continuous
biogas fermentation and acidification by overloading. Journal of Applied
Microbiology, 109(2), 44150.
21 Pandey, P. K., Ndegwa, P. M., Soupir, M. L., Alldredge, J. R., & Pitts, M. J.
(2011). Efficacies of inocula on the startup of anaerobic reactors treating dairy
manure under stirred and unstirred conditions. Biomass and Bioenergy, 35(7),
27052720.
22 Kim, M., & Speece, R. E. (2002). Aerobic waste activated sludge (WAS) for start-
up seed of mesophilic and thermophilic anaerobic digestion. Water Research,
36(15), 38606.
23 Muha, I., Zielonka, S., Lemmer, A., Schnberg, M., Linke, B., Grillo, A., &
Wittum, G. (2013). Do two-phase biogas plants separate anaerobic digestion
phases? - a mathematical model for the distribution of anaerobic digestion phases
among reactor stages. Bioresource Technology, 132, 4148.
24 Fezzani, B., & Ben Cheikh, R. (2010). Two-phase anaerobic co-digestion of olive
mill wastes in semi-continuous digesters at mesophilic temperature. Bioresource
Technology, 101(6), 162834.
25 Jeong, E., Kim, H.-W., Nam, J.-Y., & Shin, H.-S. (2010). Enhancement of
bioenergy production and effluent quality by integrating optimized acidification
with submerged anaerobic membrane bioreactor. Bioresource Technology, 101
Suppl(1), S7S12.
26 Siegert, I., & Banks, C. (2005). The effect of volatile fatty acid additions on the
anaerobic digestion of cellulose and glucose in batch reactors. Process
Biochemistry, 40(11), 34123418.
27 Blonskaja, V., Menert, a., & Vilu, R. (2003). Use of two-stage anaerobic
treatment for distillery waste. Advances in Environmental Research, 7(3), 671
678.
28 Long, J. H., Aziz, T. N., Reyes, F. L. D. L., & Ducoste, J. J. (2012). Anaerobic co-
digestion of fat, oil, and grease (FOG): A review of gas production and process
limitations. Process Safety and Environmental Protection, 90(3), 231245.

19
29 Wilson, C. A., Novak, J., Takacs, I., Wett, B., & Murthy, S. (2012). The kinetics
of process dependent ammonia inhibition of methanogenesis from acetic acid.
Water Research, 46(19), 624756.
30 Schlter, A., Bekel, T., Diaz, N. N., Dondrup, M., Eichenlaub, R., Gartemann, K.-
H, Goesmann, A. (2008). The metagenome of a biogas-producing microbial
community of a production-scale biogas plant fermenter analysed by the 454-
pyrosequencing technology. Journal of Biotechnology, 136(12), 7790.
31 Gallert, C., & Winter, J. (2008). Propionic acid accumulation and degradation
during restart of a full-scale anaerobic biowaste digester. Bioresource Technology,
99(1), 170178.
32 Senthilkumar, M., Gnanapragasam, G., Arutchelvan, V., & Nagarajan, S. (2011).
Treatment of textile dyeing wastewater using two-phase pilot plant UASB reactor
with sago wastewater as co-substrate. Chemical Engineering Journal, 166(1), 10
14.
33 Gnanapragasam, G., Senthilkumar, M., Arutchelvan, V., Velayutham, T., &
Nagarajan, S. (2011). Bio-kinetic analysis on treatment of textile dye wastewater
using anaerobic batch reactor. Bioresource Technology, 102(2), 62732.
34 Brito, A. G., Rodrigues, A. C., & Melo, L. F. (1997). Granulation during the start-
up of a UASB reactor used in the treatment of low strength wastewaters.
Biotechnology Letters, 19(4), 363367.
35 Dereli, R. K., Ersahin, M. E., Ozgun, H., Ozturk, I., Jeison, D., van der Zee, F., &
van Lier, J. B. (2012). Potentials of anaerobic membrane bioreactors to overcome
treatment limitations induced by industrial wastewaters. Bioresource Technology,
122, 16070.
36 Bertin, L., Lampis, S., Todaro, D., Scoma, A., Vallini, G., Marchetti, L., Fava,
F. (2010). Anaerobic acidogenic digestion of olive mill wastewaters in biofilm
reactors packed with ceramic filters or granular activated carbon. Water Research,
44(15), 453749.
37 De Bok, F. A. M., Plugge, C. M., & Stams, A. J. M. (2004). Interspecies electron
transfer in methanogenic propionate degrading consortia. Water Research, 38(6),
13681375.
38 Fdez-Gelfo, L. a., lvarez-Gallego, C., Sales, D., & Romero, L. I. (2012). New
indirect parameters for interpreting a destabilization episode in an anaerobic
reactor. Chemical Engineering Journal, 180, 3238.

20
39 Klocke, M., Nettmann, E., Bergmann, I., & Mundt, K. (2008). Characterization of
the methanogenic Archaea within two-phase biogas reactor systems operated with
plant biomass, 31, 190205.
40 Verrier, D., Roy, F., & Albagnac, G. (1987). Two-phase methanization of solid
vegetable wastes. Biological Wastes, 22(3), 163177.
41 Gani, K. M., Singh, J., Singh, N. K., Ali, M., Rose, V., & Kazmi, A. A. (2016).
Nitrogen and carbon removal efficiency of a polyvinyl alcohol gel based moving
bed biofilm reactor system. Water Science and Technology, 73(7), 15111519.
42 Hamjinda, N. S., Chiemchaisri, W., & Chiemchaisri, C. (2016). Upgrading two-
stage membrane bioreactor by bioaugmentation of Pseudomonas putida
entrapment in PVA/SA gel beads in treatment of ciprofloxacin. International
Biodeterioration & Biodegradation.
43 Ali, M., Oshiki, M., Rathnayake, L., Ishii, S., Satoh, H., & Okabe, S. (2015).
Rapid and successful start-up of anammox process by immobilizing the minimal
quantity of biomass in PVA-SA gel beads. Water research, 79, 147-157.
44 El-Naas, M. H., Mourad, A. H. I., & Surkatti, R. (2013). Evaluation of the
characteristics of polyvinyl alcohol (PVA) as matrices for the immobilization of
Pseudomonas putida. International Biodeterioration & Biodegradation, 85, 413-
420.
45 Singh, N. K., Singh, J., Bhatia, A., & Kazmi, A. A. (2016). A pilot-scale study on
PVA gel beads based integrated fixed film activated sludge (IFAS) plant for
municipal wastewater treatment. Water Science and Technology, 73(1), 113123.
46 Banihani, Q., Hadadin, N., & Jamrah, A. (2012). Start-up of Anaerobic
Ammonium Oxidation (Anammox) from Conventional Return Activated Sludge
in Up-flow Anaerobic Sludge Blanket ( UASB ) Reactor for Autotrophic Nitrogen
Removal from Wastewater. Jordan Journal of Civil Engineering, 6(1), 1727.
47 Zhang, W., Xie, Q., Rouse, J. D., Qiao, S., & Furukawa, K. (2009). Treatment of
high-strength corn steep liquor using cultivated polyvinyl alcohol gel beads in an
anaerobic fluidized-bed reactor. Journal of Bioscience and Bioengineering,
107(1), 4953.
48 Norstedt, R. A., Thomas, M.V. (1985). Start-up characteristics of anaerobic fixed-
bed reactors Trans. ASAE, 28, pp. 12421247.
49 Taylor, P., Chaikasem, S., Jacob, P., & Visvanathan, C. (2014). Performance
improvement in a two-stage thermophilic anaerobic membrane bioreactor using

21
 PVA-gel as biocarrier bioreactor using PVA-gel as biocarrier, Desalination and
Water Treatment, 3741.
50 Chaikasem, S., Abeynayaka, A., & Visvanathan, C. (2014). Effect of polyvinyl
alcohol hydrogel as a biocarrier on volatile fatty acids production of a two-stage
thermophilic anaerobic membrane bioreactor. Bioresource Technology, 168, 100
5.
51 Sharma, M. K., & Kazmi, A. A. (2015). Anaerobic onsite treatment of black water
using filter-based packaged system as an alternative of conventional septic tank.
Ecological Engineering, 75, 457461.
52 Zekker, I., Rikmann, E., Tenno, T., Seiman, A., Loorits, L., Kroon, K., & Tenno,
T. (2014)-a. Nitritating-anammox biomass tolerant to high dissolved oxygen
concentration and C/N ratio in treatment of yeast factory wastewater.
Environmental technology, 35(12), 1565-1576.
53 Zekker, I., Rikmann, E., Tenno, T., Seiman, A., Loorits, L., Kroon, K., & Tenno,
T. (2014)-b. Nitritating-anammox biomass tolerant to high dissolved oxygen
concentration and C/N ratio in treatment of yeast factory wastewater.
Environmental technology, 35(12), 1565-1576.
54 Rikmann, E., Zekker, I., Tomingas, M., Tenno, T., Loorits, L., Vabame, P., &
Tenno, T. (2016). Sulfate-reducing anammox for sulfate and nitrogen containing
wastewaters. Desalination and Water Treatment, 57(7), 3132-3141.
55 Falk, H. M., Reichling, P., Andersen, C., & Benz, R. (2015). Online monitoring of
concentration and dynamics of volatile fatty acids in anaerobic digestion processes
with mid-infrared spectroscopy. Bioprocess and biosystems engineering, 38(2),
237-249.
56 APHA, 2005 APHA Standard Methods for the Examination of Water and
Wastewater (21st ed.) American Public Health Association, American Water
Works Association, Water Environment Federation, Washington, DC (2005).
57 Gopal, K.G.V.T., Kumar, P., Kumar, P., 2009. Treatment of low-strength soluble
wastewater using an anaerobic baffled reactor (ABR). J. Environ. Manage. 90,
166176.
58 Speece, R. E., Boonyakitsombut, S., Kim,M., Azbar, N.& Ursillo, P., 2006.
Overview of anaerobic treatment: thermophilic and propionate implications.
Water Environ. Res. 78(5), 460473.

22
59 Marchaim, U., & Krause, C. (1993). Propionic to acetic acid ratios in overloaded
anaerobic digestion. Bioresource Technology, 43(3), 195203.
60 Hill, D. T., Cobb, S. A., Bolte J. P., (1987). Using volatile fatty acid relationships
to predict anaerobic digester failure. transac. of the ASAE. 30 (2): 0496 -0501.
61 Komemoto, K., Lim, Y. G., Nagao, N., Onoue, Y., Niwa, C., & Toda, T. (2009).
Effect of temperature on VFAs and biogas production in anaerobic solubilization
of food waste. Waste Management (New York, N.Y.), 29(12), 29505.
62 Wilson, C. A., Murthy, S. M., Fang, Y., & Novak, J. T. (2008). The effect of
temperature on the performance and stability of thermophilic anaerobic digestion.
Water Science and Technology, 57(2), 297304.
63 Hasan, S. D. M., Giongo, C., Fiorese, M. L., Gomes, S. D., Ferrari, T. C., &
Savoldi, T. E. (2015). Volatile fatty acids production from anaerobic treatment of
cassava waste water: effect of temperature and alkalinity. Environmental
Technology, 36(20), 263746.
64 Zekker, I., Rikmann, E., Mandel, A., Kroon, K., Seiman, A., Mihkelson, J., &
Tenno, T. (2016). Step-wise temperature decreasing cultivates a biofilm with high
nitrogen removal rates at 9 C in short-term anammox biofilm tests. Environmental
technology, 37(15), 1933-1946.
65 Raudkivi, M., Zekker, I., Rikmann, E., Vabame, P., Kroon, K., & Tenno, T.
(2016). Nitrite inhibition and limitationthe effect of nitrite spiking on anammox
biofilm, suspended and granular biomass. Water Science and Technology,
wst2016456.
66 Lin, J., Ortiz, R., Steele, T. W. J., & Stuckey, D. C. (2014). Toxicants inhibiting
anaerobic digestion: A review. Biotechnology Advances, 32(8), 15231534.
67 Jenicek, P., Koubova, J., Bindzar, J., & Zabranska, J. (2010). Advantages of
anaerobic digestion of sludge in microaerobic conditions. Water Science and
Technology: A Journal of the International Association on Water Pollution
Research, 62(2), 42734.
68 Seetha, N., Bhargava, R., & Kumar, P. (2010). Bioresource Technology Effect of
organic shock loads on a two-stage activated sludge-biofilm reactor. Bioresource
Technology, 101(9), 30603066.
69 Appels, L., Baeyens, J., Degrve, J., & Dewil, R. (2008). Principles and potential
of the anaerobic digestion of waste-activated sludge. Progress in Energy and
Combustion Science, 34(6), 755781.
23
Figure 1. Schematic of the packed-bed reactor system consisting of PVA gel beads

24
 b
a )
)

c d

Figure 2. (a) Photograph of PVA gel beads in pure (on the right) condition and after
enrichment with microorganism (left); SEM image of interior (b) pure and (c) enriched PVA
gel bead; (d) SEM image of dried suspended biomass from AnPBR-1 (acidogenic reactor).

25
Figure 3. COD concentration profiles of influent and effluent along with COD removal
efficiency of the overall reactor system over time during enrichment period.

26
Figure 4. Optimization of hydraulic retention time (HRT) of individual reactors on the basis
of generation and conversion of acetic acid; acedogenic reactor (top) showing generation
(positive change) and methanogenic reactor (bottom) showing conversion (negative change)
of acetic acid. Methanogenic reactor was fed with effluent from acidogenic reactor running at
optimum HRT of 36 hours

27
Figure 5a. Variation of total volatile fatty acid (VFA) and individual VFA species (acetic
acid, propionic acid and butyric acid) concentrations and pH in the effluent from the
acidogenic reactor over time.

28
Figure 5b. Time history profile of biogas generation rate and methanogenic productivity of
the methanogenic reactor

29
Figure 6. Profile of propionic acid to acetic acid ratio in the effluent from the acidogenic
reactor over time.

30
Figure 7. Variation of overall COD removal efficiency of the reactor system with time.

31
Figure 8. Average concentration of different VFA species at different points of the reactor
system during steady-state operation.

32
Table 1- Characteristics of the synthetic wastewater used in the study
Parameters Values (Mean S.D.)
pH 6.9 0.2
Temperature (C) 35 3
COD (mg/L) 1468.21 14.9
BOD (mg/L) 1041.74 16.8
TS (mg/L) 18.3 4.6
SS (mg/L) 7.3 1.4
TKN (mg/L) 86.83 4.9
NH4+ (mg/L) 9.83 1.1
TP (mg/L) 3.28 0.2

Table 2. Optimization of HRT of two-stage packed bed reactor

Experiment Hydraulic Retention Time Overall COD
Number (HRT) removal
efficiency (%)
(h)

AnPBR-1 AnPBR-2
1 72 72 89
(enrichment)
2 54 75 87
3 54 60 88
4 36 75 88
5 36 60 89
6 36 45 78
7 36 30 75
8 18 45 75
9 18 30 68
10 18 15 61

33