American Journal of Microbiological Research, 2015, Vol. 3, No.

1, 14-24
Available online at http://pubs.sciepub.com/ajmr/3/1/3
© Science and Education Publishing
DOI:10.12691/ajmr-3-1-3

Relationship between Temperature, Ph and Population

of Selected Microbial Indicators during Anaerobic
Digestion of Guinea Grass (Panicum Maximum)
Ogbonna. C. B.*, Berebon. D. P., Onwuegbu. E. K.

Department of Microbiology, Faculty of Biological Science, College of Natural and Applied Sciences, University of Port Harcourt,
P.M.B. 5323 Port Harcourt, Nigeria
*Corresponding author: chukwukaogbonna@gmail.com
Received December 01, 2014; Revised December 31, 2014; Accepted January 08, 2015
Abstract In this study, the relationship between process temperature, process pH and population of selected
microbial indicators during anaerobic digestion of guinea grass (Panicum maximum) at ambient condition was
investigated. A one stage batch-type mesophilic anaerobic digestion system was configured using rumen fluid (RF)
as inoculums (ADRF) and a low solid loading of approximately 7.0% total solid (TS). Physicochemical parameters
such as process temperature (PTMRF), process pHRF and volatile fatty acid (VFARF) were monitored with time.
Selected indicator microbial populations were monitored by standard cultural techniques based on metabolic
capacity and oxygen sensitivity with respect to time. Result showed that average PTMRF increased from 27.5°C to
35.2°C, average process pHRF ranged from 6.5 to 7.9 and VFARF ranged from 1,080.00 mg/L to 4,800.33 mg/L with
time. In terms of metabolic capacity and oxygen sensitivity, the populations of cellulolytic bacteria (CBRF), lactose
and glucose fermenting (acidogenic) bacteria (LFBRF and GFBRF), propionate and ethanol oxidizing (acetogenic)
bacteria (POBRF and EOBRF), acetate oxidizing methanogens (AOMRF), obligate anaerobic bacteria (OABRF) and
total facultative bacteria (FAABRF) increased (about 10-fold) respectively with time. Correlation analysis showed
positive relationships between the process temperature (PTMRF) and the population of selected microbial indicators
with time. However, there were negative relationships between the process pHRF and the population of selected
microbial indicators with time. Furthermore, there were positive relationships between the populations of selected
microbial indicators with time. Rumen fluid significantly (P < 0.05) affected the dynamics of the process
temperature (PTMRF) and process pHRF inside the ADRF system with time respectively. These kinds of relationships
between biotic factors and between biotic and abiotic factors could be used to monitor the state of anaerobic
digestion process with respect to time.
Keywords: relationship, microbial populations, temperature, pH, anaerobic digestion, guinea grass
Cite This Article: Ogbonna. C. B., Berebon. D. P., and Onwuegbu. E. K., “Relationship between
Temperature, Ph and Population of Selected Microbial Indicators during Anaerobic Digestion of Guinea Grass
(Panicum Maximum).” American Journal of Microbiological Research, vol. 3, no. 1 (2015): 14-24. doi:
10.12691/ajmr-3-1-3.

et al., 1990; Preeti Rao et al., 1993; Zhou et al., 2009).

1. Introduction
Anaerobic digestion (AD) of organic matter has been
recognized as a valuable resource that can be converted
into useful products via microbial mediated
transformations (Chanakya et al., 2007; Guermoud et al.,
2009; Schnurer and Jarvis, 2010). There are four stages
involved in the AD process for biogas production. These
include hydrolysis, acidogenesis, acetogenesis and
methanogenesis. These processes are carried out by
different microbial populations (or communities) which,
have to work together for the AD process to proceed
efficiently (Dassonville and Renault, 2002; Ljupka, 2010;
Schink, 1997; Zhou et al., 2009). The nature of a substrate
can determine the type and extent of bacteria populations
present in an anaerobic digester (Zinder, 1984; Ramasamy
Anaerobic digestion of cellulosic (or lignocellulosic)
materials is limited mainly by the rate of hydrolysis of the
polymeric compounds and to be efficiently fermented,
require suitable inoculation for biogas production (Labat
and Garcia, 1986; Lopes et al., 2004; Forster-Carneiro et
al., 2007; Dong et al., 2009; Uzodinma and Ofoefole,
2009). The potential application of rumen microorganisms
in anaerobic digestion systems for the conversion of
lignocellulosic materials have been investigated by several
researchers (Allison and Leek, 1993; Ogbonna et al.,
2014).
The microorganisms that participate in anaerobic
digestion process may be specific for each degradation
step and thus could have different environmental (e.g.,
temperature and pH) requirements (Azeem et al., 2011).
Various researchers have reported significant effects of
 American Journal of Microbiological Research
temperature on microbial communities during anaerobic
digestion (AD) process (Dela-Rubia et al., 2002;
Bouallagui et al., 2009b; Riau et al., 2010; Kim et al.,
2006; Trzcinski and Stuckey, 2010; Kashyap et al., 2003;
Briski et al., 2007; Fezzani and Cheikh, 2010; Ward et al.,
2008). Generally, the AD process is carried out at
mesophilic temperatures (El-Mashad et al., 2003) because
operation in the mesophilic range is more stable and
requires a smaller energy expense (Fernandez et al., 2008;
Ward et al., 2008). Overall, a temperature range between
35 and 37°C is considered suitable for the production of
methane (Azeem et al., 2011). A number of researchers
such as Agdag and Sponza (2007),Ward et al., (2008), Lee
et al., (2009b), Kim et al., (2003) and Liu et al., (2008)
have also reported a range of pH values suitable for
anaerobic digestion but the optimal pH for
methanogenesis has been found to be around 7.0 (Huber et
al., 1982; Yang and Okos, 1987).
The objective of this study was to determine the
relationship between process temperature, process pH and
population of selected microbial indicators during
anaerobic digestion of guinea grass (Panicum maximum)
at ambient condition.
2. Materials and Methods
2.1. Anaerobic Digestion Set-Up
One-stage anaerobic digestion (AD) systems were
conFigured for batch-type mesophilic reactors as
described by Ogbonna et al., (2014). The anaerobic
digestion system with rumen fluid inoculation (ADRF) was
the experimental set-up, while the anaerobic digestion
system without rumen fluid inoculation (ADNRF) was the
control set-up. Anaerobic digestion (AD) of the feed was
performed (at ambient conditions) inside the mesophilic
fermentors with a retention time of 105 days. The feed for
anaerobic digestion was also prepared as described by
Ogbonna et al., (2014).
2.2. Sample Collection and Determination of
Physicochemical/Microbial Parameters
To monitor the AD process, digester slurry samples
were collected with respect to time. Daily ambient
temperature (ATM), process temperature (PTM) and
weekly process pH were measured using thermometer
(SCT-lilliput, Scichem Tech.) and a general purpose pH
meter (SCT-lilliput, Scichem Tech.), respectively. Process
temperature and pH were respectively determined by
dipping the thermometer and the pH meter into the
digester slurry samples immediately after collection in
beakers. Volatile fatty acid (VFA) was determined using
the titrimetric method described by Buchauer (1998).
Bacteria populations were monitored by enumerating
selected indicator groups based on metabolic capacity and
oxygen sensitivity with respect to time (Ogbonna et al.,
2014). According to metabolic capability, the microbial
populations that were selected included cellulolytic
bacteria (ACB), lactose fermenting (acidogenic) bacteria
(LFB), glucose fermenting (acidogenic) bacteria (GFB),
propionate oxidizing (acetogenic) bacteria (POB), ethanol
oxidizing (acetogenic) bacteria (EOB) and acetate
oxidizing methanogens (AOM). The O2-sensitive
15
populations that were selected included obligate anaerobic
bacteria (OAB) and total facultative anaerobic bacteria
(FAAB) respectively.
2.3. Statistical Analyses
Using SPSS software (version 20), multiple correlation
analysis was performed to determine the relationships
among microbial populations, process temperature (PTM)
and process pH inside the anaerobic digester with rumen
fluid inoculation (ADRF) and the anaerobic digester
without rumen fluid inoculation (ADNRF) with respect to
time. Furthermore, the effect of rumen fluid on the
dynamics of process temperature and process pH with
time was determined using one – way ANOVA. Finally,
Chi – square (Goodness of fit) analysis was used to
determine the stability of the process temperature and
process pH with respect to time.
3. Result and Discussion
3.1. Microbial and Physicochemical Analysis
The population dynamics of selected indicator
microbial groups observed during 105 days retention time
of anaerobic digestion of Panicum maximum have been
presented in Ogbonna et al., (2014). Generally, these
microbial populations increased about 10-fold with respect
to time inside the anaerobic digester with and without
rumen fluid inoculation (ADRF and ADNRF) respectively.
Average ambient temperature (ATM) around the
anaerobic digesters ranged from 27.5°C to 29.5°C with
time (Figure 1a). In the anaerobic digester with rumen
fluid inoculation (ADRF), average process temperature
(PTMRF) increased from 27.5°C (at day 0) to 35.2°C (at
day 70) and then dropped to 33.3°C at day 105. In the
anaerobic digester without rumen fluid inoculation
(ADNRF), average process temperature (PTMNRF) increased
from 27.5°C (at day 0) to 34.4°C at day 105 (Figure 1a).
This result showed that the anaerobic digester with rumen
fluid inoculation (ADRF) was slightly more heated than the
anaerobic digester without rumen fluid inoculation
(ADNRF) with time. Nevertheless, the temperatures
observed in both anaerobic digesters (ADRF and ADNRF)
lie within the operational mesophilic temperature
requirement (i.e., 20°C to 45°C) for biogas production
during anaerobic digestion of organic matter with the
optimum at around 35°C to 37°C (Schnurer and Jarvis,
2010; Azeem et al., 2011). Average process pH (pHRF and
pHNRF) of the feed inside the anaerobic digest er with
rumen fluid inoculation (ADRF) and anaerobic digester
without rumen fluid inoculation (ADNRF) dropped from
7.9 and 8.1 (at day 0) to 6.5 and 6.8 (at day 42 and day 56)
and then increased again to 7.4 and 7.3 at day 105
respectively (Figure 1b). These pH ranges that were
observed in both anaerobic digesters (ADRF and ADNRF)
lie within the operational pH requirement (i.e., 6.0 to 8.5)
for biogas production during anaerobic digestion of
organic matter with the optimum at around 7.0 (Huber et
al., 1982; Yang and Okos, 1987; Ward et al., 2008; Lee et
al., 2009b; Kim et al., 2003; Liu et al., 2008).
In the anaerobic digester with rumen fluid inoculation
(ADRF), the concentration of volatile fatty acid (VFARF)
increased from 1,080.00 mg/L (at day 0), peaked at
16 American Journal of Microbiological Research

4,800.33 mg/L (at day 42) and then dropped to 1,630.53
mg/L at day 105. Likewise, in the anaerobic digester
without rumen fluid inoculation (ADNRF), the
concentration of volatile fatty acid (VFANRF) increased
from 729.34 mg/L (at day 0), peaked at 4,632.04 mg/L (at
day 56) and then dropped to 2,694.70 mg/L at day 105
(Figure 2). This result correlates with findinds of Claudia
et al., (2009) who also reported significant increase (and
decrease) in the concentration of volatile fatty acid during
anaerobic digestion of municipal soild waste (MSW).

Figure 1. (a) Ambient (ATM) and process temperature dynamics (PTMRF and PTMNRF) and (b) Process pH dynamics (pHRF and pHNRF) inside the
anaerobic digester with and without rumen fluid inoculation (ADRF and ADNRF)

Figure 2. Volatile fatty acid dynamics (VFARF and VFANRF) inside the anaerobic digester with and without fluid inoculation (ADRF and ADNRF)

inside the anaerobic digester with rumen fluid inoculation

3.2. Relationship between Process (ADRF) and a moderate positive relationship (r = 0.54)
Temperature and Microbial Population with between ambient temperature(ATM) and the process
Respect to Time temperature (PTMNRF) inside the anaerobic digester
without rumen fluid inoculation (ADNRF) with time
Generally, the process temperature (PTMRF and
(Figure 3). This was predicted because the temperature of
PTMNRF) inside both anaerobic digesters(ADRF and ADNRF)
the environment where any biodigester is situated may
increased with time (Figure 1a). This underscored the
affect the temperature inside the biodigester to some
strong positive relationship (r = 0.79 and r = 0.99)
degree with time (Svahn, 2006; Geradi, 2003; Schnurer
between the process temperatures (PTMRF and PTMNRF)
and Jarvis, 2010; Azeem et al., 2011). Generally, there
and the retention time (Figure 3). There was a strong
were positive relationships(with varying degrees) between
positive relationship (r = 0.76) between ambient
the process temperatures (PTMRF and PTMNRF) and the
temperature(ATM) and the process temperature(PTMRF)
 American Journal of Microbiological Research 17

populations of selected indicator microbial groups inside
the anaerobic digester with rumen fluid inoculation (ADRF)
and the anaerobic digester without rumen fluid inoculation
(ADNRF) with time (Figure 3). This may be further
underscored by the near sigmoid pattern exhibited by the
process temperatures (PTMRF and PTMNRF) with time
(Ogbonna et al., 2014). This was also predicted because
anaerobic degradation of organic matter by microbes leads
to the production of little amount of heat energy which
may accumulate with time and thus increase the
temperature of the anaerobic digester environment
(Schnurer and Jarvis, 2010). Our result also suggested that
the process temperatures (PTMRF and PTMNRF) inside
both anaerobic digesters (ADRF and ADNRF) were likely to
be more favourable to the other microbial populations than
the population of the methanogens (Geradi, 2003; Azeem
et al., 2011). This is because the positive relationship
between the process temperature (PTMRF and PTMNRF)
and the population of the methanogens (AOMRF and
AOMNRF) inside the anaerobic digesters (ADRF and ADNRF)
was the weakest (Figure 3).
There was a moderate positive relationship(r = 0.67 and
r = 0.60) between the process temperature (PTMRF and
PTMNRF) and the concentration of volatile fatty acids
(VFARF and VFANRF) inside the anaerobic digester with
and without rumen fluid inoculation (ADRF and ADNRF)
with respect to time (Figure 3). An increase in the
concentration of volatile fatty acids to some degree have
been associated with a decrease in process pH (see Figure
4) especially when alkalinity of the AD process was
significantly consumed by accumulation of these acids
(Schnurer and Jarvis, 2010; Claudia et al., 2009; Azeem et
al., 2011). Figure 3 underscores this phenomenon because
it showed that there was a strong negative relationship (r =
0.80 and r = 0.79) between the process temperature
(PTMRF and PTMNRF) and the process pH (pHRF and
pHNRF) inside the anaerobic digester with and without
rumen fluid inoculation (ADRF and ADNRF) with time. In
other words, as process temperature inside the anaerobic
digester with and without rumen fluid inoculation (ADRF
and ADNRF) increased to some degree with time, the
microbial populations and consequently degree of
digestion of the substrate also increased and “vice versa”
(Schnurer and Jarvis, 2010; Geradi, 2003; Azeem et al.,
2011). This may have led to a corresponding rise in the
concentration of volatile fatty acids (VFARF and VFANRF)
to some degree and a consequent fall in the process pH
(see Figure 3 and Figure 4) inside both anaerobic digesters
(ADRF and ADNRF) with time and “vice versa” (Schnurer
and Jarvis, 2010; Kim et al., 2003).
Chi-square (goodness of fit) analysis suggested that
there were no significant (p > 0.05) highs or lows in the
process temperature inside the anaerobic digester with and
without rumen fluid inoculation (ADRF and ADNRF) with
time. However, one-way ANOVA suggested that there
was a significant difference between the temperature
dynamics (PTMRF) inside the anaerobic digester with
rumen fluid inoculation (ADRF) and the temperature
dynamics (PTMNRF) inside the anaerobic digester without
rumen fluid inoculation (ADNRF) with time. The process
temperature (PTMRF) of the ADRF system was always
higher than the process temperature (PTMNRF) of the
ADNRF system during the anaerobic digestion process
(Figure 1a). This may be attributed to the influence of
rumen fluid inoculation inside the ADRF system. Rumen
fluid contains various microbial groups that are
specialized in anaerobic digestion of cellulosic substrates
for biogas production and was added for the purpose of
increasing the microbial populations inside the ADRF
system (Aurora, 1983; Allison and Leek, 1993; Budiyono
et al., 2009). As discussed earlier, there were positive
correlations between the process temperature and the
microbial populations inside ADRF and ADNRF systems
with time (Figure 3). Therefore, the anaerobic digester
(ADRF) with higher microbial populations should exhibit
higher (or more efficient) degree of digestion of the
substrate. This should lead to the production of higher
amount of heat energy and thus, a higher process
temperature with time (Schnurer and Jarvis, 2010; Levén
et al., 2007).

Figure 3. Relationship between process temperature (PTMRF and PTMNRF) and ambient temperature (ATM), process pH, volatile fatty acid (VFA),
cellulolytic bacteria (ACB), lactose fermenting bacteria (LFB), glucose fermenting bacteria (GFB), propionate oxidizing bacteria (POB), ethanol
oxidizing bacteria (EOB), acetate oxidizing methanogens (AOM), obligate anaerobic bacteria (OAB) and total facultative bacteria (FAAB) inside the
anaerobic digester with and without rumen fluid inoculation (ADRF and ADNRF) with respect to time
18 American Journal of Microbiological Research
3.3. Relationship between Process pH and
Microbial Population with Respect to Time
The addition of rumen fluid may have affected the
dynamics of the process pHRF inside the anaerobic digester
with rumen fluid inoculation (ADRF) because one-way
ANOVA showed a significant difference (P < 0.05)
between the dynamics of the process pHRF inside the
anaerobic digester with rumen fluid inoculation (ADRF)
and the process pHNRF inside the anaerobic digester
without rumen fluid inoculation (ADNRF).The pHRF inside
the ADRF system dropped at a faster rate than the pHNRF
inside the ADNRF system with time (Figure 1b). This may
have been due to the higher microbial populations (and
consequently higher degree of digestion of the substrate)
inside the digester with rumen fluid (ADRF) than the
digester without rumen fluid (ADNRF) (Ogbonna et al.,
2014; Azeem et al., 2014). In the anaerobic digester with
rumen fluid inoculum (ADRF), the population of lactose
and glucose fermenting (acidogenic) bacteria (LFBRF and
GFBRF) and cellulolytic bacteria (ACBRF)groups were
strongly negatively associated with the process pHRF than
the populations of ethanol and propionate oxidizing
(acetogenic) bacteria (EOBRF and POBRF) and acetate
oxidizing methanogens (AOMRF), respectively (Figure 4).
In terms of O2-sensitivity, the population of total
facultative bacteria (FAABRF) were more strongly
negatively associated with the process pHRF than the
population of obligate anaerobic bacteria (OABRF) with
time (Figure 4). However, in the anaerobic digester
without rumen fluid inoculum (ADNRF), apart from the
population of lactose and glucose fermenting (acidogenic)
bacteria (LFBNRF and GFBNRF) and cellulolytic bacteria
(ACBNRF), the population of ethanol and propionate
oxidizing (acetogenic) bacteria (EOBNRF and POBNRF)
were also strongly negatively associated with the process
pHNRF with time. In terms of O2-sensitivity, the population
of total facultative bacteria (FAABNRF) and obligate
anaerobic bacteria (OABNRF) were strongly negatively
associated with the process pHNRF with respect to time
(Figure 4).
However, Figure 4 showed that the negative
relationship between the process pH and the population of
acidogenicbacteria (LFB and GFB) was the strongest in
both anaerobic digesters (ADRF and ADNRF) with time.
This suggested (to a higher degree) that the
acidogenicbacteria groups may have been responsible for
the drop in process pH (pHRF and pHNRF) inside the
anaerobic digester with rumen fluid inoculum (ADRF) and
the anaerobic digester without rumen fluid inoculum
(ADNRF) with time (Ogbonna et al., 2014; Schnurer and
Jarvis, 2010; Kim et al., 2003; Ike et al., 2010). This is
underscored by the fact that the period when the
population of acidogenic bacteria (LFBRF and LFBNRF and
GFBRF and GFBNRF) peaked as reported in Ogbonna et al.,
(2014), correlated with the same period when volatile fatty
acid (VFARF and VFANRF) production peaked inside the
anaerobic digesters (ADRF and ADNRF) as shown in Figure
2. This also correlated with the same period when the
process pH (pHRF and pHNRF) inside the anaerobic
digesters (ADRF and ADNRF) dipped respectively (Figure
1b). This period may in fact be the peak period of the
acidogenic phase of the anaerobic digestion process inside
the ADRF and the ADNRF systems respectively (Ogbonna et
al., 2014; Schnurer and Jarvis, 2010; Ike et al., 2010).
During the AD process, it may have been possible that
some of the bacteria species belonging to the population
of cellulolytic bacteria (ACBRF and ACBNRF) inside the
anaerobic digester with and without rumen fluid
inoculation (ADRF and ADNRF) partook in sugar
fermenting acidogenesis (Schnurer and Jarvis, 2010). This
may be a possible reason why they were also strongly
negatively associated with the process pH in both AD
systems with respect to time (Figure 4). Moreover, the
population of acetogenic bacteria (EOB and POB) convert
alcohols and fatty acid such as propionic acid to acetic
acid (Sousa et al., 2007; Drake et al., 2008). These may
also make the process pH of both AD systems drop
especially if the acetic acid accumulates with time (Ike et
al., 2010; Kim et al., 2006). This could also be a possible
reason why the population of acetogenic bacteria (EOBNRF
and POBNRF) were strongly negatively correlated with the
process pH (pHNRF) inside the digester without rumen
fluid inoculum (ADNRF) with time (Figure 4).
In terms of O2-sensitivity, the population of total
facultative bacteria (FAABRF and FAABNRF) was strongly
negatively associated with the process pH (pHRF and
pHNRF) inside the anaerobic digester with and without
rumen fluid inoculation (ADRF and ADNRF) with time
(Figure 4). Also, because they may be facultative in nature,
some of them may be able to thrive in obligate anaerobic
condition to some degree (Agdag and Sponza, 2004;
Schnurer and Jarvis, 2010). This may be a possible reason
why the population of obligate anaerobic bacteria
(OABNRF) was strongly negatively associated with the
process pHNRF inside the anaerobic digester without rumen
fluid inoculation (ADNRF) with time (Figure 4). There was
a weak negative relationship between the process pH
(pHRF and pHNRF) and the population of acetotrophic
methanogens (AOMRF and AOMNRF) inside the anaerobic
digester with and without rumen fluid inoculation (ADRF
and ADNRF) with time (Figure 4). This was predicted
because when acetic acid (which is the main substrate for
acetotrophic methanogenesis) was being produced, it was
expected that the methanogens present at the time would
metabolize the acid and convert it to biogas (Zinder, 1993;
Liu and Whitman, 2008; Garcia et al., 2000; Schnurer and
Jarvis, 2010). As the acetic acid accumulated, more of it
would have been available to the methanogens as substrate
and as they utilized the substrate for conversion into
biogas, their population should have increased to some
degree with time (Claudia et al., 2009; Labat and Garcia,
1986; Schnurer and Jarvis, 2010). This may have been a
possible reason why there was a weak negative
relationship between the process pH (pHRF and pHNRF)
and the population of acetate oxidizing methanogens
(AOMRF and AOMNRF) in both AD systems (ADRF and
ADNRF) with respect to time (Figure 4).
Moreover, it was observed in Figure 1b that the process
pH (pHRF and pHNRF) inside the anaerobic digester with
and without rumen fluid inoculum (ADRF and ADNRF)
decreased from day zero, dipped at day 42 and day 56 and
then increased again until day 105 respectively. If it was
possible that accumulation of volatile fatty acid (VFA),
especially acetic acid led to a drop in the process pH with
time, it may also have been possible that its removal by
the methanogens (via conversion to biogas) caused a
corresponding rise in the process pH. Although,
 American Journal of Microbiological Research
metabolites such as CO2 and NH3 may have also
contributed to the rise in process pH with time (Schnurer
and Jarvis, 2010; Zinder, 1993; Liu and Whitman, 2008;
Garcia et al., 2000).
Chi-square analysis suggested that there were no
significant highs or lows in the process pH (pHRF and
pHNRF) inside the anaerobic digester with and without
rumen fluid (ADRF and ADNRF) with time respectively.
This has been predicted for most substrates (or feedstock)
with low or moderate degradability (Schnurer and Jarvis,
2010). Matured guinea grass (used as substrate in this
study) is not readily biodegradable due to its
Figure 4. Relationship between process pH (pHRF and pHNRF) and volatile fatty acid (VFA), cellulolytic bacteria (ACB), lactose fermenting bacteria
(LFB), glucose fermenting bacteria (GFB), propionate oxidizing bacteria (POB), ethanol oxidizing bacteria (EOB), acetate oxidizing methanogens
(AOM), obligate anaerobic bacteria (OAB) and total facultative bacteria (FAAB) inside the anaerobic digester with and without rumen fluid inoculation
(ADRF and ADNRF) with respect to time
3.4. Relationship between Microbial
Populations with Respect to Time
The interaction (or relationship) among microbial
populations in anaerobic digestion(AD) process may
either be positive, neutral or negative with time (Schnurer
and Jarvis, 2010). Correlation analysis (in Figure 5 to
Figure 7) showed that there were positive relationships
between the microbial populations selected to indicate
various metabolic and O2-sensitive stages of the AD
process inside the anaerobic digester with and without
rumen fluid inoculation (ADRF and ADNRF)with time
(Ogbonna et al., 2014). However, the degree (or strength)
of these relationships among the microbial populations
may not have been equal. For example, the population of
cellulolytic bacteria (ACBRF and ACBNRF) was very
strongly and positively associated with the population of
lactose and glucose fermenting acidogens (LFBRF and
GFBRF and LFBNRF and GFBNRF) followed by the
population of propionate and ethanol oxidizing acetogens
(POBRF and EOBRF and POBNRF and EOBNRF) and the
population of acetateoxidizing methanogens (AOMRF and
AOMNRF) to a lesser degree inside the anaerobic digester
with and without rumen fluid inoculation (ADRF and
ADNRF) respectively with time (Figure 5a). This may be
because of the fact that the populations of acetogens and
methanogens are not usually directly associated
metabolically with the population of polymer hydrolysing
19
lignocellulose content. Because of this, the rate of organic
acid production during anaerobic biodegradation of the
grass may have been slow or moderate (Susan, 1995).
Therefore, the effect on the process pH may not have been
as significant as it would if the substrate was easily
degradable (Schnurer and Jarvis, 2010). An easily
degradable substrate degrades faster and may rapidly
produce higher concentration of VFAs which may lead to
a significant drop in the process pH and thus, negatively
affecting the anaerobic digestion process by slowing or
inhibiting methanogenesis (Ogbonna et al., 2014; Kim et
al., 2008; Azeem et al., 2011; Schnurer and Jarvis, 2010).
(or cellulolytic) bacteria as does the population of
acidogens along theanaerobic digestion food chain
(Schnurer and Jarvis, 2010; Yassar, 2011; Chanakya and
Sreesha, 2012). However, this positive correlation was
still strong and moderate for the acetogens and
methanogens, respectively (Figure 5a). Generally during
anaerobic digestion process, hydrolysis must first occur to
some degree before acidogenesis, acetogenesis and
methanogenesisin this order (Ljupka, 2010; Schnurer and
Jarvis, 2010; Yassar, 2011; Chanakya and Sreesha, 2012).
Moreover, it is possible that some (or most) of the
microbial populations responsible for hydrolysis may also
be the same ones taking part in acidogenesis inside the
anaerobic digesters (ADRF and ADNRF) with time
(Schnurer and Jarvis, 2010). This may also be a reason
why the populations of cellulolytic bacteria (ACBRF and
ACBNRF) and acidogenic bacteria (LFBRF and GFBRF and
LFBNRF and GFBNRF) were very strongly and positively
associated with one another in both AD systems (ADRF
and ADNRF) with time. In terms of O2-sensitivity, the
population of cellulolytic bacteria (ACBRF and ACBNRF)
was very strongly positively related to (or associated with)
the population of total facultative bacteria (FAABRF and
FAABNRF) than the population of obligate anaerobic
bacteria (OABRF and OABNRF) which was also strong,
inside both AD systems (ADRF and ADNRF) with respect to
time (Figure 5a). This was predicted because cellulose
hydrolysis can occur in either aerobic, microaerobic or
anaerobic conditions (Schnurer and Jarvis, 2010).
20 American Journal of Microbiological Research
Furthermore, it is highly likely that oxygen was present
inside the biodigesters (ADRF and ADNRF) because it may
not have been possible to evacuate all the air (oxygen)
from the 500L-capacity anaerobic digestion tanks before
loading.
The population of propionate oxidizing acetogens
(POBRF and POBNRF) was very strongly and positively
related to (or associated with) the populations of ethanol
oxidizing acetogens (EOBRF and EOBNRF) and acetate
oxidizing methanogens (AOMRF and AOMNRF) with time
inside both AD systems (ADRF and ADNRF), respectively
(Figure 6b). The population of these acetogens (POBRF
and EOBRF and POBNRF and EOBNRF) may have increased
or decreased together because they may have been
positively influenced by the population of the acidogens
(LFBRF and GFBRF and LFBNRF and GFBNRF) during
acidogenesis as indicated in Figure 5b and Figure 6a
which showed that there were very strong positive
relationships between the acetogens and the acidogens
inside the anaerobic digester with rumen fluid inoculum
(ADRF) and the anaerobic digester without rumen fluid
inoculum (ADNRF) with time(Labat and Garcia, 1986;
Claudia et al., 2009; Schnurer and Jarvis, 2010; Yassar,
2011). The population of propionate oxidizing acetogens
(POBRF and POBNRF) may have correlated strongly and
positively with the population of acetate oxidizing
methanogens (AOMRF and AOMNRF) inside the anaerobic
digesters (ADRF and ADNRF) as shown in Figure 6b
because propionate oxidation usually produces CO2, H2
and acetic acid which is the substrate for acetotrophic
methanogenesis (Schnurer and Jarvis, 2010; Yassar, 2011;
Chanakya and Sreesha, 2012).
In terms of O2-sensitivity, the population of propionate
oxidizing acetogens (POBRF and POBNRF) was very
strongly and positively related to (or associated with) the
population of obligate anaerobic bacteria (OABRF and
OABNRF) than with the population of total facultative
bacteria (FAABRF and FAABNRF) inside the anaerobic
digester with and without rumen fluid inoculation (ADRF
and ADNRF) respectively with time (Figure 6b). This may
be because acetogenesis (via propionate oxidation) may
only proceed under obligate anaerobic condition (Schnurer
and Jarvis, 2010). Furthermore, if propionate oxidation
(for acetogenesis) may only occur under anaerobic
conditions, then the population of facultative bacteria
(FAABRF and FAABNRF) which are capable of consuming
any oxygen that may have been present inside the
biodigesters (ADRF and ADNRF) at the beginning of the
AD process must increase to some degree with time
(Claudia et al., 2009; Labat and Garcia, 1986; Schnurer
and Jarvis, 2010). Therefore, as the concentration of the
oxygen inside the digester reduced with time (because of
an increase in the population of total facultative bacteria),
the population of obligate anaerobic bacteria like the
propionate oxidizing acetogens (POB) may also begin to
increase and become established with time (Schnurer and
Jarvis, 2010). This may have been one of the reasons why
the population of propionate oxidizing acetogens (POBRF
and POBNRF) increased together with the population of
obligate anaerobic bacteria (OABRF and OABNRF) and total
facultative bacteria(FAABRF and FAABNRF) inside the
anaerobic digesters (ADRF and ADNRF) to some degree
with time (Figure 6b).
Likewise, the population of acetate oxidizing
methanogens (AOMRF and AOMNRF) was very strongly
and positively related to (or associated with) the
population of obligate anaerobic (OABRF and ADNRF) than
with the population of facultative aerobes and anaerobes
(FAABRF and FAABNRF) inside the anaerobic digesters
with and without rumen fluid inoculation (ADRF and
ADNRF) respectively with time (Figure 7b).
Methanogenesis can only occur in an obligate anaerobic
environment (Schnurer and Jarvis, 2010). This may
explain (to some degree) why their population was
strongly positively related to the population of obligate
anaerobic bacteria (Figure 7b). Their population may have
also increased together with the population of total
facultative bacteria because they may have needed the
population of the facultative groups to create the anaerobic
environment that enabled them increased with respect to
time (Ogbonna et al., 2014; Schnurer and Jarvis, 2010).
Facultative bacteria are capable of consuming any oxygen
which may have been present inside the anaerobic
digesters (ADRF and ADNRF) at the start of the AD process
thus, eliminating all the oxygen inside the anaerobic
digesters with time (Schnurer and Jarvis, 2010). This
would have promoted the growth of obligate (or strict)
anaerobic bacteria population such as the methanogens to
some degree with time (Schnurer and Jarvis, 2010).
This fact may be further underscored by Figure 7c
which showed that as the population of total facultative
bacteria (FAABRF and FAABNRF) increased, the
population of obligate anaerobic bacteria (OABRF and
OABNRF) also increased to some degree inside the
anaerobic digesters (ADRF and ADNRF) with time and
“vice versa”. In fact some authors like Labat and Garcia
(1986) and Schnurer and Jarvis (2010) have suggested that
an AD process may not be seriously negatively affected if
there was an accidental introduction of air (or oxygen)
inside the bio-digesters because the facultative bacteria
groups may quickly consume any oxygen that was
introduced before it significantly harms the population of
obligate (or strict) anaerobes. Of course there may have
been other factors (such as the process temperature and
pH) which may have also affected the populations of these
microbial groups in such a way that would have made
them to increase or decrease together to some degree with
time (Figure 5 to Figure 7).
It is very important to note that an increase in the
population of methanogens may also positively affect the
populations of hydrolytic bacteria and acid (and hydrogen)
forming (i.e., acidogenic and acetogenic) bacteria groups
to some degree with time (Figure 5 to Figure 7). This is
because when the respective populations of hydrolytic,
acidogenic and acetogenic bacteria increased with time,
hydrolysis, acidogenesis and acetogenesis also increased
to some degree (Ogbonna et al., 2014). Some of the
products of acidogenesis and acetogenesis include
alcohols, carbon dioxide, hydrogen, organic acids (or
acetic acids in the case of acetogenesis), etc. Therefore, as
acidogenesis and acetogenesis increase with respect to
time, the concentrations of these by-products may also
increase with time. The accumulation of hydrogen and
organic acids (and/or acetic acid) to some degree may be
inhibitive to the respective populations of the hydrolytic,
acidogenic, acetogenic and even the methanogenic groups
with time (Hickey et al., 1987; Labib et al., 1992). Thus,
 American Journal of Microbiological Research 21

sufficient system assimilation capacity for hydrogen and
acetate removal must be maintained to ensure continuous
acid production (Yassar, 2011). The methanogens usually
convert the hydrogen (with carbon dioxide) and acetate to
biogas. As their populations increase with time, this
conversion rate may also increase to some degree and thus,
continuously keeping the concentration of these by-
products below inhibitory levels (Parkin and Owen, 1986;
Yassar, 2011; Schnurer and Jarvis, 2010). This metabolic
relationship between the population of methanogens and
the population of other bacteria groups monitored in this
study may be one of the reasons why their populations
increased together to some degree with time and “vice
versa” (Figure 5 to Figure 7).

Figure 5. (a) Relationship between the population of cellulolytic bacteria (ACBRF& ACBNRF) and the populations of lactose fermenting acidogens
(LFBRF& LFBNRF), glucose fermenting acidogens (GFBRF& GFBNRF), propionate oxidizing acetogens (POBRF& POBNRF), ethanol oxidizing acetogens
(EOBRF& EOBNRF), acetate oxidizing methanogens (AOMRF& AOMNRF), obligate anaerobic bacteria (OABRF& OABNRF) and total facultative bacteria
(FAABRF& FAABNRF) inside the anaerobic digester with and without rumen fluid inoculation (ADRF& ADNRF) respectively with time at ambient
condition. (b) Relationship between the population of lactose fermenting acidogens (LFBRF& LFBNRF) and the populations of glucose fermenting
acidogens (GFBRF& GFBNRF), propionate oxidizing acetogens (POBRF& POBNRF), ethanol oxidizing acetogens (EOBRF& EOBNRF), acetate oxidizing
methanogens (AOMRF& AOMNRF), obligate anaerobic bacteria (OABRF& OABNRF) and total facultative bacteria (FAABRF& FAABNRF) inside the
anaerobic digester with and without rumen fluid inoculation (ADRF& ADNRF) respectively with time at ambient condition

Figure 6. (a) Relationship between the population of glucose fermenting acidogens (GFBRF& GFBNRF) and the populations ofpropionate oxidizing
acetogens (POBRF& POBNRF), ethanol oxidizing acetogens (EOBRF& EOBNRF), acetate oxidizing methanogens (AOMRF& AOMNRF), obligate anaerobic
bacteria (OABRF& OABNRF) and total facultative bacteria (FAABRF& FAABNRF) inside the anaerobic digester with and without rumen fluid inoculation
(ADRF& ADNRF) respectively with time at ambient condition.(b) Relationship between the population of propionate oxidizing acetogens (POBRF&
POBNRF) and the populations of ethanol oxidizing acetogens (EOBRF& EOBNRF), acetate oxidizing methanogens (AOMRF& AOMNRF), obligate
anaerobic bacteria (OABRF& OABNRF) and total facultative bacteria (FAABRF& FAABNRF) inside the anaerobic digester with and without rumen fluid
inoculation (ADRF& ADNRF) respectively with time at ambient condition
22 American Journal of Microbiological Research

Figure 7. (a) Relationship between the population of ethanol oxidizing acetogens (EOBRF& EOBNRF) and the populations of acetate oxidizing
methanogens (AOMRF& AOMNRF), obligate anaerobic bacteria (OABRF& OABNRF) and total facultative bacteria (FAABRF& FAABNRF) inside the
anaerobic digester with and without rumen fluid inoculation (ADRF& ADNRF) with time at ambient condition.(b) Relationship between the population of
acetate oxidizing methanogens (AOMRF& AOMNRF) and the populations of obligate anaerobic bacteria (OABRF& OABNRF) and total facultative bacteria
(FAABRF& FAABNRF) inside the anaerobic digester with and without rumen fluid inoculation (ADRF& ADNRF) respectively with time at ambient
condition.(c) Relationship between the populations of obligate anaerobic bacteria (OABRF& OABNRF) and the populations of total facultative anaerobic
bacteria (FAABRF& FAABNRF) inside the anaerobic digester with and without rumen fluid inoculation (ADRF& ADNRF) respectively with time at
ambient condition

GFBRF: glucose fermenting bacteria in ADRF

4. Conclusion
The biogas generation process is a natural biological
process that requires co-operation among different
microbial populations. This co-operation is underscored
by the positive correlations among the microbial indicator
populations selected and monitored with respect to time
(Figure 5 to Figure 7). There were positive correlations
between the process temperature and the microbial
populations inside the anaerobic digesters (ADRF and
ADNRF) with time (Figure 3). However, there were
negative correlations between the process pH and the
microbial populations inside the anaerobic digesters
(ADRF and ADNRF) with time (Figure 4). These
relationships amongbiotic factors and between biotic and
abiotic factors could indicate bad running of the AD
process. Specific correlations (or relationships) with time
could possibly explain process failure. Therefore, the best
result will only be obtained when a total understanding of
the microbial ecological processes inform and guide
applications of biogas production technology.
Nomenclature
AD: anaerobic digestion
RF: rumen fluid
NRF: no (or without) rumen fluid
ADRF: anaerobic digester with RF inoculation
ADNRF: anaerobic digester without RF inoculation
ACBRF: cellulolytic bacteria inside ADRF
ACBNRF: cellulolytic bacteria inside ADNRF
LFBRF: lactose fermenting bacteria in ADRF
LFBNRF: lactose fermenting bacteria in ADNRF
GFBNRF: glucose fermenting bacteria in ADNRF
POBRF: propionate oxidizing bacteria in ADRF
POBNRF: propionate oxidizing bacteria in ADNRF
EOBRF: ethanol oxidizing bacteria in ADRF
EOBNRF: ethanol oxidizing bacteria in ADNRF
AOMRF: acetate oxidizing methanogens in ADRF
AOMNRF: acetate oxidizing methanogens in ADNRF
FAABRF: facultative aerobic and anaerobic bacteria in
ADRF
FAABNRF: facultative aerobic and anaerobic bacteria in
ADNRF
OABRF: obligate anaerobic bacteria inside ADRF
OABNRF: obligate anaerobic bacteria inside ADNRF
MPN: most probable number
CFU: colony forming unit
ML: millimetre
TS: total solid
PTMRF: process temperature in ADRF
PTMNRF: process temperature in ADNRF
pHRF: process pH inside ADRF
pHNRF: process pH inside ADNRF
VFARF: volatile fatty acid inside ADRF
VFANRF: volatile fatty acid inside ADNRF
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