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

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 (AD_RF) was the experimental set-up, while the anaerobic digestion system without rumen fluid inoculation (AD_NRF) 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).

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 populations that were selected included obligate anaerobic bacteria (OAB) and total facultative anaerobic bacteria (FAAB) respectively.

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 (AD_RF) and the anaerobic digester without rumen fluid inoculation (AD_NRF) 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

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 (AD_RF and AD_NRF) 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 (AD_RF), average process temperature (PTM_RF) 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 (AD_NRF), average process temperature (PTM_NRF) 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 (AD_RF) was slightly more heated than the anaerobic digester without rumen fluid inoculation (AD_NRF) with time. Nevertheless, the temperatures observed in both anaerobic digesters (AD_RF and AD_NRF) 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 (pH_RF and pH_NRF) of the feed inside the anaerobic digest er with rumen fluid inoculation (AD_RF) and anaerobic digester without rumen fluid inoculation (AD_NRF) 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 (AD_RF and AD_NRF) 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 (AD_RF), the concentration of volatile fatty acid (VFA_RF) increased from 1,080.00 mg/L (at day 0), peaked at 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 (AD_NRF), the concentration of volatile fatty acid (VFA_NRF) 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 (PTM_RF and PTM_NRF) and (b) Process pH dynamics (pH_RF and pH_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF and AD_NRF)

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Figure 2. Volatile fatty acid dynamics (VFA_RF and VFA_NRF) inside the anaerobic digester with and without fluid inoculation (AD_RF and AD_NRF)

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Generally, the process temperature (PTM_RF and PTM_NRF) inside both anaerobic digesters(AD_RF and AD_NRF) increased with time (Figure 1a). This underscored the strong positive relationship (r = 0.79 and r = 0.99) between the process temperatures (PTM_RF and PTM_NRF) and the retention time (Figure 3). There was a strong positive relationship (r = 0.76) between ambient temperature(ATM) and the process temperature(PTM_RF) inside the anaerobic digester with rumen fluid inoculation (AD_RF) and a moderate positive relationship (r = 0.54) between ambient temperature(ATM) and the process temperature (PTM_NRF) inside the anaerobic digester without rumen fluid inoculation (AD_NRF) with time (Figure 3). This was predicted because the temperature of the environment where any biodigester is situated may affect the temperature inside the biodigester to some degree with time (Svahn, 2006; Geradi, 2003; Schnurer and Jarvis, 2010; Azeem et al., 2011). Generally, there were positive relationships(with varying degrees) between the process temperatures (PTM_RF and PTM_NRF) and the populations of selected indicator microbial groups inside the anaerobic digester with rumen fluid inoculation (AD_RF) and the anaerobic digester without rumen fluid inoculation (AD_NRF) with time (Figure 3). This may be further underscored by the near sigmoid pattern exhibited by the process temperatures (PTM_RF and PTM_NRF) 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 (PTM_RF and PTM_NRF) inside both anaerobic digesters (AD_RF and AD_NRF) 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 (PTM_RF and PTM_NRF) and the population of the methanogens (AOM_RF and AOM_NRF) inside the anaerobic digesters (AD_RF and AD_NRF) was the weakest (Figure 3).

There was a moderate positive relationship(r = 0.67 and r = 0.60) between the process temperature (PTM_RF and PTM_NRF) and the concentration of volatile fatty acids (VFA_RF and VFA_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF and AD_NRF) 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 (PTM_RF and PTM_NRF) and the process pH (pH_RF and pH_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF and AD_NRF) with time. In other words, as process temperature inside the anaerobic digester with and without rumen fluid inoculation (AD_RF and AD_NRF) 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 (VFA_RF and VFA_NRF) to some degree and a consequent fall in the process pH (see Figure 3 and Figure 4) inside both anaerobic digesters (AD_RF and AD_NRF) 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 (AD_RF and AD_NRF) with time. However, one-way ANOVA suggested that there was a significant difference between the temperature dynamics (PTM_RF) inside the anaerobic digester with rumen fluid inoculation (AD_RF) and the temperature dynamics (PTM_NRF) inside the anaerobic digester without rumen fluid inoculation (AD_NRF) with time. The process temperature (PTM_RF) of the AD_RF system was always higher than the process temperature (PTM_NRF) of the AD_NRF system during the anaerobic digestion process (Figure 1a). This may be attributed to the influence of rumen fluid inoculation inside the AD_RF 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 AD_RF 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 AD_RF and AD_NRF systems with time (Figure 3). Therefore, the anaerobic digester (AD_RF) 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 (PTM_RF and PTM_NRF) 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 (AD_RF and AD_NRF) with respect to time

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The addition of rumen fluid may have affected the dynamics of the process pH_RF inside the anaerobic digester with rumen fluid inoculation (AD_RF) because one-way ANOVA showed a significant difference (P < 0.05) between the dynamics of the process pH_RF inside the anaerobic digester with rumen fluid inoculation (AD_RF) and the process pH_NRF inside the anaerobic digester without rumen fluid inoculation (AD_NRF).The pH_RF inside the AD_RF system dropped at a faster rate than the pH_NRF inside the AD_NRF 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 (AD_RF) than the digester without rumen fluid (AD_NRF) (Ogbonna et al., 2014; Azeem et al., 2014). In the anaerobic digester with rumen fluid inoculum (AD_RF), the population of lactose and glucose fermenting (acidogenic) bacteria (LFB_RF and GFB_RF) and cellulolytic bacteria (ACB_RF)groups were strongly negatively associated with the process pH_RF than the populations of ethanol and propionate oxidizing (acetogenic) bacteria (EOB_RF and POB_RF) and acetate oxidizing methanogens (AOM_RF), respectively (Figure 4). In terms of O₂-sensitivity, the population of total facultative bacteria (FAAB_RF) were more strongly negatively associated with the process pH_RF than the population of obligate anaerobic bacteria (OAB_RF) with time (Figure 4). However, in the anaerobic digester without rumen fluid inoculum (AD_NRF), apart from the population of lactose and glucose fermenting (acidogenic) bacteria (LFB_NRF and GFB_NRF) and cellulolytic bacteria (ACB_NRF), the population of ethanol and propionate oxidizing (acetogenic) bacteria (EOB_NRF and POB_NRF) were also strongly negatively associated with the process pH_NRF with time. In terms of O₂-sensitivity, the population of total facultative bacteria (FAAB_NRF) and obligate anaerobic bacteria (OAB_NRF) were strongly negatively associated with the process pH_NRF 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 (AD_RF and AD_NRF) with time. This suggested (to a higher degree) that the acidogenicbacteria groups may have been responsible for the drop in process pH (pH_RF and pH_NRF) inside the anaerobic digester with rumen fluid inoculum (AD_RF) and the anaerobic digester without rumen fluid inoculum (AD_NRF) 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 (LFB_RF and LFB_NRF and GFB_RF and GFB_NRF) peaked as reported in Ogbonna et al., (2014), correlated with the same period when volatile fatty acid (VFA_RF and VFA_NRF) production peaked inside the anaerobic digesters (AD_RF and AD_NRF) as shown in Figure 2. This also correlated with the same period when the process pH (pH_RF and pH_NRF) inside the anaerobic digesters (AD_RF and AD_NRF) dipped respectively (Figure 1b). This period may in fact be the peak period of the acidogenic phase of the anaerobic digestion process inside the AD_RF and the AD_NRF 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 (ACB_RF and ACB_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF and AD_NRF) 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 (EOB_NRF and POB_NRF) were strongly negatively correlated with the process pH (pH_NRF) inside the digester without rumen fluid inoculum (AD_NRF) with time (Figure 4).

In terms of O₂-sensitivity, the population of total facultative bacteria (FAAB_RF and FAAB_NRF) was strongly negatively associated with the process pH (pH_RF and pH_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF and AD_NRF) 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 (OAB_NRF) was strongly negatively associated with the process pH_NRF inside the anaerobic digester without rumen fluid inoculation (AD_NRF) with time (Figure 4). There was a weak negative relationship between the process pH (pH_RF and pH_NRF) and the population of acetotrophic methanogens (AOM_RF and AOM_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF and AD_NRF) 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 (pH_RF and pH_NRF) and the population of acetate oxidizing methanogens (AOM_RF and AOM_NRF) in both AD systems (AD_RF and AD_NRF) with respect to time (Figure 4).

Moreover, it was observed in Figure 1b that the process pH (pH_RF and pH_NRF) inside the anaerobic digester with and without rumen fluid inoculum (AD_RF and AD_NRF) 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, 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 (pH_RF and pH_NRF) inside the anaerobic digester with and without rumen fluid (AD_RF and AD_NRF) 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 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).

Figure 4. Relationship between process pH (pH_RF and pH_NRF) 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 (AD_RF and AD_NRF) with respect to time

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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 (AD_RF and AD_NRF)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 (ACB_RF and ACB_NRF) was very strongly and positively associated with the population of lactose and glucose fermenting acidogens (LFB_RF and GFB_RF and LFB_NRF and GFB_NRF) followed by the population of propionate and ethanol oxidizing acetogens (POB_RF and EOB_RF and POB_NRF and EOB_NRF) and the population of acetateoxidizing methanogens (AOM_RF and AOM_NRF) to a lesser degree inside the anaerobic digester with and without rumen fluid inoculation (AD_RF and AD_NRF) 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 (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 (AD_RF and AD_NRF) with time (Schnurer and Jarvis, 2010). This may also be a reason why the populations of cellulolytic bacteria (ACB_RF and ACB_NRF) and acidogenic bacteria (LFB_RF and GFB_RF and LFB_NRF and GFB_NRF) were very strongly and positively associated with one another in both AD systems (AD_RF and AD_NRF) with time. In terms of O₂-sensitivity, the population of cellulolytic bacteria (ACB_RF and ACB_NRF) was very strongly positively related to (or associated with) the population of total facultative bacteria (FAAB_RF and FAAB_NRF) than the population of obligate anaerobic bacteria (OAB_RF and OAB_NRF) which was also strong, inside both AD systems (AD_RF and AD_NRF) 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). Furthermore, it is highly likely that oxygen was present inside the biodigesters (AD_RF and AD_NRF) 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 (POB_RF and POB_NRF) was very strongly and positively related to (or associated with) the populations of ethanol oxidizing acetogens (EOB_RF and EOB_NRF) and acetate oxidizing methanogens (AOM_RF and AOM_NRF) with time inside both AD systems (AD_RF and AD_NRF), respectively (Figure 6b). The population of these acetogens (POB_RF and EOB_RF and POB_NRF and EOB_NRF) may have increased or decreased together because they may have been positively influenced by the population of the acidogens (LFB_RF and GFB_RF and LFB_NRF and GFB_NRF) 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 (AD_RF) and the anaerobic digester without rumen fluid inoculum (AD_NRF) with time(Labat and Garcia, 1986; Claudia et al., 2009; Schnurer and Jarvis, 2010; Yassar, 2011). The population of propionate oxidizing acetogens (POB_RF and POB_NRF) may have correlated strongly and positively with the population of acetate oxidizing methanogens (AOM_RF and AOM_NRF) inside the anaerobic digesters (AD_RF and AD_NRF) 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 (POB_RF and POB_NRF) was very strongly and positively related to (or associated with) the population of obligate anaerobic bacteria (OAB_RF and OAB_NRF) than with the population of total facultative bacteria (FAAB_RF and FAAB_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF and AD_NRF) 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 (FAAB_RF and FAAB_NRF) which are capable of consuming any oxygen that may have been present inside the biodigesters (AD_RF and AD_NRF) 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 (POB_RF and POB_NRF) increased together with the population of obligate anaerobic bacteria (OAB_RF and OAB_NRF) and total facultative bacteria(FAAB_RF and FAAB_NRF) inside the anaerobic digesters (AD_RF and AD_NRF) to some degree with time (Figure 6b).

Likewise, the population of acetate oxidizing methanogens (AOM_RF and AOM_NRF) was very strongly and positively related to (or associated with) the population of obligate anaerobic (OAB_RF and AD_NRF) than with the population of facultative aerobes and anaerobes (FAAB_RF and FAAB_NRF) inside the anaerobic digesters with and without rumen fluid inoculation (AD_RF and AD_NRF) 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 (AD_RF and AD_NRF) 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 (FAAB_RF and FAAB_NRF) increased, the population of obligate anaerobic bacteria (OAB_RF and OAB_NRF) also increased to some degree inside the anaerobic digesters (AD_RF and AD_NRF) 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, 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 (ACB_RF& ACB_NRF) and the populations of lactose fermenting acidogens (LFB_RF& LFB_NRF), glucose fermenting acidogens (GFB_RF& GFB_NRF), propionate oxidizing acetogens (POB_RF& POB_NRF), ethanol oxidizing acetogens (EOB_RF& EOB_NRF), acetate oxidizing methanogens (AOM_RF& AOM_NRF), obligate anaerobic bacteria (OAB_RF& OAB_NRF) and total facultative bacteria (FAAB_RF& FAAB_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF& AD_NRF) respectively with time at ambient condition. (b) Relationship between the population of lactose fermenting acidogens (LFB_RF& LFB_NRF) and the populations of glucose fermenting acidogens (GFB_RF& GFB_NRF), propionate oxidizing acetogens (POB_RF& POB_NRF), ethanol oxidizing acetogens (EOB_RF& EOB_NRF), acetate oxidizing methanogens (AOM_RF& AOM_NRF), obligate anaerobic bacteria (OAB_RF& OAB_NRF) and total facultative bacteria (FAAB_RF& FAAB_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF& AD_NRF) respectively with time at ambient condition

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Figure 6. (a) Relationship between the population of glucose fermenting acidogens (GFB_RF& GFB_NRF) and the populations ofpropionate oxidizing acetogens (POB_RF& POB_NRF), ethanol oxidizing acetogens (EOB_RF& EOB_NRF), acetate oxidizing methanogens (AOM_RF& AOM_NRF), obligate anaerobic bacteria (OAB_RF& OAB_NRF) and total facultative bacteria (FAAB_RF& FAAB_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF& AD_NRF) respectively with time at ambient condition.(b) Relationship between the population of propionate oxidizing acetogens (POB_RF& POB_NRF) and the populations of ethanol oxidizing acetogens (EOB_RF& EOB_NRF), acetate oxidizing methanogens (AOM_RF& AOM_NRF), obligate anaerobic bacteria (OAB_RF& OAB_NRF) and total facultative bacteria (FAAB_RF& FAAB_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF& AD_NRF) respectively with time at ambient condition

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Figure 7. (a) Relationship between the population of ethanol oxidizing acetogens (EOB_RF& EOB_NRF) and the populations of acetate oxidizing methanogens (AOM_RF& AOM_NRF), obligate anaerobic bacteria (OAB_RF& OAB_NRF) and total facultative bacteria (FAAB_RF& FAAB_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF& AD_NRF) with time at ambient condition.(b) Relationship between the population of acetate oxidizing methanogens (AOM_RF& AOM_NRF) and the populations of obligate anaerobic bacteria (OAB_RF& OAB_NRF) and total facultative bacteria (FAAB_RF& FAAB_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF& AD_NRF) respectively with time at ambient condition.(c) Relationship between the populations of obligate anaerobic bacteria (OAB_RF& OAB_NRF) and the populations of total facultative anaerobic bacteria (FAAB_RF& FAAB_NRF) inside the anaerobic digester with and without rumen fluid inoculation (AD_RF& AD_NRF) respectively with time at ambient condition

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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 (AD_RF and AD_NRF) with time (Figure 3). However, there were negative correlations between the process pH and the microbial populations inside the anaerobic digesters (AD_RF and AD_NRF) 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

AD_RF: anaerobic digester with RF inoculation

AD_NRF: anaerobic digester without RF inoculation

ACB_RF: cellulolytic bacteria inside AD_RF

ACB_NRF: cellulolytic bacteria inside AD_NRF

LFB_RF: lactose fermenting bacteria in AD_RF

LFB_NRF: lactose fermenting bacteria in AD_NRF

GFB_RF: glucose fermenting bacteria in AD_RF

GFB_NRF: glucose fermenting bacteria in AD_NRF

POB_RF: propionate oxidizing bacteria in AD_RF

POB_NRF: propionate oxidizing bacteria in AD_NRF

EOB_RF: ethanol oxidizing bacteria in AD_RF

EOB_NRF: ethanol oxidizing bacteria in AD_NRF

AOM_RF: acetate oxidizing methanogens in AD_RF

AOM_NRF: acetate oxidizing methanogens in AD_NRF

FAAB_RF: facultative aerobic and anaerobic bacteria in AD_RF

FAAB_NRF: facultative aerobic and anaerobic bacteria in AD_NRF

OAB_RF: obligate anaerobic bacteria inside AD_RF

OAB_NRF: obligate anaerobic bacteria inside AD_NRF

MPN: most probable number

CFU: colony forming unit

ML: millimetre

TS: total solid

PTM_RF: process temperature in AD_RF

PTM_NRF: process temperature in AD_NRF

pH_RF: process pH inside AD_RF

pH_NRF: process pH inside AD_NRF

VFA_RF: volatile fatty acid inside AD_RF

VFA_NRF: volatile fatty acid inside AD_NRF

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