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Microbial Population Dynamics during Anaerobic Digestion of Guinea Grass (Panicum maximum)

Ogbonna C. B.*, Ibiene A. A., Stanley H. O.

Department of Microbiology, Faculty of Biological Science, College of Natural and Applied Sciences, University of Port Harcourt, P.M.B. 5323 Port Harcourt, Nigeria

Abstract: The effect of rumen fluid on microbial population dynamics during anaerobic digestion of Guinea grass (Panicum maximum) at ambient condition with respect to time was investigated. A one stage batch-typemesophilic 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, chemical oxygen demand (CODRF) and volatile fatty acid (VFARF) were monitored with time. Selected indicator microbial populations were monitored by standard cultural enumerations based on metabolic capacity and oxygen sensitivity with respect to time. Furthermore, their respective growth rates and population proportions were determined. Result showed that the average PTMRF increased from 27.5°C to 35.2°C while average process pHRF ranged from 6.5 to 7.9 with time, respectively. The CODRF decreased from 11,250.60 mg/L to 2,865.20 mg/L, while VFARF ranged from 1,080.00 mg/L to 4,800.33 mg/L with time, respectively. In terms of metabolic capacity, the populations of cellulolytic bacteria (ACBRF), lactose fermenting bacteria (LFBRF) and glucose fermenting bacteria (GFBRF) ranged from 3.6 x 10⁴ MPN/ml to 2.9 x 10⁵ MPN/ml, 3.4 x 10⁴ MPN/ml to 2.9 x 10⁵ MPN/ml and 4.4 x 10⁴ MPN/ml to 4.6 x 10⁵ MPN/ml respectively with time. The populations of propionate oxidizing bacteria (POBRF), ethanol oxidizing bacteria (EOBRF) and acetate oxidizing methanogens (AOMRF) ranged from 2.9 x 10⁴ MPN/ml to 2.4 x 10⁵ MPN/ml, 2.7 x 10⁴ MPN/ml to 2.1 x 10⁵ MPN/ml and 1.4 x 10⁴ MPN/ml to 2.1 x 10⁵ MPN/ml respectively with time. In terms of O₂-sensitivity, the populations of obligate anaerobic bacteria (OABRF) and facultative aerobic and anaerobic bacteria (FAABRF) ranged from 2.12 x 10⁵ CFU/ml to 4.53 x 10⁶ CFU/ml and 4.6 x 10⁵ CFU/ml to 4.74 x 10⁶ CFU/ml respectively with time. The population of GFBRF had the highest growth rate of 0.057 day⁻¹ while the population of EOBRF had the lowest growth rate of 0.021 day⁻¹. In terms of O₂-sensitivity, the population of FAABRF had the highest growth rate of 0.051 day⁻¹ compared to the population of OABRF with growth rate of 0.040 day⁻¹. The population of GFBRF predominated (26.3%), while the population of AOMRF were the minority (10.44%). In terms of O₂-sensitivity, the population of FAABRF predominated (56.73%) compared to the population of OABRF (43.23%). Rumen fluid significantly (p < 0.05) increased the microbial populations inside ADRF with respect to time. Therefore, rumen fluid could be used to boost the microbial population in anaerobic digesters as this could enhance depolymerisation, obtain higher degradation rates of cellulosic (or lignocellulosic) substrates and thus higher energy (biogas/methane) benefits.

Keywords: Anaerobic digestion (AD), Guinea grass, Rumen fluid, microbial population dynamics

Received July 26, 2014; Revised November 04, 2014; Accepted November 26, 2014

Citation: Ogbonna C. B., Ibiene A. A., Stanley H. O., Microbial Population Dynamics during Anaerobic Digestion of Guinea Grass (Panicum maximum). Journal of Applied & Environmental Microbiology, 2 (6). 294-302. January 2014. doi:10.12691/jaem-2-6-5

1. Introduction

One of the most important agricultural crops in Nigeria is Guinea Grass (Panicum maximum). It is a native of Africa, widely distributed and readily available in the country. It has leaves that are fine and soft and contains good levels of protein (13-21%) and can be grown for uses such as; pasture, silage, hay, and rough grazing. Bio-energy, especially biogas production can provide a new prospect for grassland use in the country without affecting the balance of the ecosystem (Gerin et al., 2008; Beatrice et al., 2009; Ahn et al., 2010; Uzodinma and Ofoefole, 2009). Anaerobic degradation of organic matter involves a series of microbial metabolic reactions such as hydrolysis, acidogensis, acetogenesis and methanogensis (Themelis and Ulloa, 2007; Schnurer and Jarvis, 2010) and it has the potential to provide useful products such as biofuel (e.g., biogas) and organic amendment for agricultural soil (Chanakya et al., 2007; Schnurer and Jarvis, 2010). Anaerobic digestion of cellulosic (or lignocellulosic) materials is limited mainly by the rate of hydrolysis of the polymeric compounds (Labat and Garcia, 1986). These substrates are usually devoid of appropriate microflora and, to be efficiently fermented, require suitable inoculation (e.g., from sludge or cow dung, etc.) 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 applications of rumen microorganisms [See Allison and Leek, 1993] in AD systems for the conversion of lignocellulosic materials were investigated (Dalhoff et al., 2003; Barnes & Keller 2003, 2004). For example, O’Sullivan et al., (2006) studied cellulose solubilisation rates in batch reactors using rumen culture and MSW leachate as sources of inocula and microcrystalline cellulose as substrate. The authors obtained about 34-50% higher cellulose solubilisation rates when rumen culture was used as an inoculum source than with the MSW leachate. Furthermore, Hu and Yu (2005), studied AD of corn stover in batch and semi-continuous cultures and obtained a high VS conversion efficiency of 65-70% after 10 days of incubation at 25-40°C. In another such study by Yue et al., (2007), a degradation efficiency of 52.3% was achieved during AD of aquatic plant such as Canna indica L. using rumen cultures in batch experiments. Total bacterial count was generally estimated to be 10¹⁰ bacteria/ml in stabilized anaerobic digesters. These results were obtained after addition of rumen fluid or biodigester juice to the enumeration media (Hobson and Shaw 1973; Iannotti et al. 1978; Ueki et al., 1978). Due to the high anaerobic bacteria population inside the rumen of Cow [Aurora, 1983; Allison and Leek, 1993] and the abundance of rumen waste disposal from slaughterhouses in Nigeria, this study focused on the effect of rumen fluid on the population dynamics of the microbial community taking part in anaerobic digestion of the Guinea grass (P. maximum) at ambient condition with respect to time using selected indicator microbial groups.

2. Materials and Methods

2.1. Anaerobic Digestion Set-up

One-stage 500 litre – capacity anaerobic digesters (AD) were configured for batch-type mesophilic reactors with useful volumes of around 310.30 litres respectively. The anaerobic digester with rumen fluid inoculation (ADRF) was the experimental set-up, while the anaerobic digester without rumen fluid inoculation (ADNRF) was the control set-up. Anaerobic digestion of the feed was performed inside the biodigestersat ambient condition and a retention time of 105 days. Cow’s rumen content was collected from an abattoir in Aluu community located in Rivers State (Nigeria). After collection, Rumen fluid was prepared and stored in an air-tight 60L – capacity gallon for a month as described by Budiyono et al., (2009). Furthermore, Budiyono et al., (2009) suggested that keeping the rumen fluid for such a period of time does not affect the microbial community inside the fluid. Matured guinea grass was harvested within Abuja Campus (University of Port Harcourt, Nigeria), chopped into smaller pieces, minced and weighed with a weighing balance. Samples were immediately taken to the laboratory for determination of water content (WC) and total solid (TS) content of the grass using the USEPA (2001) method 1684. The feed inside both AD systems (ADRF and ADNRF) was prepared as presented in Table 1 using the formula below (Yadvika, et al., 2004);

Table 1. Composition of the Feed for Anaerobic digestion

2.2 Sample Collection and Determination of Physicochemical Parameters

To monitor the anaerobic digestion (AD) process, digester slurry samples were collected anaerobically at two-week intervals from the anaerobic digesters into air tight sterile sample bottles by not allowing any head space in the bottles and transported to the laboratory in a PVC Box for physicochemical and microbiological analyses. However, the last sample was collected at three-weeks interval (i.e., day 0, day 14, day 28, day 42 day 56, day 70, day 84 and day 105, respectively).Process temperature (PTM) and pH were measured using water thermometer (SCT-lilliput, Scichem Tech.) and a general purpose pH meter (SCT-lilliput, Scichem Tech.), respectively. Chemical oxygen demand (COD) and total volatile fatty acid (VFA) were determined using the titrimetric methods described by Reaffirmed (2006) and Buchauer (1998), respectively.

2.3. Enumeration of Selected Microbial Population

Immediately after collection and homogenization of biodigester slurry samples, the samples were serially diluted (anaerobically) from 10⁻¹ to 10⁻⁶ using the reduced mineral solution of Ralph (2011) as described by Labat and Garcia, (1986) and Claudia et al. (2009) respectively. Bacteria populations were evaluated by counting selected groups based on metabolic capacity and oxygen sensitivity, respectively. The metabolic groups selected included the populations of cellulolytic bacteria (ACB), lactose fermentative bacteria (LFB), glucose fermentative bacteria (GFB), propionate oxidizing bacteria (POB), ethanol oxidizing bacteria (EOB) and acetate oxidizing methanogens (AOM), respectively. They were enumerated anaerobically by the Most Probable Number (MPN) technique (n=3) described by Labat and Garcia, (1986) and Claudia et al. (2009) respectively and incubated at 35°C with incubation times depending on the metabolic group (Diaz et al., 2002). The MPN results were interpreted with appropriate tables from Oblinger and Koburger (1975) and reported asmost probable number per millimetre (MPN/ml) of the digester slurry sample. The growth of POB, EOB and AOM was respectively reported as positive tube(s) in biogas production (Claudia et al., 2009). The growth of GFB and LFB was respectively evaluated for acidification (i.e., change in medium colour from blue to yellow) and gas production, while the growth of cellulolytic bacteria (ACB) was evaluated for yellow or brown pigmentation (or deposits) and or gas production (Claudia et al., 2009).

The oxygen-sensitive groups selected included obligate anaerobic bacteria (OAB) and facultative aerobic and anaerobic bacteria (FAAB) populations, respectively. They were enumerated by the pour plate count method described by Bryant (1972) and incubated at 35°C for 72 and 48 hours, respectively. Their growth was reported as colony forming unit per millimetre (CFU/ml) of digester slurry sample. After enumeration, the growth rate and the proportion of all selected microbial populations were estimated from their respective growth curves using the formulae stated in Dubey and Maheshwari (2008);

Where;

T₁ = Initial time, T₂ = Final time, N₁ = Cell number at T₁, N₂ = Cell number at T₂

2.4. Statistical Analysis

Within the Microsoft Excel (2013) environment, the two-factor analysis of variance (2-Way ANOVA) was used to determine if there was a significant difference between the population of microbes inside the anaerobic digester with rumen fluid inoculation (ADRF) and the population of microbes inside the anaerobic digester without rumen fluid inoculation (ADNRF) system.

3. Result and Discussion

3.1. Physicochemical Analysis

The average process temperature (PTMRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) increased from 27.5°C to 35.2°C with time while the average process temperature (PTMNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) increased from 27.5°C to 34.4°C with time. The average process pH (pHRF and pHNRF) inside ADRF and ADNRF ranged from 7.9 to 7.4 and 8.1 to 7.3 respectively with time. The CODRFinside ADRF decreased from 11,250.60 mg/L to 2,865.20 mg/L while the CODNRF inside ADNRF decreased from 11,250.20 mg/L to 4,538.90 mg/L with time. The concentration of volatile fatty acid (VFARF) inside ADRF increased from 1,080.00 mg/L to 4,800.33 mg/L whilethe concentration of volatile fatty acid (VFANRF) inside ADNRFincreased from 729.34 mg/L to 4,632.04mg/L with time.

3.2. Microbiological Analysis

Figure 1. Population dynamics of cellulolytic bacteria (ACBRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) and population dynamics of cellulolytic bacteria (ACBNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) during anaerobic digestion of Panicum maximum

Generally, the indicator bacteria populations enumerated during anaerobic digestion of Panicum maximum increased with respect to time as shown in Figure 1 to Figure 8. The population of cellulolytic bacteria (ACBRF) insidethe anaerobic digester with rumen fluid inoculation (ADRF) increased from 3.6 x 10⁴ MPN/ml at day 0 to6.4 x 10⁴ MPN/ml and 9.3 x 10⁴ MPN/mlatday 14 and day 28, respectively. The population peaked at 2.9 x 10⁵ MPN/ml on day 42 and day 56 respectively. However,at day 70, day 84 and day 105, this population decreased slightly to 2.4 x 10⁵ MPN/ml and 2.1 x 10⁵ MPN/ml respectively (Figure 1). Likewise, the population of cellulolytic bacteria (ACBNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) increased from 1.5 x 10⁴ MPN/ml at day 0 to3.5 x 10⁴ MPN/ml, 4.4 x 10⁴ MPN/ml, 1.2 x 10⁵ MPN/ml and 1.6 x 10⁵ MPN/ml at day 14, day 28, day 42 and day 56, respectively.The population peaked at2.1 x 10⁵ MPN/ml on day 70 however, at day 84 and day 105, this population decreased slightly to 1.6 x 10⁵ MPN/ml and 1.5 x 10⁵MPN/ml respectively (Figure 1). The periods when the populations of CBRF and CBNRF increased, peaked and then decreased during anaerobic digestion of the grass (Panicum maximum) indicated thatcellulose hydrolysis (or cellulolytic activity) may have increased, peaked and then decreased at those times inside ADRF and ADNRF, respectively (Claudia et al., 2009; Labat and Garcia, 1986).

The population of lactose fermenting (acidogenic) bacteria (LFBRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) increased from 3.4 x 10⁴ MPN/ml at day 0 to7.5 x 10⁴ MPN/ml, 1.2 x 10⁵ MPN/ml and 2.1 x 10⁵ MPN/ml at day 14, day 28 and day 42 respectively.The population peaked at 2.9 x 10⁵ MPN/ml on days 56 and 70, respectively. However at day 84 and day 105, this population decreased to 2.4 x10⁵MPN/ml and 2.1 x 10⁵ MPN/ml respectively (Figure 2). Likewise, population of lactose fermenting (acidogenic) bacteria (LFBNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) increased from 1.6 x 10⁴ MPN/ml at day 0 to 3.6 x 10⁴ MPN/ml, 6.4 x 10⁴ MPN/ml, 1.2 x 10⁵ MPN/ml and 1.5 x 10⁵ MPN/ml at day 14, day 28, day 42 and day 56 respectively. The population peaked at 1.6 x 10⁵ MPN/ml on day 70 however, this population decreased to 1.5 x 10⁵ MPN/ml and 1.2 x10⁵MPN/ml at day 84 and day 105 respectively (Figure 2).

Figure 2. Population dynamics of lactose fermentative bacteria (LFBRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) and population dynamics of lactose fermentative bacteria (LFBNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) during anaerobic digestion of Panicum maximum.

The population of glucose fermenting (acidogenic) bacteria (GFBRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) increased from 4.4 x 10⁴ MPN/ml at day 0 to 9.3 x 10⁴ MPN/ml and 1.6 x 10⁵ MPN/ml at day 14 an day 28 respectively.The population peaked at 4.6 x 10⁵ MPN/ml (on day 42 and day 56, respectively. However,this population decreased to 2.9 x 10⁵MPN/ml and 2.4 x 10⁵ MPN/ml at day 70, day 84 and day 105 respectively (Figure 3). Likewise, the populationof glucose fermenting (acidogenic) bacteria (GFBNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) increased from 2.6 x 10⁴ MPN/ml at day 0 to4.4 x 10⁴ MPN/ml, 7.5 x 10⁴ MPN/ml, 1.6 x 10⁵ MPN/ml and 2.1 x 10⁵ MPN/ml at day 14, day 28, day 42 and day 56 respectively. The population peaked at 2.4 x 10⁵ MPN/ml on day 70 however, this population decreased to 2.1 x 10⁵MPN/ml at day 84 and day 105 respectively (Figure 3).The periods when the respective populations of LFBRF and GFBRF and LFBNRF and GFBNRF increased, peaked and then decreased during anaerobic digestion of the grass (Panicum maximum) indicated that fermentation(or acidogenic activity) may have increased, peaked and then decreased at those times inside ADRF and ADNRF, respectively (Claudia et al., 2009; Labat and Garcia, 1986). This is because fermentation of sugars in anaerobic digestion processes have been associated with acidogenesis (Ike et al., 2010; Schnurer and Jarvis, 2010; Chanakya and Sreesha, 2012; Ljupka, 2010; Yassar, 2011).

Figure 3. Population dynamics of glucose fermentative bacteria (GFBRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) and population dynamics of glucose fermentative bacteria (GFBNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) during anaerobic digestion of Panicum maximum

Figure 4. Population dynamics of propionate oxidizing bacteria (POBRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) and population dynamics of propionate oxidizing bacteria (POBNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) during anaerobic digestion of Panicum maximum

The population of propionate oxidizing (acetogenic) bacteria (POBRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) increased from 2.9 x 10⁴ MPN/ml at day 0 to5.3 x 10⁴ MPN/ml, 7.5 x 10⁴ MPN/ml, 1.2 x 10⁵ MPN/ml and 1.5 x 10⁵ MPN/ml at day 14, day 28, day 42 and day 56 respectively. The population peaked at 2.4 x 10⁵ MPN/ml on day 70 and day 84 respectively however, this population decreased to 2.1 x10⁵MPN/ml at day 105 (Figure 4). Likewise, the population of propionate oxidizing (acetogenic) bacteria (POBNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) increased from 3.0 x 10³ MPN/ml at day 0 to2.7 x 10⁴ MPN/ml, 3.6 x 10⁴ MPN/ml, 6.4 x 10⁴ MPN/ml, 7.5 x 10⁴ MPN/ml and 1.5 x 10⁵ MPN/ml at day 14, day 28, day 42, day 56 and day 70 respectively. The population peaked at 1.6 x 10⁵ MPN/ml on day 84 however, it decreased to 1.5 x10⁵ MPN/ml at day 105 (Figure 4).

Figure 5. Population dynamics of ethanol oxidizing bacteria (EOBRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) and population dynamics of ethanol oxidizing bacteria (EOBNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) during anaerobic digestion of Panicum maximum

The population of ethanol oxidizind bacteria (EOBRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) increased from 2.7 x 10⁴ MPN/ml at day 0 to 6.4 x10⁴ MPN/ml, 7.5 x10⁴ MPN/ml, 9.5 x 10⁴ MPN/ml and 1.6 x 10⁵ MPN/ml at day 14, day 28, day 42 and day 56respectively. The population peaked at 2.1 x 10⁵ MPN/ml on day 70 and day 84 respectively however, at day 105, it decreased to 1.6 x 10⁵ MPN/ml (Figure 5). Likewise, the population of ethanol oxidizind bacteria (EOBNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) increased from 6.0 x 10⁴ MPN/ml at day 0 to 3.4 x 10⁴ MPN/ml, 3.9 x 10⁴ MPN/ml, 5.3 x 10⁴ MPN/ml, 9.3 x 10⁴ MPN/ml and 1.5 x 10⁵ MPN/ml at day 14, day 28, day 42, day 56 and 70 respectively. This population peaked at 1.6 x 10⁵MPN/ml on day 84 and day 105 respectively (Figure 5). The periods when the respective populations of POBRF and EOBRF and POBNRF and EOBNRF increased, peaked and then decreased during the anaerobic digestion process indicated that anaerobic oxidation (or acetogenic activity) may have increased, peaked and then decreased at those times inside ADRF and ADNRF, respectively (Claudia et al., 2009; Labat and Garcia, 1986). This is because anaerobic oxidation of short chain fatty acids (e.g., butyrate, propionate, etc.) and alcohols (e.g., ethanol, propanol, etc.) in an anaerobic digestion process have been associated with acetogenesis (Claudia et al., 2009; Schnurer and Jarvis, 2010; Chanakya and Sreesha, 2012; Ljupka, 2010; Yassar, 2011).

Figure 6. Population dynamics of acetate oxidizing methanogens (AOMRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) and population dynamics of acetate oxidizing methanogens (AOMNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) during anaerobic digestion of Panicum maximum

Figure 7. Population dynamics of facultative aerobic and anaerobic bacteria (FAABRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) and population dynamics of facultative aerobic and anaerobic bacteria bacteria (FAABNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) during anaerobic digestion of Panicum maximum

The population of acetate oxidizingmethanogens (AOMRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) increased from 1.4 x 10⁴ MPN/ml at day 0to 3.6 x 10⁴ MPN/ml and 4.4 x 10⁴ MPN/ml at day 14 and day 28 respectively. Their population decreasedslightly to 4.2 x 10⁴ MPN/ml at day 42.However, at day 56, day 70, day 84 and day 105, the population increased again to 9.3 x 10⁴ MPN/ml, 1.6 x 10⁵ MPN/ml and 2.1 x 10⁵ MPN/ml respectively (Figure 6). The population ofacetate oxidizingmethanogens (AOMNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) increased from 3.0 x 10³ MPN/ml at day 0 to 1.6 x 10⁴ MPN/ml, 2.4 x 10⁴ MPN/ml and 2.9 x 10⁴ MPN/ml at day 14, day 28 and day 42 respectively. Their population decreased slightly to 2.0 x 10⁴ MPN/ml at day 56 and then increasing again to 7.5 x 10⁴ MPN/ml, 1.5 x 10⁵ MPN/ml and 1.6 x 105 MPN/ml at day 70, day 84 and day 105 respectively (Figure 6). This implied that acetotrophic methanogenesis (or acetate oxidation byacetotrophic methanogens) may have increased from day 0 to day 28 and day 42 inside ADRF and ADNRF respectively, but dropped slightly at around day 42 (inside ADRF) and day 56 (inside ADNRF) and then increased again at around day 56 and day 70 to day 105 inside ADRF and ADNRF respectively. (Schnurer and Jarvis, 2010; Chanakya and Sreesha, 2012; Ljupka, 2010; Yassar, 2011).

Figure 8. Population dynamics of obligate anaerobic bacteria (OABRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) and population dynamics of obligate anaerobic bacteria (OABNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) during anaerobic digestion of Panicum maximum

The population offacultative aerobic and anaerobic bacteria (FAABRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) increased from 4.6 x 10⁵ CFU/ml at day 0 to 1.08 x 10⁶ CFU/ml,2.21 x 10⁶ CFU/ml and 4.52 x 10⁶ CFU/ml at day 14, day 28 and day 42 respectively. The population peaked at 4.74 x 10⁶ CFU/ml on day 56 however, on day 70, day 84 and day 105, this population decreased to 4.56 x 10⁶ CFU/ml, 4.24 x 10⁶ CFU/ml and 4.05 x 10⁶ CFU/ml respectively (Figure 7). Likewise, the population of facultative aerobic and anaerobic bacteria (FAABNRF) inside the anaerobic digester without rumen fluid inoculation (ADNRF) increased from 9.7 x 10⁴ CFU/ml at day 0 to 2.37 x 10⁵ CFU/ml, 6.85 x 10⁵ CFU/ml,1.47 x 10⁶ CFU/ml, 2.42 x 10⁶ CFU/ml and 3.19 x 10⁶ CFU/ml at day 14, day 28, day 42, day 56 and day 70 respectively. The population peaked at 3.34 x 10⁶ CFU/ml on day 84 and then decreased slightly to 3.18 x 10⁶ CFU/ml at day 105 (Figure 7). The population of obligate anaerobic bacteria (OABRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) increased from 2.12 x 10⁵ CFU/ml at day 0 to6.5 x 10⁵ CFU/ml, 1.14 x 10⁶ CFU/ml, 2.01 x 10⁶ CFU/ml, 2.81 x 10⁶ CFU/ml and 3.92 x 10⁶ CFU/ml at day 14, day 28, day 42, day 56, and day 70 respectively. The population peaked at 4.53 x 10⁶ CFU/ml on day 84 and then decreased slightly to 4.45 x 10⁶ CFU/ml at day 105 (Figure 8). Likewise, the population of obligate anaerobic bacteria (OABNRF) inside the anaerobic digester with rumen fluid inoculation (ADNRF) increased from 4.6 x 10⁴ CFU/ml at day 0 to 2.12 x 10⁵CFU/ml, 5.6 x 10⁵ CFU/ml, 9.45 x 10⁵ CFU/ml, 1.36 x 10⁶ CFU/ml, 1.95 x 10⁶ CFU/ml, 2.61 x10⁶ CFU/ml and 2.76 x 10⁶ CFU/ml at day 14, day 28, day 42, day 56, day 70, day 84 and day 105 respectively (Figure 8).

The pattern of growth exhibited by these indicator microbial populations with respect to time inside the anaerobic digester with rumen fluid inoculation (ADRF) and the anaerobic digester without rumen fluid inoculation (ADNRF) tend to obey the sigmoid function (S curve) as generally occurred in batch culture. Their respective populations increased (about 10-fold) with respect to time (see Figure 1 to Figure 8). This Observation is in line with the result of other authors who also reported significant increase in the population of various bacteria and archaea groups based on metabolic capacity and oxygen sensitivity during anaerobic digestion of Municipal solid waste (MSW), Sugar beets and Cassava peels respectively (Claudia et al., 2009; Labat and Garcia, 1986; Cuzin et al., 1992). Furthermore, statistical analysis (one – way ANOVA) showed that there was a significantly (P < 0.05) difference between the microbial population inside the anaerobic digester with rumen fluid inoculation (ADRF) and the microbial population inside the anaerobic digester without rumen fluid inoculation (ADNRF) with respect to time.

This was most likely due to the addition of rumen fluid inside the ADRF system. Some author(s) have also shown that rumen fluid inoculation may be capable of positively affecting the microbial population in an anaerobic digestion system (Dalhoff et al., 2003; Barnes & Keller 2003, 2004; Hu and Yu, 2005; O’Sullivan et al., 2006; Yue et al., 2007; Jagadabhi et al., 2010). This is because rumen fluid contains high population of bacteria that are specialized in biogas production through anaerobic digestion of lignocellulosic substrates such as grasses (Allison and Leek, 1993; Barnes and Keller 2003). Rumen fluid may also contain some growth factors which may have been beneficial to these bacteria populations with time (Hobson and Shaw 1973; Iannotti et al. 1978; Ueki et al., 1978). These may have been some of the reasons why the bacteria and archaea populations inside the ADRF system were higher than the bacteria and archaea populations inside the ADNRF system at any given period during anaerobic digestion of the guinea grass (Figure 1 to Figure 8).

In the anaerobic digester with rumen fluid inoculation (ADRF), the population of cellulolytic bacteria (ACBRF), lactose fermenting bacteria (LFBRF), glucose fermenting bacteria (GFBRF), propionate oxidizing bacteria (POBRF), ethanol oxidizing bacteria (EOBRF), acetate oxidizing methanogens (AOMRF), facultative aerobic and anaerobic bacteria (FAABRF) and obligate anaerobic bacteria (OABRF) had growth rates of 0.054 day⁻¹, 0.038 day⁻¹, 0.057 day⁻¹, 0.030 day⁻¹, 0.021 day⁻¹, 0.028 day⁻¹, 0.051 day⁻¹ and 0.040 day⁻¹ respectively. In the anaerobic digester without rumen fluid inoculation (ADNRF), the population of cellulolytic bacteria (ACBNRF), lactose fermenting bacteria (LFBNRF), glucose fermenting bacteria (GFBNRF), propionate oxidizing bacteria (POBNRF), ethanol oxidizing bacteria (EOBNRF), acetate oxidizing methanogens (AOMNRF), facultative aerobic and anaerobic bacteria (FAABNRF) and obligate anaerobic bacteria (OABNRF) had growth rates of 0.044 day⁻¹, 0.043 day⁻¹, 0.046 day⁻¹, 0.031 day⁻¹, 0.022 day⁻¹, 0.028 day⁻¹, 0.056 day⁻¹ and 0.053 day⁻¹ respectively (Figure 9).

This result suggested that the bio-degraders inside the anaerobic digesters (ADRF and ADNRF) were slow growers (see Figure 9). Nevertheless, the cellulolytic bacteria populations (i.e., ACBRF and ACBNRF) and the sugar fermenting (acidogenic) bacteria populations (i.e., LFBRF and GFBRF and LFBNRF and GFBNRF) grew at a faster pace compared to the propionate and ethanol oxidizing (acetogenic) bacteria populations (i.e., POBRF and EOBRF and POBNRF and EOBNRF) and the acetate oxidizing methanogenic populations (i.e., AOMRF and AOMNRF) inside the respective anaerobic digesters (ADRF and ADNRF) as observed in Figure 9. This was predicted because of the metabolic relationships among these microbial populations in anaerobic digestion food chains. Usually, hydrolysis followed by acidogenesis must first occur to some degree before acetogenesis and methanogenesis (Schnurer and Jarvis, 2010; Chanakya and Sreesha, 2012; Ljupka, 2010; Yassar, 2011).

Figure 9. Population growth rates of selected indicator bacteria groups based on metabolic capacity and oxygen sensitivity inside the anaerobic digester with rumen fluid inoculation (ADRF) andthe anaerobic digester without rumen fluid inoculation(ADNRF) respectively

Generally, methanogens are usually the slowest growers, while the acidogens are usually the fastest growers compared to other bacteria groups in most anaerobic digestion food chains (Schnurer and Jarvis, 2010). The reason why the population of ethanol oxidizing bacteria (EOBRF and EOBNRF) groups inside both anaerobic digesters (ADRF and ADNRF) may have had the slowest growth rate as observed in Figure 9, may be linked to the metabolic pathways by which the acetogens generated the acetate which was converted to biogas bythe acetotrophic methanogens (Ljupka, 2010). It may have been that the propionate oxidation pathway was favoured (than the ethanol oxidation pathway) during Acetogenesis. In terms of O₂-sensitivity, the population of facultative bacteria groups (FAABRF and FAABNRF) grew at a faster pace compared to the population of obligate anaerobic bacteria (OABRF and OABNRF) inside the anaerobic digesters (ADRF and the ADNRF) as observed in Figure 9. This was predicted because obligate anaerobes are generally slower growers compared to facultative aerobes and anaerobes respectively (Schnurer and Jarvis, 2010; Labat and Garcia, 1986; Claudia et al., 2009).

Inside the anaerobic digester with rumen fluid inoculation (ADRF), the population of sugar fermenting acidogens (LFBRF and GFBRF) predominated with a proportion of 45.26%. This was followed by the population of propionate and alcohol oxidizing acetogens (POBRF and EOBRF) with a proportion of 25.41%. Next, was the population of cellulolytic bacteria (ACBRF) with a proportion of 18.89% and the population of acetate oxidizing methanogens (AOMRF) were the minority with a proportion of 10.44% (Figure 10). Inside the anaerobic digester without rumen fluid inoculation (ADNRF), the population of sugar fermenting acidogens (LFBNRF and GFBNRF) predominated with a proportion of 42.16%. This was followed by the population of propionate and alcohol oxidizing acetogens (POBNRF and EOBNRF) with a proportion of 28.82%. The population of cellulolytic bacteria (ACBNRF) followed with a proportion of 18.93%, while the population of acetate oxidizing methanogens (AOMNRF) were the minority with a proportion of 10.0% (Figure 10). This result is in line with the result of other studies (Labat and Garcia, 1986; Claudia et al., 2009; Schnurer and Jarvis, 2010) which suggested that the population of the acidogens and the methanogens may be the majority and the minority populations in most anaerobic digestion (AD) systems, respectively. This may be linked to their growth rates during the AD process as suggested by Figure 9 above (Schnurer and Jarvis, 2010).

In terms of O₂-sensitivity, the population of facultative aerobic and anaerobic bacteria (FAABRF) inside the anaerobic digester with rumen fluid inoculation (ADRF) predominated with a proportion of 56.73% compared with the population of obligate anaerobic bacteria (OABRF) which had a proportion of 43.23%. Inside the anaerobic digester without rumen fluid inoculation (ADNRF), the population of facultative aerobic and anaerobic bacteria (FAABNRF) predominated with a proportion of 58.34% compared with the population of obligate anaerobic bacteria (OABRF) which had a proportion of 41.66% (Figure 10). This may also be linked to their respective growth rates as discussed earlier (Figure 9). It is very important to monitor the proportion (%) of important microbial indicator groups during anaerobic digestion processes (Claudia et al., 2009; Labat and Garcia, 1986; Cuzin et al., 1992). This is because for example, in most AD systems treating cellulosic or lignocellulosic substrates (e.g., matured grass) that are not easily biodegradable, hydrolysis is usually the rate limiting phase especially when the proportion (%) of hydrolytic bacteria population that would attack the biopolymer in the initial substrate was not high enough to accelerate hydrolysis (Susan, 1995; Schnurer and Jarvis, 2010). However, if the substrate was easily (or readily) biodegradable, methanogenesis may become the rate limiting phase of the AD process especially when the proportion of methanogens that would convert acetic acid, hydrogen and carbon dioxide into biogas was insufficient at the time (Kim et al., 2008; Azeem et al., Schnurer and Jarvis, 2010; Susan, 1995).

Figure 10. Proportion (%) of selected indicator microbial populationsinside the anaerobic digester with rumen fluid inoculation (ADRF) and the anaerobic digester without rumen fluid inoculation(ADNRF) respectively

4. Conclusion

Anaerobic digestion process is said to be divided into four (4) stages (or phases) namely, Hydrolysis, Acidogenesis, Acetogenesis and Methanogenesis (Schnurer and Jarvis, 2010; Chanakya and Sreesha, 2012). However, in reality, these phases may be difficult to distinguish. Therefore, if the populations of some metabolically important microbial indicators were monitored with respect to time (as was done in this study), it may be possible to use their population dynamics to at least indicate (or monitor) the occurrence or progress of these processes to some degree (Claudia et al., 2009; Labat and Garcia, 1986; Ike et al., 2010; Cuzin et al., 1992). Furthermore, analysis of various bacterial groups during anaerobic digestion (AD) process could indicate bad running of the process. Two or three specific counts could possibly explain process failure, but cannot contribute to optimization of the biogas yield. However, the possibility of increasing the performance of an AD process by controlling its microflora appears possible. Our result suggested that rumen fluid significantly (P < 0.05) increased the microbial population inside the anaerobic digester with rumen fluid inoculation (ADRF) compared to the microbial population inside the anaerobic digester without rumen fluid inoculation (ADNRF) with time. Therefore, rumen fluid could be used to boost the microbial population inside anaerobic digesters which could enhance depolymerisation, obtain higher degradation rates of lignocellulosic substrates and thus higher energy (biogas/methane) benefits.

Nomenclature

AD: anaerobic digestion

RF: 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

GFBRF: glucose fermenting bacteria in ADRF

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

WS: wet solid

WA: water

PTMRF: process temperature in ADRF

PTMNRF: process temperature in ADNRF

pHRF: process pH inside ADRF

pHNRF: process pH inside ADNRF

CODRF: chemical oxygen demand in ADRF

CODNRF: chemical oxygen demand in ADNRF

VFARF: volatile fatty acid inside ADRF

VFANRF: volatile fatty acid inside ADNRF

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