Microbial inoculum (sludge) was collected from a mesophilic operating anaerobic bioreactor from the bio-methanation plant at TERI GRAM, India. Food waste and grass were the substrates used. Mechanical pre-treatment (Chipping) was carried out to reduce the size of grass for better digestion [18] [19]. After chipping the final particle size of grass was usually between 10 - 30 mm [19]. Grass was collected in a polythene bag and transferred to the laboratory. The pre-treatment used for the food waste was grinding [20] - [24]. Substrate ratio for food waste and grass was varied to maintain the C/N ratio. No chemical treatment was carried out on the materials before use.

Batch experiments were carried out in the laboratory. The digestion test was carried out using 300 mL glass bottles with a working volume of 225 mL. The experiment was operated at 37˚C ± 2˚C for twenty (20) days. The experimental conditions are given in Table 1. The experimental conditions were carried out to examine the influence of co-digestion ratio (R1 to R5) on biogas yield. The concentrations of the substrate were based on organic loading mass [25]. The amount of volatile solids (VS) of the inoculum and the substrate were calculated based on the predetermined co-digestion ratio. For all experiments, fresh water

was added, and no additional nutrients/trace elements were added to the reactors as it was assumed that they are provided by the inoculum (anaerobic sludge).

Studing the reactors (R1, R2, R3, R4, and R5), the influence of the different co-digestion of food waste and grass on biogas production, food waste was used as the sole substrate and inoculum was added for R1, while grass was the sole substrate in R5 and the inoculum was added. The experimental conditions of the batch test were chosen based on trail preliminary experiments in the laboratory. The designed percentages (see experimental set-ups) of waste were put in the digester and water was added at a ratio of 1:2. This was placed in a BOD incubator for six (6) days without the addition of culture. This acidification stage was carried out for 6 days. The pH was adjusted with Sodium Hydroxide (NaOH). The methanogenic culture was added after the sixth day. Biogas production started the next day and the monitoring of the gas was done for twenty (20) days. The daily gas production was recorded as Daily Gas Yields (DGY) by measurement of displaced water every morning. This is done by noting the quantity of water displaced by the gas collected in the measuring cylinder.

Table 2 shows the composition of raw substrate used in the digestion process. It is clear from the table that grass has a low pH which can be attributed to the high concentration of volatile fatty acids (VFAs) in the waste of above 800 mg/l which was also reported by Abu-Dahrieh J. K. et al. [29]. The increased VFA content could be as a result of the fermentation of the grass. The VFAs in the waste are then used directly in the acetogenesis stage to produce acetic acid, which is then used by the methanogens to produce methane.

All the experiments were carried out under daily mean temperature range of 37˚C ± 2˚C throughout the period of biogas production. The results of the experiment carried out for the twenty (20) days indicated that blending of field grass with the food waste affected the total biogas yield and onset of gas flammability for each of the biogas systems. Biogas production from all the rectors commenced within 24 hours after the acidification process of six (6) days. During the acidification process, pH dropped to 5 on the third day and it was reset to 7. This could be due to the fact that Volatile Fatty Acid (VFA) is possibly high. If the VFAs are not utilized at the rate they are produced, then it can kill the methanogenic activity due to lower pH. Feng et al. [30] reported that the possible acidogenic biological pathways that lead to a varied distribution of the VFAs, mainly is a function of pH. Maximum biogas was produced on the first day in all rectors. Daily biogas production from the grass and food waste is graphically shown in Figure 1.

It is observed in Figure 1 that the reactor 1 (R1) i.e. 100% FW produced the highest daily biogas yield of 233 mL. Grass only (R5) produced the lowest daily biogas yield of 125 mL. Food waste produced 50% more than grass only (R5). This is probably due to the lignocellulose structure of grass. Cellulose and hemicellulose are polysaccharides that can be hydrolyzed into simple sugars. Lignin which acts as a support to the cell structure, embedding cellulose and hemicellulose, hinders the susceptibility to microbial attack during hydrolysis process [31]. The main aim of the pretreatment is to break the lignin layer that protects the cellulose and hemicellulose, in order to make the biomass more accessible for digestion [31]. Co-digestion at 75% FW and 25% GR (R2) resulted in 6% more than food waste only. Figure 1 illustrates daily biogas production for all reactors over a period of twenty (20) days. For all of the reactors, there is a sharp increase in the amount of biogas produced during the first 1 - 2 days of the experiment. This is then followed by a sharp decrease following the near-complete consumption of the different substrates in the reactors. This was due to the increased availability of the biomass during the initial stages, leading to subsequent growth of the anaerobic organisms. After 3 days the rate of biogas production began to decrease due to a reduction in nutrient content. The samples show that there was a decrease in pH from pH 7.0 - 5.0 over the course of the experiment for all reactors. The dropped pH was reset to 7.

At the beginning of gas production, the anaerobes present in the organic material became active and began increasing in population [31]. When gas production began to rise, they were fully established and were acting on more substrate. At the peak of production, they were acting on the maximum amount of organic matter possible. After this point, gas production began to drop because the excess substrates were being converted to methane. At this point also, there is a steady decline in the amount of substrate available to the bacteria to act on [31]. There is also a decrease in either carbon or nitrogen available for use. When one becomes exhausted, the process slows gradually to a stop. This decline continues until gas production gradually comes to an end. While the process progresses, the condition becomes more appropriate for the methane forming bacteria. This led to an increase in the percentage composition of methane in the biogas produced until the maximum level is reached. Thus as fermentation approaches the end, the burning characteristics of the biogas improve [32].

Figure 2 shows the commutative biogas yield for the experimental setup R1 to R5. Cumulative Biogas Yield for R4 made up of 25% food waste and 75% grass recorded 243 mL. This may be due to the high cellulose content of the grass. The small amount of anaerobic bacteria present will take more time to establish itself and begin biogas production. The sample made of 50% grass and 50% food waste (R3), and those made up of 25% grass and 75% food waste (R2), 100% food waste (R1) and 100% grass (R5) recorded 485, 840, 805 and 418 mL of biogas yield respectively.

Co-digestion of 25% grass and 75% food waste (R2) produced the maximum biogas. This is similar to the study of Okonkwo et al. [33]. They reported that a maximum volume of biogas, 809 cm³ was produced by the sample containing 50% poultry dropping and 50% weeds. This indicates that this sample possesses the best C/N ratio of all the samples prepared. Different materials have their C/N

ratio, but mixture of different materials can alter the overall C/N ratio of the total feedstock. The result shows that this sample contains the C/N ratio which approaches the optimum C/N ratio of 30:1. R4 produced the least of the biogas which is similar to the studies by Okonkwo et al. [33]. They reported that the sample containing 25% poultry dropping and 75% groundnut shell had the least desirable value of C/N ratio. The bacteria responsible for the anaerobic process required both elements, as do all living organisms, but they consume carbon roughly 30 times faster than nitrogen. Assuming all other conditions are favourable for biogas production, a carbon-nitrogen ratio of about 30:1 is ideal for the raw material fed into a biogas plant. A higher ratio will leave carbon still available after the nitrogen has been consumed, starving some of the bacteria of this element. These will in turn die, returning nitrogen to the mixture, but slowing the process. Too much nitrogen will cause this to be left over at the end of digestion (which stops when the carbon has been consumed). The correct ratio of carbon to nitrogen will prevent the loss of methane content [33].