2.1 Substrate, co-substrate and inoculum

Wastewater from Pontificia Bolivariana University (UPB by its acronym in Spanish) and aerobic sludge from secondary treatment were used as substrate and co-substrate, respectively. Anaerobic sludge from wastewater treatment plant of soft drinks was used as inoculum. Physico-chemical parameters were evaluated to substrate, co-substrate and inoculum according to standard methods [⁸]. In addition, the following activities were performed to the inoculum: acidogenic (AA), hydrolytic (HA) and specific methanogenic activity (SMA).

Glucose and starch were used as substrate for AA and HA tests, respectively. The tests were performed using amber bottles with 100 mL of working volume, which were filled with inoculum (1.5 gVSS/L) and substrate (0.15 gCOD/L). All the experiments (AA, HA and SMA) were carried out under the addition of macro-nutrients, micro-nutrients, yeast extract and bicarbonate [⁹] and were incubated at 37°C. Samples of the supernatant were taken to determine the remaining glucose or starch concentrations in the flasks. The starch concentration was measured as the difference between total sugars and reducing sugars. Glucose concentration was assumed as reducing sugars concentration. Reducing sugars were determined by spectrophotometry at 540 nm according to the DNS method [¹⁰] and the total sugar with Dubois method [¹¹]. AA and HA were calculated with the Equation (1), where P is the maximum slope consumption, CF is conversion factor (gCOD/g glucose), V and gVSS are inoculum volume and mass added [¹²].

In the SMA test, a volatile fatty acids solution composed of acetic, propionic and butyric acid, was used as substrate. The test was performed using the Oxitop® system (WTW Germany) with 500 mL capacity glass bottles and the working volume was 400 mL. Bottles were filled with inoculum (1.5 gVSS/·L), substrate [¹³], macro-nutrients, micro-nutrients, sulfide solution, yeast extract and bicarbonate [⁹]. The pH was adjusted to a value of 7 after the mixing. The bottles were flushed with N₂ gas for 3 minutes to maintain an anaerobic condition and NaOH pellets were dosed inside a plastic holder inside the OxiTop flask, just underneath the screw-cap to absorb carbon dioxide from biogas. The bottles were incubated in a water bath at 37°C and manual stirring (three times a day) was used. Biogas production was measured as pressure increase every two hours during the first week and gradually decrease until the end of the test. The pressure measured by the head was converted in accumulated volumetric methane production (L) with Equation (2).

Where:

𝑉_{𝐶𝐻4𝐶𝐸}: Volume of 𝐶𝐻₄ at standard conditions (L)

𝑃: Pressure recording obtained in the OxiTop (atm)

𝑉𝑙: Free volume of the reactor (L)

𝑇𝑒: Temperature of the experiment (K)

𝑇_𝐶𝐸: Temperature at standard conditions (K)

𝑃_𝐶𝐸: Pressure at standard conditions (atm)

SMA was calculated with the maximum slope of the accumulated methane production curve (mL/d), divided by the amount of VSS introduced in the bottle using the proper conversion factor to report it as gCOD_CH4/gVSS·d.

2.2 Experimental Conditions

To evaluate the influence of the pH and C/N ratio on the biochemical methane potential a 3² factorial design was used. C/N ratio used were 4.9 (C₁), 8.2 (C₂) and 14.2 (C₃) and pH values were 9.0 (pH₁), 7.5 (pH₂) and 6.9 (pH₃) units. C₁ and pH₁ are the operating conditions of the wastewater treatment plant at UPB. C/N ratio was changed with the co-substrate addition, simulating the aerobic sludge recirculation to the anaerobic reactor, and the pH with hydrochloric acid 0.1N. Table 1 shows the operating conditions used.

Table 1 Operating conditions 

2.3 Biochemical methane potential tests

The BMP assay was performed using the Oxitop® system (WTW Germany) with 250 mL capacity glass bottles and the working volume was 200 mL. Bottles were filled with inoculum (1.5 gVSS/L), substrate and co-substrate, macronutrients and micronutrients. The bottles were incubated at 37°C and manual stirring was used. Biogas production was measured as pressure increase. Control test without substrate/co-substrate was used to measure the amount of methane produced by inoculum. The BMP was calculated by Equation (3), where V_TCH4CE is the methane volume at standard conditions (L) and gVS is the volatile solids of the substrate. Three replicates were used for each operating condition [Table 1]. The BMP was calculated by subtracting the control results to each trial.

The influence of the pH and C/N ratio on the biochemical methane potential was evaluated applying the modified Gompertz model, according to Equation (4) [¹⁴, ¹⁵]:

Where C is the cumulative methane production (ml) obtained at time t (d), B is the methane production potential (ml), R_b is the maximum methane production rate (mL·d^-1), λ = lag phase time (d) and e is a mathematical constant (2.718).

3.1 Substrate, co-substrate and inoculum characterization

The chemical characterization of substrate, co-substrate and inoculum is shown in Table 2. The pH value in the substrate was 9.03 units which is related to high urea content and the use of alkaline detergents in the institution. High values of pH cause instability of anaerobic digestion due to the rapid conversion rate of ionized ammonia nitrogen into free ammonia nitrogen. For limiting the inhibitory effect of free ammonia nitrogen on the anaerobic digestion process, it is suggested to keep the pH values close to 7 [⁶].

Table 2 Characteristics of substrate, co-substrate and inoculum 

*n.a. not available

According to the results, the wastewater has low volatile solids (45% TS) and COD content. The COD/BOD was 1.95 showing that the biodegradable fraction is high and it is a good indication for biological treatment [¹⁶]. Total Kjeldahl nitrogen (TKN) was 118.08 mg/L and ammonium nitrogen was between 50 and 200 mg/L contributing to the necessary nutrients for the growth of microorganisms [⁶]. However, the C/N ratio was very low (4.9). The optimal C/N ratio for anaerobic degradation of organic waste is between 20 and 35. The low C/N ratio is regarded as an important factor limiting anaerobic digestion [⁷]. Because the substrate has a very low C/N ratio (4.9), co-digestion with carbon-rich co-substrate is widely recommended. The aerobic sludge from secondary treatment was used as co-substrate to increase the C/N ratio. The total COD, TKN and C/N ratio were 40000 mg/L, 180 mg/L and 220.

For inoculum, the VS/TS ratio (0.9) shows the presence of active biomass useful for the degradation of organic matter and the pH and alkalinity values has good buffer capacity [¹⁷, ¹⁸]. To evaluate the different anaerobic consortia in the inoculum, hydrolytic, acidogenic and methanogenic activity were made. Starch and glucose consumption were analyzed for 72 hours to determinate the HA and AA of inoculum, respectively. The maximum consumption rate of starch was in the first 4 hours resulting in an HA value of 2.79 gCOD/gVSS·d showing the ability of inoculum for degrading the substrate used. For glucose, the maximum consumption rate was between 7 and 12 hours and the AA was 2.8 gCOD/gVSS·d. According to [¹⁹], the hydrolytic and methanogenic activities depend on the type of biomass, type of substrate and/or the operational conditions. They found, for biomass from sugar industry AA and HA values similar to those found in this study. The SMA obtained was 0,14 gCOD_CH4/gVSS·d, indicating good capacity as inoculum [²⁰]. However, hydrolytic and acidogenic activities were higher than the specific methanogenic activity, due probably to an imbalance in the syntrophic groups of inocula by the origin of the sludge.

3.2 Influence of pH and C/N ratio on the methane production

The comparison of experimental and predicted cumulative biogas production using the modified Gompertz model for each C/N ratio is shown in Figure 1. According to the results, the highest values of methane production were reached with pH₂ reaching values of 27.3, 49.0 and 61.0 mL CH₄ for C₁, C₂ and C₃, respectively. Furthermore, the effect of pH on the methane production for C₁ was not as strong as on C₂ and C₃ due to C₁ is the ratio to which the treatment plant works. For C_1, the methane production achieved with pH₂ was 49% and 12% higher than those reached with pH₃ and pH₁, respectively. Conversely, the C₂pH₂ combination obtained methane production 98% and 238% higher than with pH₃ and pH₁, respectively. However, for C_1, the lowest methane yields were obtained.

The ideal pH range for methanogenic and acidogenic microorganisms is different. Methanogenesis is most efficient at pH 6.5-8.2, being the optimal pH 7.0 and the activity of methanogenic bacteria decreased at a higher or lower pH [²¹]. Although pH₃ was close to 7.0, the methane production was low for all C/N ratios, probably because the alkalinity was not enough to buffer the volatile fatty acid production, inhibiting the activity of methanogens. Furthermore, the optimal pH depends on the type of substrate, as well as on the type of microorganisms involved in the digestion process [¹].

Table 3 shows the kinetic parameters obtained with the Gompertz model. The pH has a direct influence on the methane production potential (B) and the maximum methane production rate (R_b). It was found that the value of R_b obtained at pH₂ was higher than the value obtained at pH₁ and pH₃. These results are in agreement with other authors who report that the optimal pH for methanogenesis is around 7.0 [⁷]. When the pH is high, the ionized ammonia nitrogen can be converted in free ammonia nitrogen and inhibiting the methanogenesis [⁶].

The lag phase of the Gompertz equation was not influenced by the pH, however, the C/N ratio was a relevant factor. For C₁, the lag phase was between 1 and 2 days while the other ratios non-lag phase was observed. These results show the inhibition of anaerobic process with substrates with a low C/N ratio.

Figure 1 Influence of pH on methane production (mL CH₄). The solid line represents the Gompertz model fit data 

Table 3 Results of kinetic study from the modified Gompertz model 

Anaerobic digestion is a process susceptible to environmental conditions, due to the interaction between fermentative and methanogenic microorganisms. C/N ratio is a relevant factor in methane production. When the C/N ratio is high in the substrate, nitrogen will be consumed rapidly by methanogens to meet their protein requirements, which results in low methane production and with low C/N ratio, nitrogen will be present in the form of ammonia which inhibits the methanogens metabolism due to its toxicity [²²].

The comparison of experimental and predicted cumulative biogas production using the modified Gompertz model for each pH value is shown in Figure 2. All the C/N ratio had TKN concentrations between 127.9 and 119.4 mg/L, according to the nutrients required for the anaerobic digestion process [⁶]. As can be seen in the figure, for all pH value the higher methane production was achieved with the C₃. Several authors suggest optimal C/N ratio between 20 and 30 [²¹]; however, other authors have reported different values for C/N ratio depending on the substrate. For the co-digestion of onion juice and wastewater sludge, it has been suggested that the C/N ratio be 15 [²³]. In addition, it has been reported that lower C/N ratio promoted the activity of the methanogens in a long time when anaerobic semi-continuous digestion of food wastes/animal slurry wastewater was performed [²⁴]. The effect of C/N ratio and organic loading rate (OLR) on the anaerobic co-digestion of high solids containing distillers grains and swine manure was evaluated [²⁵]. The authors reported that maximum methanogenesis was achieved with the C/N ratio of 30/1. Other reports have shown that the benefits of improving methane yield from anaerobic digestion of multi-component substrates, based on optimized feeding composition and C/N ratio [²⁶]. C/N ratios of 25:1 and 30:1 had similar highest cumulative biogas production, about threefold higher than that from material with a C/N ratio of 15:1.

According to the results, there was a first stage during the first 7 days of operation, in which the methane production rate was the highest in all the digestion time, and then those values decreased gradually to become almost zero. The average methane production rate in the first stage were 2.86, 2.01 and 4.83 for C₁, C₂ and C₃, respectively, indicating a greater conversion of organic matter into methane for the highest C/N ratio evaluated. Considering the pH, the methane production rate in the first stage were 2.82, 4.57 and 2.32 for pH₁, pH₂ and pH₃, respectively. The results suggest that the optimum conditions for maximum methane production were with pH₂ (7.5) and C₃ ratio (14.2). In this condition, the highest methane production reached was 61 mL CH₄.

Figure 2 Influence of C/N ratio on methane production (mL CH₄): (A) pH 9.0±0.5; (B) pH 7.5± 0.1; (C) pH 6.9±0.1. ). The solid line represents the Gompertz model fit data 

The biochemical methane potential (BMP) test is the definition of the methane (or biogas) produced from a given weight of a certain substrate [²⁷] and this has been used to determinate the energy efficiency of different wastes or substrates. Figure 3 shows the BMP results for each C/N ratio and pH evaluated.

Figure 3 Biochemical methane potential for each C/N ratio and pH 

Figure 3 shows that for pH₁ and pH₃ the BMP value obtained was lower than for pH₂ for all C/N ratios. The maximum BMP value was 318.1 L CH₄ /kg VSS with initial pH₂ and C₂, with a COD removal of 79.2%, is a viable option to improve the anaerobic treatment. Furthermore, although the maximum methane production was presented for the C₃pH₂ condition (61 mL CH₄), the BMP in this condition (217.9 L CH₄ /kg VSS) was lower than the other C/N ratios. However, the COD removal was higher (85.2%) than other conditions as can be seen in Figure 4. This behavior is due to the carbon source used as co-substrate to rise the C/N ratio of the reactors since the use of aerobic sludge increased the VS content. For C₃, the VS was 1.99 g/L while for C₁ and C₂ were 0.58 g/L and 1.08g/L, respectively, therefore the BMP value decreased causing the substrate loses energy efficiency [²⁸].

According to Figure 4, the COD removal was above 70% for C₂ and C₃, however, the methane production rate of C₃pH₂ was twice higher than C₂pH₂ indicating that this condition is better to the wastewater treatment plant. Conversely, when the methane production of C₃pH₂ (61.02mL CH₄) is compared with C₃pH₁ and the COD removal obtained (85.23% and 66.30%, respectively), it is evident that the anaerobic processes inside the reactor were affected due to an inhibition in pH₁ condition [²⁹].

Figure 4 COD removal for each operating condition