Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

Following the protocol, the system was built, tested, and inoculated. The conditions were measured and stored, and the samples were taken and analyzed. The protocol was performed a year, starting in October 2019 and lasting until October 2020. It is important to mention that from here onwards, the HRAPs will be referred to as RT3 and RT4.

Biomethane productivity
In order to determine the conditions that promote the highest H₂S and CO₂ removal and, consequently, the highest concentration of methane, several recirculation liquid/biogas ratios (L/G) were tried in a range from 0.5 to 3.4. These results were obtained for experiments with a duration of at least 60 min (1 h) of continuous biogas bubbling in the period between the 25^th of September and the 28^th of September. During these tests, the microalgae fixed the CO_2, and the bacteria oxidized the H₂S, concentrating the methane (CH₄) and, essentially, purifying the gas mixture.

Considering the averaged CO₂ elimination capacity of the whole system (HRAP volume + Tank volume = 24.75 m³) and a stable biomass concentration of 0.8 g/L, then a specific fixation rate was estimated, resulting in 65 mgCO₂/gbiomass h, which is lower than the maximum theoretical reported (300 mgCO₂/gbiomass h). This denotes that the biogas purification process based on microalgae-bacteria is suitable to be enhanced.

Generally, biogas purification had increased efficacy in higher L/G values, maintaining removal efficiencies at or above 98% for H₂S and less than 7.5%vol content values for CO₂ (Figure 5, Figure 6, and Figure 7). However, O₂ biomethane contamination due to photosynthetic production of this gas was much higher at higher L/G values, which can be a potential problem for commercial use as O₂ concentrations, by law, must remain quite low to reduce the risk of explosion²⁰. Another reason is linked to avoiding diminishing its calorific value by O₂ dilution. Instead, it could be argued that the L/Gs 1.6 and 2.5 represent the most efficient results overall, with CO₂ concentrations between 6.6%vol and 6.8%vol, CH₄ at 87%vol, and O₂ at less than 1.5%vol, as well as presenting H₂S removal efficiencies of above 98.5% (Figure 5, Figure 6, and Figure 7). A comparison between obtained percentages and what is accepted by law can be found in Table 1.

It is interesting to note that the recirculation liquid/biogas ratio of 2 has a higher CO₂ concentration (7.4%vol) even if the value sits between the most efficient L/Gs; this could be attributed to the fact that it was tested in RT3 instead of RT4. In this case, the conditions were less favorable for CO₂ removal, possibly due to a lower biomass concentration. Overall, the mean biomethane produced in these conditions came up to 20.68 m³/day, with an average flow rate of 4.14 m³/h.

Results may vary depending on the growth conditions, the type of biogas (synthetic or real) and the algae; for example, Serejo et al.²¹ used two synthetic biogas mixtures simulating an all CO₂ and N₂ gas mixture to compare with a regular biogas made of 70%vol CH₄, 29.5%vol CO₂ and 0.5%vol H₂S, purifying it through a 180 L HRAP-absorption column system cultivating Chlorella vulgaris. In this paper, Serejo tests different L/G ratios as well, ranging from 0.5 to 67, in a smaller but similar system at lower pH values and artificial lighting. Full removal of H₂S and an average removal percentage of 80% in the best ratios (above 15) was achieved. These removal efficiencies increased linearly with the ratio; however, oxygen contamination also increased, which could pose problems in the overall quality of the resulting biomethane. The increase in our CO₂ removal efficiencies was not linear; nonetheless, a better CO₂ elimination can be seen with larger ratios. The explanation is multicausal, involving pH, culture nutrient conditions, and biomass growth, as well as biogas bubbling.

The effect of the L/G ratio on the performance of the biogas upgrading system was evaluated without repetition. It was justified since the assays were performed at a diurnal period from 10:00 to 13:00 h (it would induce stable solar irradiation and outdoor temperature); therefore, it induced nearly optimal growing conditions for photosynthetic microorganisms, then pH could be assumed to be the most influential parameter on the CO₂ absorption²² where also very little standard deviation lower than 2% was reported for the assays assessing the effect of L/G ratio on the CO₂ absorption removal efficiency.

The reactors are cultivated on the outside, which means that, even if the inoculum was a pure culture of Arthrospira maxima algae, the probability of contamination with other organisms that can survive in the harsh pH conditions within the culture is high. Such is the case for sulfur-oxidizing bacteria^23,24. However, this contamination proves to be beneficial for the final purpose of the experiment since these bacteria help remove the H₂S from the biogas, essentially taking charge of this task and aiding in the quality of the resulting biomethane.

Under the environmental and ionic strength conditions prevailing during the system operation, the dissolved H₂S was being oxidized to polysulfides and thiosulfate by oxic-abiotic reactions, where, after some days, it should be completely oxidized to sulfate²⁵. The H₂S removal by precipitation with cations in the aqueous nutritive medium is insignificant due to the insufficient amount of cations fed to the system compared with the H₂S loading rate (reaching Cations/H₂S molar ratios much lower than 2). The absence of precipitates was confirmed by our visual inspection during the performance of the biogas upgrading process. The biological sulfide oxidation was not verified at this moment since the system is open to the environment.

System conditions
Dissolved oxygen (DO) and pH variations were measured in both light and dark conditions. During the day (light conditions), DO increased due to the photosynthetic production of oxygen by the microalgae, while at night (dark conditions), it decreased both due to lack of photosynthesis and because of heterotrophic metabolism, which utilizes respiration (Figure 8).

Levels of pH also varied with the presence of CO₂ within the liquid (Figure 8), increasing in value when less CO₂ was dissolved and decreasing when less CO₂ was removed; notably, there are smaller peaks around the times when no more CO₂ was being provided, which will be discussed further on. During the mornings, pH hit its peak at around 11:00 AM and the lowest values at around 18:00 PM, which is also consistent with algae photosynthetic activity. It is important to bring attention to the major drop around day 2; the short exploratory test using the L/G of 1.64 was performed on the 29^th of September, supplying continuous biogas by around 24 h (at around day 1) and it provoked a massive destabilization in the system, requiring the supply of urea to aid in the nitrogen recovery. The other short exploratory test using 1.58 was performed on the 5^th of October (at around day 7), but at better system conditions (biogas supply during daylight period), which is why the pH only strayed slightly from the regular peaks for two days before returning to normal behavior.

The smaller peaks in pH in Figure 8 can be attributed to a period of self-regulation of the algae to the environment while changing from photosynthesis to respiration.

Referring to the short exploratory tests to relate pH and L/G with CO₂ removal percentages (Figure 9), we tested two ratios, 1.64 and 1.58, as was mentioned previously. These are both averages from the recorded L/Gs during the experiments. Two distinct behaviors can be noted, where the removal percentage and the pH at a ratio of 1.58 were remarkably less stable and much lower than the ones recorded for the ratio of 1.64.

This is supported in the biogas upgrading performed by Bahr et al.¹⁵, through the use of an HRAP-column system with a species of Arthrospira maxima algae. Bahr assessed the removal efficiencies of CO₂ at different pH conditions and media liquid flow rates, as well as the removal of H₂S and O₂ contamination, on several synthetic gas compositions ranging from simply CO₂-N₂ to biogas compositions with varying H₂S concentrations (up to 0.5%vol). They concluded that at higher pH values (ranging 9-10) and higher culture media liquid flow rate (80 mL/min), the CO₂ removal percentages were close to 100% but suffered higher O₂ contamination, while at higher pH values (ranging 9-10), and lower culture media liquid flow rate (20 mL/min), the CO₂ removal percentages remained close to 100% and much less O₂ contamination was observed. They also reported full H₂S removal in these conditions.

Similarly, DO oscillation (Figure 8) can be attributed to the photosynthetic activity of the algae since, during the day, DO increased due to the photosynthetic production of oxygen by the microalgae, while at night, it decreased both due to lack of photosynthesis and because of heterotrophic metabolism, which utilizes respiration.

The temperature in the HRAP photobioreactor (RT4) varied due to the time of day and autumn weather, peaking most days between 23 °C and 28 °C at around 17:00 and hitting the lowest values between 11 °C and 15 °C at around 6:00 (Figure 10). The temperature at the inlet and outlet of the absorption tank was occasionally measured, resulting in an average temperature of 30.1 °C and 32.5 °C, respectively. Therefore, the water content (vapor) after treatment shall be slightly higher (13.5%) than before biogas treatment, assuming that in both cases, moisture in biogas achieved saturation. It is highly recommended to install a biogas dryer for optimal management and further use of purified biogas.

The average L/G that was intended for the period between the 28^th of September and the 10^th of October was 1.6 since the short tests suggested that this ratio would promote better results; however, it was not possible to maintain it during the nights due to the excessive acidification of the microalgae culture caused by a poor pH buffering capacity of the aqueous culture media. Therefore, only during daylight hours, biogas was fed to the absorption tank, adjusting the L/G values to around 1.5.

Biomass productivity
The inoculation on RT3 was performed on the 20^th of May 2020 and on RT4 on the 27^th of May 2020; the time in between the tests (September) and the inoculation served to stabilize the culture and solve operational issues that arose, such as plagues and malfunctions in the system, considering the COVID global pandemic.

Biomass growth was measured in two ways: sampling and harvesting. For the purposes of this article, sampling refers to the concentration of biomass at any given time in the reactor, while harvesting refers to the production efficiency of the biomass, meaning the amount of biomass that was recovered during the process to avoid growth inhibition. The testing was done from the 29th of September to the 9th of October, at an average L/G of 1.5, even though a ratio of 1.6 was preferred; the reason for it resulting lower was due to the 1.15 ratio recorded around day 11.

Sampling (Figure 11) was done regularly from day 1 to day 11 (from the 29^th of September to the 9^th of October), where the growth trend in both reactors was very similar: it started with a higher concentration, hitting the lowest value for the experiment at days 4 and 5, steadily recovering in RT4 and with some variation in RT3, finally dropping again. The very same behavior is seen in Harvesting, which then suggests that an event (most likely an outside factor) affected the growth of both cultures simultaneously.

Harvesting (Figure 12) was done semi-regularly, alternating one harvest for RT3 and the next harvest for RT4. However, the scale must be considered; in both sampling and harvesting, the variation between numbers is very low, indicating that the event that affected both reactors was not critical. The red dotted line in Figure 8 denotes the period of time when the reactors were not harvested; this was due to two factors: a few days were during the weekend, when, unfortunately, the reactors were not accessible for sampling or harvesting (which can also be corroborated in Figure 11), and the methodology calls for harvesting of the reactor that has the highest concentration. In the complex, there were four reactors, of which only two (RT3 and RT4) participated in this study, making the days after the weekend, days when the other two reactors (RT1 and RT2) were harvested by the team and resulting in no harvesting data from RT3 and RT4. The harvesting data was around 50% less than the sampling data; this could be because the methodology's efficiency is lower.

The variation between values each day was small (Figure 11), which alludes to a resilient culture that allows for change in system conditions and remains stable. Arthrospira maxima preferentially grows in highly carbonated media at high pH and is highly sensitive to NH₃ inhibition¹⁵, which is consistent with the results shown in Figure 8. The calibration performed in August 2020 is shown in Figure 13.

Post-production review and byproducts
In order to review the potential of this gas to reduce harmful emissions to the environment, a full report by an outside company was performed, where the findings stated that the biomethane produced with this technology reduced the total direct CO₂ emissions by 84%, compared to using the unpurified biogas directly from the anaerobic digester. Additionally, when taken through a life cycle analysis of electricity generated by both the raw biogas and the purified biomethane, the overall heat capacity that the biomethane was able to provide was 23,000 kJ higher than the heat capacity of the raw biogas.

Finally, a byproduct of this purification process is the harvested microalgae, which, once dry, has a myriad of applications in other industries, which could add more value to the method and make the process cost-effective²⁶. For instance, a study was performed on basil crops to evaluate parameters such as number of leaves, shoot fresh and dry weight, and leaf fresh weight when using dried Scenedesmus biomass versus a regular inorganic fertilizer; they found comparable results in these criteria in both biomass and fertilizer²⁷. Similar results were found in another study where they compared the growth of four commercial crop plants while using different concentrations of a fertilizer made of algal biomass suspended in water; even at low concentrations (20%) of the fertilizer, the crops reached maximum growth, comparably with chemical fertilizers²⁸.

Figure 1: Visual representation of the biological process happening in biogas purification using microalgae Please click here to view a larger version of this figure.

Figure 2: P&ID diagram for the system described in the protocol. Please click here to view a larger version of this figure.

Figure 3: Photograph of HRAPs that were used during experimentation. Please click here to view a larger version of this figure.

Figure 4: Absorption tank. (A) Photographs of culture medium and biogas inlets to Absorption tank. (B) Front and back view of Absorption tank. Please click here to view a larger version of this figure.

Figure 5: Short exploratory tests in RT3 to determine L/G efficiency. Dark green corresponds to CH₄, green corresponds to CO₂, light pink corresponds to O₂, and dark pink corresponds to N₂. Average pH 9.2435; Liquid inlet 60-100 L/min; Gas inlet 50-120 L/min. Please click here to view a larger version of this figure.

Figure 6: Short exploratory tests in RT4 to determine L/G efficiency. Dark pink corresponds to N₂, light pink corresponds to O₂, dark green corresponds to CO₂, and light green corresponds to CH₄. Average pH 9.95; Liquid inlet 116-118 L/min; Gas inlet 35-75 L/min. Please click here to view a larger version of this figure.

Figure 7: Comparison of all removal percentages for H₂S in each L/G during the short exploratory tests. The L/Gs of 0.5, 1, 1.5, and 2 correspond to RT3, and 1.6, 2.5, 3.3, and 3.4 to RT4. Please click here to view a larger version of this figure.

Figure 8: pH and DO profile. pH (dark green) and DO (light green) profile for RT4 between the 28^th of September and the 10^th of October 2020. Liquid inlet 75-118 L/min; Gas inlet 57-75 L/min. Average feed concentrations for each gas: CH₄– 60%vol, H₂S – 2400 ppmv, CO₂– 34%vol, O₂– 0.6%vol. Please click here to view a larger version of this figure.

Figure 9: Removal percentage profiles for CO₂ depending on pH levels and L/G. Green corresponds to the CO₂ removal percentages at the L/G ratios: 1.58 (dark green triangles) and 1.64 (light green circles). Pink corresponds to the pH values at the L/G ratios: 1.58 (dark pink triangles) and 1.64 (light pink circles). Liquid inlet 75-118 L/min; Gas inlet 57-75 L/min. Average feed concentrations for each gas: CH₄– 60%vol, H₂S – 2400 ppmv, CO₂– 34%vol, O₂– 0.6%vol. Please click here to view a larger version of this figure.

Figure 10: Temperature profile for RT4 between the 28^th of September and the 10^th of October 2020. Please click here to view a larger version of this figure.

Figure 11: Sampling results for RT4 (light green squares) and RT3 (dark green circles) between the 28^th of September and the 10^th of October 2020. The L/G ratios are indicated with arrows. Please click here to view a larger version of this figure.

Figure 12: Harvesting results for RT4 (light green squares) and RT3 (dark green circles) between the 28^th of September and the 10^th of October 2020. The L/G ratios are indicated with arrows. In red dotted lines, the period where there was no harvest for either reactor is shown. Please click here to view a larger version of this figure.

Figure 13: The calibration curve performed in August 2020, correlating the concentration of the algal culture in grams per liter to the absorbance at 750 nm. Please click here to view a larger version of this figure.

Table 1: Comparative compositions of biogas