Contribution of prokaryotes and eukaryotes to CO₂ emissions in the wastewater treatment process

Introduction

When considering carbon dioxide (CO₂) production in the wastewater treatment process, a distinction should be made between indirect and direct emissions (Massara et al., 2017). The energy consumption of devices working in the wastewater treatment plant (WWTP) causes the indirect emissions. All these devices require an electric power supply. Most WWTPs use electric motors for screen cleaning systems and transport of screenings and sand/gravel from grit chambers, sludge scrapers in primary and secondary settling tanks, drive of sludge treatment devices and above all, supplying system blowers for the aeration of oxygen zones within bioreactors (USEPA, 2013; Bao, Sun & Sun, 2015; Drewnowski et al., 2019). Direct emissions are caused by organisms found in the wastewater purification process. There are many groups of organisms that take part in this process, however, taking into account that popular technologies are based on activated sludge in the form of flocs, mainly prokaryotes and eukaryotes can be considered (Jaromin-Gleń et al., 2013; Kong et al., 2016). Wastewater treatment—regardless of the technology used – causes CO₂ emissions. However, the largest amounts of this gas are released into the atmosphere during the aerobic phase. This is connected with the respiration of both the prokaryotes and eukaryotes present in activated sludge during the decomposition of organic matter in the wastewater. During this process, prokaryotes are mainly involved in removing carbon (C) compounds and biogenic substances in dissolved forms, whereas eukaryotes remove bigger suspended particles—also not flocculated prokaryotes. Eukaryotes collect (take up) suspended particles, decompose them and incorporate them into their own biomass; in this way, eukaryotes cause removal, decomposition and transformation of pollution. Eukaryotes, in addition to removing suspended particles, actively (mostly by grazing) prey on living within and on flock prokaryotes (flocculated ones). Eukaryotes, therefore, exert ecological pressure on prokaryotes and stimulate them to grow, helping them to form flocks of proper size, shape and density parameters, indispensable for proper sedimentation. The above-mentioned ecological pressure and the removal of suspension are the main tasks of eukaryotes in a normally functioning activated sludge in the formation of flocks. During these biological activities, eukaryotes emit CO₂ (Čech, Hartman & Macek, 1994; Pajdak-Stós et al., 2010; Kong et al., 2016; Babko et al., 2017)_.

Sequencing batch reactors (SBRs) are not as popular as flow treatment plants, however, the lower investment cost of a single reactor and greater operational flexibility, makes SBR technology a reasonable alternative in every scale of WWTP (Shingh & Srivastava, 2011). They are often used for industry factories (for instance in food industry), housing estates or small towns (Fernandes et al., 2013; Quan & Gogina, 2019).

From a biochemical point of view, processes occurring at flow-mode and sequencing reactors with activated sludge are similar. The main difference is that in the flow system, the subsequent processes occur in a series of chambers and in SBRs aerobic, anoxic and anaerobic processes are carried out in one chamber. Therefore, the SBR wastewater treatment process has the following successive stages: (i) filling the chamber, (ii) mixing (the anoxic and anaerobic processes start at this stage), (iii) aeration (intensification of the aerobic processes), (iv) settling (sedimentation of activated sludge, when at this same time the aerobic, anoxic and sometimes anaerobic processes occur), (v) decantation and (vi) idle phase (smooth mixing to condition the biomass and sludge wasting). The consequence of different ways of wastewater treatment is different dynamics and amount of gas emissions.

While it is known that activated sludge microorganisms (prokaryotes and eukaryotes) carry out the decomposition of organic substances—and prokaryotes are responsible for most of this process—to the best of the authors’ knowledge, there is no research identifying how total emissions of CO₂ are divided, that is, estimating the parts emitted by prokaryotes and eukaryotes. The aim of this work is to determine the quantitative contribution in emissions of CO₂ by each group. The emission of CO₂ caused by prokaryotes and eukaryotes over the course of a year (taking into account seasonal differences) in a laboratory-scale model SBR is presented in this study. Use of a small scale enabled us to accurately investigate the CO₂ emission, because the space above the wastewater in a full-scale WWTP is open, which causes a problem with the representative and reproducible sampling of gases emitted from the reactor.

Materials and Methods

Results

All parameters measured, demonstrated that the process of wastewater purification was lasting and effective. pH was stable and was in the range of 7.5–8.5. DO was kept within the assumed scope that is, 2.0 and 2.5 mg O₂∙dm⁻³. Redox potential during the aeration phase was in the range of −150 mV to +200 mV. COD of purified wastewater was between 10 and 25 mg∙dm⁻³, while total suspending solids (TSS) were 0.5–5.0 mg∙dm⁻³ and turbidity was 1.0–4.0 NTU.

A total of 32 taxa of ciliates (10 crawling, 17 attached and five free-swimming), two taxa of testate amoebas, two taxa of naked amoebas, one taxon of flagellates, five taxa of rotifers and one taxon of nematodes were identified through composition analysis of the activated sludge. The greatest abundance of eukaryotic species occurred in the spring and summer period (25 species) and the smallest number was recorded in the autumn (19 species). However, the largest number of individuals was noted during the autumn period—5472 (SD = 419) indiv∙cm⁻³ and the smallest number was found in the spring—1,688 (SD = 40) indiv∙cm⁻³. Thus, the qualitative composition of eukaryotic organisms in experiments had statistically significant seasonal differences (p ≤ 0.05).

In order to assess the intensity of oxygen consumption and, accordingly, the emission of CO₂ by ciliated protozoa, data on the composition of the species and abundance of each species are necessary, since the respiration rate of these eukaryotic organisms is a function of their mass, which differs greatly among different species. Having this information and taking into account the temperature, on the basis of formula (2), we calculated the intensity of oxygen consumption and CO₂ emissions by this group of activated sludge. Assuming that CO₂ during the purification process is emitted as a result of biological processes and therefore, only by prokaryotes and eukaryotes, and knowing both the total amount of CO₂ emitted and the amount of CO₂ emitted by eukaryotes, we could evaluate the contribution of these two groups of organisms to the total production of CO₂. The seasonal changes are shown in (Fig. 1).

For eukaryotes, all seasons are characterized by significant differences. The season with the highest CO₂ emissions was summer, with about 0.7 × 10⁻⁵·g·m⁻²·min⁻¹, followed by autumn 0.3 × 10⁻⁵·g·m⁻²·min⁻¹, spring 0.2 × 10⁻⁵·g·m⁻²·min⁻¹ and winter 0.1 × 10⁻⁵·g·m⁻²·min⁻¹.

On the other hand, in the case of prokaryotes, there were no significant differences between seasons. The season with the highest emissions was autumn (1.3 g·m⁻²·min⁻¹), followed by summer (1.2 g·m⁻²·min⁻¹), spring (1.0 g·m⁻²·min⁻¹) and winter (0.93 g·m⁻²·min⁻¹). It should be noted that statistically significant differences in CO₂ emissions between the seasons were observed for the eukaryotes (p ≤ 0.05) but not for prokaryotes.

The contribution made by the eukaryotic organisms to the total CO₂ emissions during the aeration phase in subsequent seasons is shown in Fig. 2. The contribution to the total CO₂ emissions made by eukaryotic organisms during the aeration phase by season is shown in Fig. 2. The season with the highest contribution from eukaryotes was summer (20 °C) with almost 10 ppm. Next were autumn and spring (both 15 °C) with about 3.3 and 2.9 ppm respectively. In winter, the season with the lowest temperature (10 °C), the lowest contribution of CO₂ emissions by eukaryotes was recorded. This suggests that these differences can probably be best explained by temperature.

Discussion

It can be seen from Fig. 1A that CO₂ emissions produced by the prokaryotes differed by season, (although differences were not statistically significant). As expected, the highest level was recorded in the summer and autumn seasons. The required temperature for most pollutant removal processes is considered to be higher than 15 °C for activated sludge (Keefer, 1962; Krishna & Van Loosdrecht, 1999; Henze et al., 2006) ); thus higher temperatures in summer and autumn may have caused higher microbial activity. The highest activity in the autumn season can be likely explained by two factors: the abundance of species and individuals of species after the summer season and the higher concentrations of nutrients in wastewater, related to inhabitants’ return from holiday. Many large cities in Poland where schools and colleges are based, are characterized by clearly visible variability/difference in wastewater inflow—both the quantity and the load of biogens. Between the holiday season and the winter semester, in Lublin, where approximately 40% of the population are non-resident pupils and students, there is a sharp increase in the amount of wastewater at the end of the holidays and return to school (September and October)—a change of around 46,000 to around 72,000 m³∙d⁻¹. At the same time, such an increase in the amount of wastewater causes not only the inflow of a larger load of biogens—containing pollutants generated at that time—but also the flushing of sediment deposits, accumulating on the bottom of the gravitational sewer network during flows at a lower velocity. This velocity in holiday months (July and August) often decreases below the velocity of the hydraulic self-cleaning of channels in many places on the wastewater network. The only bigger and long lasting shift in quantity and quality of wastewater within the WWTP analysed, is noted during the summer in July and August (Jaromin, Szaja & Łagód, 2012).

In natural water bodies at temperatures below 10 °C, the intensity of biochemical processes decreases significantly, which is also manifested in a decrease in the formation of gases by microorganisms and other hydrobionts (Brylinsky & Mann, 1973; Sun et al., 2017).

In conditions of wastewater treatment facilities where the temperature even in winter does not drop below 10 °C and the inflow of organic substances (energy subsidies) does not stop, differences in CO₂ emissions between seasons were not statistically significant (p > 0.05), what is important in terms of their influence onto atmosphere composition.

Discussion of the results is based on the available data, which can only be compared in an indirect way. The authors are not familiar with any publications reporting data for laboratory-scale SBR, which consider emissions from prokaryotes and eukaryotes and differences by season. Therefore, only total CO₂ emissions were compared. Bao, Sun & Sun (2015) report that the annual average value of emissions from SBR chambers (in full-scale WWTPs) is about 2.8 g·m⁻²·min⁻¹, which is more than twice that from the laboratory-scale SBR (1.12 g·m⁻²·min⁻¹).

Additionally, our studies refer to the aerobic phase and therefore the results should be compared with the literature data for aerobic WWTP chambers. Yan, Li & Liu (2014) report the average CO₂ emission fluxes in the aerobic areas of the reversed A2O (anoxic/anaerobic/aerobic) process and A2O (anaerobic/anoxic/aerobic) process were 0.7 and 1.14 g·m⁻²·min⁻¹ respectively (both in the full-scale WWTP). This is similar to the results obtained by the authors for the laboratory-scale SBR. Furthermore, (Yan, Li & Liu, 2014) present the results for nine months throughout the year (from March to November) however, analysing their results in terms of seasonality, it is difficult to notice significant differences between subsequent seasons, similar to the presented in our work.

Significantly larger differences in CO₂ production (p ≤ 0.05) were observed in the case of the eukaryotic organisms (Fig. 1B). The results from this study thus indicate that eukaryotes reacted more strongly to temperature changes than prokaryotes. The temperature seemed to have had a substantially greater influence here, as CO₂ emissions in the summer were nearly three times greater than those in the autumn. The slightly higher emissions in the autumn (compared with the spring when the temperature was the same as during the autumn) can be explained in the same way as in the case of the prokaryotes—the favourable conditions prevailing during the Summer season and the larger load of pollutants in raw wastewater had an influence on the structure of the activated sludge in the autumn. As in the case of prokaryotes, the activity of the eukaryotes was lowest in the winter period.

As could be expected, the contribution of eukaryotic organisms to the total CO₂ emissions is very small—at a level of several ppm (Fig. 2). The main role of eukaryotic organisms in the process of wastewater purification, is not to remove dissolved organic matter (this is the main role of the prokaryote), that is, a process responsible for the majority of CO₂ emissions, as eukaryotes are predominantly bacteriophages. Eukaryotic organisms in activated sludge mainly have the function of regulators, maintaining bacterial communities in the phase of exponential growth and helping to form flocks with proper size, shape and density parameters (Čech, Hartman & Macek, 1994).

The results presented in this study should be treated as an estimation of the order of magnitude, rather than the exact evaluation, mainly because the investigations were carried out in the laboratory model. This process carried out on an industrial scale can be slightly different. It should be noted that scaling in SBR technology is a common problem in many investigations, as this kind of WWTP has a very wide range of applications: from a plant of several cubic meters in a small housing estate or a small production facility to the order of hundreds of thousands of cubic metres (Neczaj et al., 2008; Marañón et al., 2008; Gürtekin, 2014; Bao, Sun & Sun, 2016).

The uncertainty of our results should be also discussed. We found two major factors that could influence the results obtained: calculation of the eukaryotic organisms and estimation of CO₂ emissions, produced by individuals of the given species. The error in the calculation of individuals of the species can primarily be caused by an unrepresentative subsample. To prevent this, the samples were taken in different places and counting was repeated. Moreover, the same operator counted the eukaryotes throughout the experiment. To minimize the human factor, the operator was rested and the light was optimized.

As shown in the Methods chapter, the calculation of CO₂ emissions (caused by one individual) was based on the assumption that the amount of CO₂ emitted is equivalent to the amount of oxygen consumed, which is burdened with an error that is, difficult to estimate. Despite all these sources of uncertainty, emissions caused by eukaryotic organisms at a level a few or several dozen ppm can be expected.

Conclusions

To the best of our knowledge, the quantitative contribution of CO₂ production by different components of activated sludge in WWTPs, has not been estimated to date. For eukaryotes, the season with the highest CO₂ emissions was summer with about 0.7 × 10⁻⁵ g·m⁻²·min⁻¹ and the lowest was winter, 0.1 × 10⁻⁵ g·m⁻²·min⁻¹. In the case of prokaryotes, the differences between seasons were not significant. The contribution of eukaryotes to the total CO₂ emissions is of the order of several or more ppm. The exact value is impossible to estimate because of seasonal changes. The vast majority of CO₂ emissions is caused by prokaryotes. This is consistent with the fact that prokaryotes are responsible for wastewater purification from dissolved C compounds.

Supplemental Information