Currently, the increasing demand for control and process optimization in wastewater treatment plants (WWTPs) requires an advanced approach to the improvement of the system, e.g., by bioaugmentation. This method is defined as the introduction of a specific strain or a consortium of organisms to enhance the biological activity of a process factor [1
]. It has effectively been used in several environmental aspects such as bioremediation of contaminated soils and groundwater treatment [3
]. In WWTP, this method has been applied in both aerobic and anaerobic systems [7
]. Bioaugmentation has been used to increase the population of nitrifiers and increase the tolerance of microorganism against various negative factors such as pH fluctuations, toxic agents, temperature changes, and shock loading [10
]. Moreover, Van Limbergen et al. [16
] indicated that bioaugmentation could improve degradation of refractory compounds as well as flocculation, which in turn affects the parameters of activated sludge (AS) floc and their composition [17
]. It was also found that this method could support the start-up of new reactors [19
]. In anaerobic systems, such a technique is involved in improvement of the process stability and biogas yields [21
] as well as odor reduction [22
Various organisms can be used in the bioaugmentation process, e.g., autotrophs, heterotrophs, facultative anaerobes and aerobes [2
]. Archaea can also be applied for this purpose [24
]. These microorganisms are frequent constituents of AS [29
]. However, their contribution to the total biomass is usually inconsiderable and most frequently does not exceed 8% of the total number of bacterial cells [32
]. In classical bioreactors for removal of C, N, and P with AS, Archaea occur mainly due to the supply of supernatants formed during the sludge treatment process in fermentation chambers. Previous studies [25
] have shown that Archaea are involved in many biochemical processes and, therefore, they can be used for removal of nutrients from various types of industrial and municipal wastewater. An important fact in this context seems to be that the Archaea domain microorganisms have been reported to play an important role in ammonia removal from wastewater [29
]. They are useful in a removal of nitrogen compounds from wastewater both in the intermittent aeration system [24
] and in the system with alternating anaerobic, anoxic, and aerobic conditions [25
]. They also exert a beneficial effect on the stability of the process, ensuring its lower sensitivity to shock pollutant loading, and help reduce the organic carbon demand during the biological processes of removal of nitrogen compounds [28
One of the parameters that can be used for characterization of AS is the oxygen uptake rate (OUR). It describes the respiration rate, i.e., the amount of oxygen per unit volume utilized per time unit by the available microorganisms [37
]. OUR can be applied to control and optimize process conditions as well as identify potential instabilities of AS systems [38
]. Furthermore, OUR has also been used to determine microbial activity and viability [42
]. In AS stabilization, this measurement presents the degree of sludge stability. This parameter is related to the main biochemical processes of biomass growth/decay and substrate removal [45
]. The exogenous oxygen uptake rate characterize the activity of heterotrophs and assessment of easily biodegradable substrate in wastewater [46
]. However, the endogenous OUR measurement (absence of substrate in wastewater) indicates the consumption for bacterial growth–decay cycle, maintenance energy production and protozoa respiration [47
The aim of this study was to determine the effect of bioaugmentation with Archaea on AS expressed by the OUR parameter, during the wastewater treatment process in a sequencing batch reactor (SBR). Moreover, the influence of external substrate depletion and aerobic stabilization in bioaugmented and non-bioaugmented AS systems was investigated.
3. Results and Discussion
The results of the study are shown in Figure 2
and Figure 3
. The values of endogenous OUR (Figure 2
) indicate the presence of the five-day-long adaptation period (Stage I) for AS transferred from a full-scale bioreactor to the laboratory-scale SBRs, in both Bioreactor A and B. This relatively short duration was related to the fact that only the scale and type of the bioreactor were changed, as the AS was transferred from a large-scale flow system to the laboratory-scale batch one. All process parameters as well as the wastewater subjected to the treatment remained unchanged. The next easily distinguishable stage of the feed-on step involved stable operation of the SBR bioreactors in standard conditions for 25 days (Stage II in Figure 2
Inconsiderable changes in the average OURendo values were found during the Step I (feed-on step) in Bioreactor B. Regarding Bioreactor A, noticeable and statistically significant OURendo increases were observed in Stages I and II. This is caused by Archaea bioaugmentation, as bioreactor performance differs only in this aspect. It is also visible that the standard deviation of the OUR measurements was lower for Bioreactor A than for B at both stages.
In the Step II (feed-off step) can be distinguished Stage I (lasting 8 days), where the OURendo decline in SBR-A was substantially lower than in SBR-B in comparison with the feed-on step; Stage II (lasting 16 days), where the OURendo decreased in SBR-A (F test showed that variances at Stages I and II are different p = 0.029, Welche test applied for comparison of average values gave the results that they were statistically different p = 2 × 10−8) and increased slightly in SBR-B relative to the previous step (variances different with p = 0.002 and differences in average values are statistically insignificant p = 0.17); and Stage III (lasting six days), where the OURendo dropped significantly in both bioreactors in comparison to the previous step (for SBR-A test F showed no differences in variances p = 0.126 and T student test showed differences in average values p = 3.306, for SBR-B test F showed differences in variances p = 0.030 and Welche test showed differences in average values p = 2.911 × 10−5). In the feed-off step, the standard deviation of the OURendo measurements was lower for SBR-A only in Stage I and comparable to the level achieved for SBR-B in the other two stages.
While averaging the results of the feed-on and feed-off steps, it was found that, in the Step I (i.e., in substrate presence), the average endogenous oxygen uptake rate was 28.70 ± 2.75 mgO2
in bioaugmented SBR-A and 21.63 ± 0.9 mgO2
in non-bioaugmented SBR-B (Figure 2
). In turn, in the Step II (when the absence of substrate and continuous aeration conditions occurred), the average endogenous oxygen uptake rate was 12.73 ± 3.93 and 10.56 ± 4.23 mgO2
in previously bioaugmented and non-bioaugmented AS, respectively (Figure 2
Given the analysis of the OURendo, values in SBR-A and SBR-B were are not stable during the specified stages, which is reflected in the standard deviations of the results. The increase in the SBR-A of the OURendo value after the adaptation period in the bioaugmented system is considerable (for SBR-A, F test showed differences in variance p = 0.030 and Welche test shows differences in average results p = 8.809 × 10−13; for SBR-B, F test showed differences in variance p = 0.012 and Welche test showed differences in average results p = 0.003), which generally yields a higher standard deviation of the results calculated for the total feed-on step (28.70 ± 2.75 mgO2·dm−3·h−1) and may indicate lesser stability of the system. However, the upward trend in the changes and the analysis of the individual stages allows concluding that bioaugmentation exerts a positive effect on the respiratory activity of AS.
The results of OURexo
measurements (Figure 3
) indicate that, just as for OURendo
, there is a visible AS adaptation period in both laboratory-scale SBRs referred to as Stage I (five days), and a subsequent stable operation Stage II (25 days). The OURexo
values achieved in SBR-B changed substantially in the feed-on step and were considerably lower in Stage II (differences in variance p
= 0.015 and differences in average value p
= 5.206 × 10−10
). In SBR-A, they were also different in Stage I (differences in variance p
= 0.054 and differences in average value p
= 2.737 × 10−4
), but the change was not as drastic as in SBR-B. The standard deviation of the measurement results for SBR-A and SBR-B in both these stages were not different (for I p
= 0.22 and for II p
In the feed-off step, three distinct stages can be distinguished, similar to Figure 2
. These involve Stage I (lasting eight days), where the OURexo
in SBR-A declines to a level similar to that achieved for SBR- B; Stage II (lasting 16 days), where OURexo
drops in SBR-A (variance not different p
= 0.46 and different averages p
= 0.0002) and in SBR-B in comparison to Stage I (variance not different p
= 0.29 and different averages p
= 1.754 × 10−11
); and Stage III (lasting six days), where OURexo
in both bioreactors falls distinctly relative to Stage II, in SBR-A (variance not different p
= 0.24 and different averages p
= 7.842 × 10−14
) and in SBR-B (variance not different p
= 0.47 and different averages p
While averaging the results of the feed-on and feed-off steps, it was found that, in the first step (in substrate presence), the average exogenous oxygen uptake rate value was 95.55 ± 11.33 and 57.15 ± 24.56 mgO2
in bioaugmented and non-bioaugmented AS, respectively (Figure 3
For the second step (characterized by an absence of the substrate and continuous aeration), the average exogenous oxygen uptake rate was 29.74 ± 10.67 mgO2
in previously bioaugmented AS, while decreasing considerably to 19.82 ± 12.42 mgO2
in non-bioaugmented Bioreactor B (Figure 3
The analysis of the OURexo
values (Figure 3
) in both stages of the experiment allows a conclusion that, similar to the OURendo
results (Figure 2
), SBR-A was characterized by a greater stability of the individual stages of both steps, which is reflected in the standard deviations exhibiting lower values.
For both OURendo and OURexo, two basic stages can be distinguished in the feed-on step and three stages in the feed-off step. At the beginning of the feed-on step, there is a ca. 5-day long adaptation period for AS transferred from a large-scale bioreactor to a laboratory-scale SBR, and the rest of the time (25 days) is a stable operation period. For both OURendo and OURexo, this parameter in the feed-on step is higher for the bioaugmented bioreactor. However, when the adaptation step is finished, the OURexo clearly declines in both SBR-A and -B, which is particularly evident for the non-bioaugmented bioreactor, where it falls by 1/3. In the feed-off step, the OURendo and OURexo are always higher for the bioaugmented Bioreactor A. Interestingly, in Stage II of the feed-off step in Bioreactor B, the OURendo increases in comparison to Stage I, which is not the case in Bioreactor A.
Similarly, the standard error of the measurements is usually lower for the bioreactor with the bioaugmented AS. Regarding OURendo in the feed-on step, it is 0.5 for the bioaugmented SBR-A and 0.16 for the non-bioaugmented SBR-B, while in the feed-off step these values are higher: 0.72 and 0.77, respectively. When OURexo is considered, the standard error in the feed-on step reaches 2.07 and 4.48 for SBR-A and -B, respectively. In the feed-off step, lower values are found: 1.95 for SBR-A and 2.27 for SBR-B.
Generalization and averaging of the results for the two feed-on and feed-off steps allows concluding that the maximum respiration rate was observed in the feed-on step of the experiment, and a decrease in both OURexo and OURendo occurred during the feed-off step. In this case, the average endogenous oxygen uptake rate was 12.73 ± 3.93 mgO2·dm−3·h−1 in bioaugmented AS, and 10.56 ± 4.23 mgO2·dm−3·h−1 in non-bioaugmented one. In the case of the exogenous oxygen uptake rate, the average values were 29.74 ± 10.67 and 19.82 ± 12.42 mgO2·dm−3·h−1 in bioaugmented and non-bioaugmented AS, respectively.
As background for OUR measurement and supplementary tools for checking the stability of processes in bioreactors during the feed on step the effectiveness of wastewater treatment in bioaugmented and non-bioaugmented SBRs was observed. Higher changeability of effectiveness was noticed in non-bioaugmented one however no significant differences were observed between level of treatment effectiveness in both of bioreactors. In addition, higher changeability in effectiveness of treatment was observed at beginning of experiment in both bioreactors which reflects the adaptation phase of AS for laboratory condition.
According to Henze et al. [49
], the low values of the respiratory rate might be caused by oxygen stabilization of sludge. The typical OURexo
values for AS range from 30.0 to 100 mgO2
]. In the study by Puig et al. [51
] similar data was obtained for SBR, i.e., the exogenous respiration rate ranged from 35 to 110 mgO2
. The results presented in this paper are mostly consistent with those reported by others. However, the endogenous oxygen uptake rate exceeds the OURendo
values given by Avcioglu et al. [52
] that varied from 2.0 to 8.0 mgO2
This suggests that AS used for the experiment had a good quality due to the presence of many microorganisms in the flock assemblages as well as a sufficient substrate [49
results in aerobically stabilized sludge (i.e., in feed-off step) were comparable to these presented by Cokgor et al. [53
]. In their study, domestic sludge was aerobically digested at room temperature of 20 °C for 35 days. A maximum OUR value of ca. 40.0 mgO2
was observed at the beginning of aerobic digestion. Then, it decreased to 18.0 and 21.0 mgO2
after 17 and 30 days, respectively. Bernard and Gray [54
] investigated aerobically digested domestic sludge at a temperature of 16.5–20 °C; they also observed a significant reduction of specific oxygen uptake rate ranging from 65.8% to 93.1% (after 35 days) (SOUR was expressed as milligram of oxygen consumed per gram of volatile suspended solids (VSS) per hour and determined using equation SOUR = OUR/VSS).
Both endogenous and exogenous oxygen uptake rates were higher in the case of bioaugmented AS, which corresponded to the research carried out by Jun et al. [24
] (SOUR was investigated) and also mentioned by Fredriksson et al. [33
]. The authors suggested a symbiotic relationship between bacteria and Archaea. However, the difference between OUR values observed in the present study for non-bioaugmented and bioaugmented AS was higher in the case of AS (feed-on step) than in stabilized sludge (feed-off step). Based on these investigations, it can be supposed that the bioaugmentation-assisted process is considerably more stable, as evidenced by the lower standard deviation value in each particular stage of the feed-on and feed-off steps. Therefore, it can be assumed that Archaea have a stabilizing effect on AS and decrease its sensitivity to changes in the quality of supplied wastewater and to disruption of substrate supply. This supports the advisability of bioaugmentation of AS and confirms the findings concerning enhancement of bioreactor stability presented in the Introduction of this paper. On the other hand, the research indicates that Archaea-bioaugmented AS is characterized by higher activity (expressed by a higher OUR) at prolonged aeration and exhibits increased resistance to oxygen stabilization, which makes this type of stabilization less effective and therefore less cost-efficient.
Summarizing, it should be stressed that aspects of the novelty in the work is the description of the influence of bioaugmentation with Archaea on oxygen uptake rate in AS system in wide range of process stages (adaptation phase, stable operation and aerobic stabilization). Moreover, the study confirmed the increasing as well as stabilizing influence of Archaea addition on respiration activity of AS described by oxygen uptake rate.