Effect of crawling ciliates on the structure and morphology of active sludge flocs and occurrence of nitrifying bacteria (Experiment I)
Hypothesis 2: Increased density of A. cicada influences the structure and size of activated sludge flocs
Detailed hypothesis: Flocs under increased density of A. cicada are larger and more compact because bacteria under increased predatory pressure exhibits higher growth rates, which is reflected in the aggregate surface and size.
Alternative detailed hypothesis: With increased density of A. cicada the flocs are smaller because the ciliates predatory pressure is so strong that the bacteria does not keep up with the population quantity.
Growth rate of bacteria population is not sufficient to contradict ciliates predatory pressure and in consequence bacteria population decreases.
Materials and methods
The experiment was conducted in laboratory bioreactors according descriptions included in chapter II (see pages 30−33). Bioreactors was filled with 1300 ml of activated sludge from Skała municipal WWTP (6960 people equivalent) localized in Małopolska voivodship, Poland. The Skała WWTP was chosen because it was during start-up phase (in fourth month of start-up) did not have a well-formed microorganism community and did not properly reduce nitrogen and phosphorous compounds. The mean value of mixed liquor suspended solids (MLSS) at the beginning of experiment in each bioreactor was 2.16 ± 0.04 g/l (Mean ± SD). The average sludge load was 0.10 gCOD/(gMLSS)*day and 0.0012g NH4-N/(gMLSS)*day. Temperature was maintained on stable level 16oC. Solid retention time (SRT) was approximately 37 days, during whole time of experiments excess sludge was not removed.
Acclimatization of sludge in bioreactors lasted four days (11 cycles). After this time, dense culture of crawling ciliate Aspidisca cicada was introduced into the three randomly chosen bioreactors so that the final density was 140 individuals/ml. A. cicada ciliates were isolated from Niegoszowice WWTP (Małopolska voivodship, Poland) and cultured under conditions described by Sudo and Aiba (1972). To the next three control bioreactors the same volume of filtered through 5 μm nylon filter (Microporous Membrane, UK) medium from the ciliated culture was added.
37 The experiment after adding ciliates lasted for five weeks. At that time, twice a week samples to evaluate the density of protozoa were taken. For measurement of mean flocs area, minimum 30 photos of flocs for each bioreactor were taken. Another sludge subsamples were fixed with 99.8% pure ethanol (POCH, Gliwice, Poland) (1:1 vol./vol.) for estimating the concentration of nitrifying bacteria by fluorescent in situ hybridization (FISH) method (see chapter II, page 32).
Once a week, the concentration of nitrogen compounds in effluent from three cycles (daily average sample) were analyzed. Chemical analysis of effluent parameters: COD, NH4-N were carried out by Biospekt Sp. z o.o. company based on the guidelines of the accredited laboratory of MPWiK Kraków.
Results
Aspidisca cicada density in treatment bioreactors increased until the 12th day of experiment and later sharply decreased (Figure 3.1). Individuals of A. cicada were also detected in control bioreactors in 5th day of experiment although any A. cicada cell was not detected at the beginning of experiment in activated sludge sample taken from treatment plant. This was probably effect of very low density of A.
cicada individuals or resting cyst, from which in advantageous conditions in laboratory bioreactors ciliates came out.
Figure 3.1. Aspidisca cicada density in experimental groups over time. Mean ± SD.
The nitrification efficiency measured as ammonia reduction, increased with time in all bioreactors (Figure 3.2).
38 Figure 3.2. Ammonia nitrogen reduction in experimental groups over time. Mean ± SD.
At the beginning in control bioreactors between 5th and 12th experimental day average ammonia reduction decrease from 54% to 36% but in 19th day reach 93% and to the end of experiment hold high level over 80%. In treatment group ammonia reduction increased consecutively from 29% in 5th to 93%
in 19th day. In last, 33rd experimental day ammonia reduction reached 83%.
Higher density of A. cicada also did not affect the abundance of ammonia oxidizing bacteria. In 5th day in bioreactors with added ciliates mean abundance of AOB bacteria was slightly higher than in control but this difference was not statistically significant. In 12th day AOB abundance was measured once again and the significant difference between experimental group were not detected. Also differences between 5th and 12th day in AOB abundance were not observed (Table 3.1 and 3.2).
Table 3.1. AOB abundance according to FISH procedure in experimental groups in two sampling days.
Mean ± SD.
Experimental day
Group 5th 12th
Control 1.96 ± 0.39 2.51 ± 0.23 Treatment 2.42 ± 0.81 2.49 ± 0.36
39 Table 3.2. Repeated measures ANOVA results of AOB abundance in experimental groups over time.
Effect SS df MS F p
Intercept 64.29 1 64.29 446.57 <0.0001
Group 0.23 1 0.23 1.62 0.27
Residuals 0.58 4 0.14
Time 0.29 1 0.29 0.83 0.15
Time*Group 0.17 1 0.17 0.48 0.53
Residuals 1.42 4 0.36
Addition of dense culture of A. cicada did not have a statistically significant effect on the size of activated sludge flocs. The flocs from the bioreactors with added ciliates and those from the control did not differ in median of the floc area (Table 3.3). Median floc area increased with time in both experimental group. In control group median floc area in 1st experimental day had 3773 µm2 and in last day increased to 24840 µm2. In treatment group median floc area in first day had 4383 µm2 and in last day increased to 21134 µm2 (Figure 3.4). Experimental groups also did not differ in growth of flocs area (F(1,4) = 0.96, p = 0.38) (Table 3.4).
Table 3.3. Repeated measures of ANOVA, flocs area in experimental groups with time.
Effect SS df MS F p
Intercept 2.95*105 1 2.95*109 993.73 < 0.0001 Group 7.15*106 1 7.15*106 2.41 0.20
Residuals 1.88*107 4 2.97*106
Time 1.53*109 5 3.07*108 64.95 < 0.0001 Time*Group 2.09*107 5 4.18*106 0.88 0.51
Residuals 9.47*107 20 4.74*106
Figure 3.3. Median floc area in experimental groups with time. Mean ± SD.
40 Table 3.4. Floc growth in experimental groups. Mean ± SD.
Group Floc growth [µm2] Control 21067 ± 6515 Treatment 16751 ± 3978
In both experimental groups the same crawling ciliates species but in different proportion was observed (Figure 3.4). In control bioreactors from 5th to 12th day visibly dominated Chilodonella sp. then from 16th to 19th day Acineria uncinata. Since 26th day dominated Trochilla minuta but in the next 6 days it disappeared completely. In treatment bioreactors Aspidisca cicada dominated from 5th to 16th day and after this period none of other crawling ciliates species achieved domination. The peak of maximum density of crawling ciliates was achieved earlier in treatment than in control bioreactors.
Figure 3.4. Density of crawling ciliates species in control bioreactors (A) and treatment bioreactors (B).
Bars represent mean value for experimental group.
We also observed clear predator – prey interaction in all treatment bioreactors (Figure 3.5). Predatory attached ciliates from Suctoria group effectively reduced prey - Aspidisca density (Figure 3.6).
41 Figure 3.5. Changes in Aspidica and Suctoria density over time in treatment bioreactors. Mean ± SD.
Figure 3.6. A. cicada captured (white arrow) by Suctoria sp.
Another interesting observation was noticed: in all bioreactors over time increased percentage of ammonia reduction and this change corresponded with a decreasing number of bacterivorous attached ciliates from genus Vorticella, Carchesium and Opercularia (Figure 3.7).
42 Figure 3.7. Changes in attached ciliates density and ammonia reduction efficiency over time in all bioreactors. Mean ± SD.
High fluctuations in density and changes in protozoa community composition were observed in control and treatment bioreactors (Figure 3.8). General trends seem to be very similar in both experimental groups. One of the most visible differences between experimental groups was mean higher density of flagellates in treatment bioreactors.
43 Figure 3.8. Changes in protozoa and metazoa community in control (A) and treatment (B) bioreactors over time. Bars and dots represent mean value for each group of microorganism.
44 Discussion
Lack of changes in floc size/(area) and AOB abundance could be explained by the fact that despite the addition of dense culture of A. cicada, ciliates pressure was still too weak to produce visible changes in nitrifying bacteria community. Another explanation could be too short period of time (11 days) when experimental groups differ in density of A. cicada and as consequence ciliates effect were not observed.
The short period of time when experimental groups differed in the density of A. cicada could be caused by two main factors: predator pressure and carrying capacity of activated sludge in laboratory scale bioreactors. The predatory ciliates species – Suctoria effectively reduced the density of A. cicada (Figure 3.5). In full scale activated sludge bioreactors, Suctoria probably could also control crawling ciliates population, and both group of ciliates are in continuous fluctuations. Fast development of large population of Suctoria in full-scale treatment plant was observed by Curds (1973b), these high density of predatory ciliates caused considerable decrease in bacterivorous ciliates population as a consequence of turbid effluent. When Suctoria disappeared, the bacterivorous ciliates reappeared, and effluent quality returned to normal (Curds 1973b).
The protozoa/ciliates community in laboratory bioreactors may had limited carrying capacity, considerably lower than in full scale bioreactors located in WWTP. This phenomenon could be explained on the basis of the ecological theory of island biogeography proposed by MacArthur and Wilson in 60’s in XXth century. According to Curtis et al. (2003) we could treat treatment plants like islands. Based on island biogeography theory carrying capacity is proportional to island area. Assuming larger bioreactors have higher carrying capacity.
Therefore, additional A. cicada individuals could not enter to the system. For the other hand added A.
cicada specimens could not find an adequate niche in the system and thus rapidly disappear either by wash-out or by predation similarly to many bacterial strains used for bioaugmentation (Daims et al.
2001).
Additionally, A. cicada influence could be also diminishing by other crawling ciliates species presented in activated sludge (Chilodonella, Trochilla, Acineria, Trithigmostoma, Cinetochilum). These species may have a similar effect on the floc structure and AOB abundance due to occupying a similar ecological niche (Figure 3.4). The graph clearly showed that treatment and control bioreactors were shifted in time relative to each other in terms of crawling ciliates density.
Visible lower density of flagellates in control bioreactors could be explained by domination of A.uncinata from 16th to 26th. According to Augustin et al. (1987) A. uncinata is capable to eat only small preys, mainly flagellates.
The results obtained in this experiment suggest that crawling ciliates Aspidisca cicada contrary to flagellates Adriamonas peritocrescens from Verhagen and Laanbroek (1992) research did not reduce the numbers of nitrifying bacteria. Our results seem to be consisted with Neubacher et al. (2008)
45 observation that ciliates had no influence on abundance and growth of nitrifying bacteria and nitrification. Similarly Winkler et al. (2012) used FISH method to analyze structure and composition of aerobic granules and flocculent sludge in pilot plant and full scale treatment plant and their observation suggests that protozoa were not selective and ingested all bacteria not only nitrifiers.
This fact could explain why in our experiment in all bioreactors percentage of ammonia reduction increased over time and this change corresponded with a decreasing number of bacterivorous attached ciliates. At the beginning probably most of the bacteria in bioreactors formed incompact flocs because activated sludge used in experiment came from WWTP in start-up phase. Mixing and aeration in bioreactors could teared off weakly connected bacteria from the flocs and they were easily consumed by dominated attached ciliates. Durability of the flocs increased over time because activated sludge process generally selects bacteria with good adhesive ability. As a consequence, attached ciliates lost their food base and their density decreased. At the same time nitrifying bacteria which survived inside the flocs slowly started to transform ammonia. Similar trend could be find in study conducted by Pajdak-Stós et al. (2010) during start-up period in full scale treatment plant. Also Winkler et al. (2012) noticed that Vorticella-like ciliates were actively grazing on GAOs, PAOs and nitrifiers bacteria, because found their individuals within ciliates vacuoles.
Taking into account this information we could suggest that the theory of reduction of nitrifying bacteria by protozoa grazing in activated sludge environment is possible especially during process start-up phase.
Our results were not clear-cut because in our experiment “true” control group were not existed.
Additionally, our estimation of AOB abundance in FISH method was semi-quantitative. This type of estimation is less precise than method with direct count proposed by (Nielsen et al. 2009).
The question whether protozoa can selectively impact population dynamics of nitrifying bacteria and other functional groups of bacteria in activated sludge is still open and additional work is needed to better understand this issue.
46