A break in air supply causes a decrease in the number of PAOs colonies because PAOs divide under aerobic conditions

Materials and Methods

The six bioreactors were filled 1300 ml of activated sludge from Chrzanów municipal WWTP (51 286 people equivalent) localized in Małopolska voivodship, Poland. The Chrzanów WWTP is effectively and stable working plant in MLE technology designed to eliminate N and P compounds.

Bioreactors worked in an eight-hour cycle divided into a four phases (chapter II see pages 30−31).

The activated sludge was acclimated to the artificial sewage and working cycle for a 20 days.

The acclimatization time was extended to verify the differences in the composition of the protozoan communities present in the six bioreactors working in the same technological regime. During acclimatization period in one of the experimental bioreactors failure of the peristaltic pump occurred. In consequence of wrongly added by the pomp volume of NaOH the level of pH raised up to 11. For that reason, before oxygen switch off simulation, activated sludge from all control and treatment bioreactors were mixed together and distributed once again to all bioreactors which was working already for one week.

After acclimatization period, in the three treatment bioreactors aeration and agitation was switched off for 24 hours. During this time the artificial sewage was added and supernatant removed according to the pre-determined technological procedure. Others three control bioreactors worked according to the original assumptions. To assess the density of protozoa, metazoan, AOB, NOB and PAO, samples of activated sludge were collected once a week during the acclimatization period, immediately before switching mixing and aeration off and promptly after switching on. Additionally, two series of the samples were collected once a week after restarting the aeration and mixing.

At least once a week sludge sample from each bioreactor was collected and two replicates were analyzed for estimation protozoa and metazoa individual’s density. Protists (ciliates, flagellates and amoebas) and metazoans (rotifers and nematodes) were counted under microscopes and were identified using the professional keys (see chapter II page 32).

To detect and estimate the density of nitrifying bacteria (AOB and NOB) and PAO, fluorescence in situ hybridization (FISH) technique was used (see chapter II page 32).

Chemical analysis of effluent parameters: COD, Ntotal, Ptotal, NH4-N were carried out by Biospekt Sp. z o.o. company based on the guidelines of the accredited laboratory of MPWiK Kraków.

75 The Sludge Biotic Index (SBI) was calculated according to Madoni (1994). The Shannon-Wiener biodiversity index (H’) was calculated according to Krebs (1999).

Results

At the beginning of experiment in all bioreactors attached ciliates from genus Vorticella, Epistylis and Opercularia dominated. During acclimatization time protozoa community was clearly rebuild – ciliates density decreased and at the same time density of naked and testacea amoebas increased. On 20^th day, just before oxygen shortage in all bioreactors dominated testacea, mainly Euglypha sp. and Cochlipodium sp. and the second most numerous group was naked amoebas. Density of flagellates was increasing steadily over time in both control and treatment bioreactors and starting from 22^nd day flagellates markedly dominated in all bioreactors. In contrary, whole density of the other protozoa and metazoa species after two weeks from starting point of experiment was decreasing steadily over time (Figure 5.1).

76 Figure 5.1. Microorganism community composition over time in control (A) and treatment (B) bioreactors. Red arrow indicates moment of oxygen shortage. Bars and points are mean values for microorganism category in experimental groups.

The applied Priciple Response Curves statistic (PRC) did not show statistically significant differences between protozoa community in experimental bioreactors.

Moreover, according to Monte Carlo permutation test the effects of oxygen shortage on protozoa and metazoan community including its interaction with time were not significant, (F = 0.9, p = 0.972) (Figure 5.2).

77 Figure 5.2. Principal response curves (PRC) with species weights for the protozoa and metazoa community data set, indicating the effects of oxygen shortage. Diagram showed 10 best fitted protozoa and metazoa representatives. Numbers on x axis mean days after oxygen shortage. Holophry – H.

discolor, AIcur – A. incurvata, CystHolo – Holophrya cysts, Bdelloid – Bdelloidea rotifers, Large naked – naked amoebas >50µm, Monogono – Monogononta rotifers, Tritigms – Thrithigmostoma sp., Acineria – A. uncinata, NonInd – non identified ciliates species, VortcSp – Vorticella sp.

Based on this results the first hypothesis: The shortage in air supply significantly rebuilds the protozoa community in activated sludge, had to be rejected.

Moreover, during experiment significant differences in Shannon-Wiener biodiversity index (H’) in protozoa communities were not observed from treatment and control bioreactors (Table 5.1 and 5.2).

The value of biodiversity index decreased visible over time in all bioreactors (Table 5.1 and 5.2).

Table 5.1. Protozoa community Shannon-Wiener index (H’) in both experimental and control bioreactors over time. Mean ± SD. Days 1 and 20 were before switching off the aeration.

Experimental day

Group 1 20 22 36

Control 2.27 ± 0.11 2.07 ± 0.06 1.98 ± 0.19 1.31 ± 0.29 Treatment 2.31 ± 0.07 2.14 ± 0.10 1.97 ± 0.21 1.17 ± 0.01

78 Table 5.2. Repeated measures ANOVA results of Shannon-Wiener index in experimental groups over time.

Effect SS df MS F p

Intercept 86.71 1 86.71 446.57 <0.0001

Group 0.001 1 0.001 0.047 0.84

Residuals 0.05 4 0.014

Time 3.84 3 1.28 46.39 <0.0001

Time*Group 0.04 3 0.01 0.45 0.72

Residuals 0.33 12 0.03

The Sludge Biotic Index (SBI) values also decreased over time from 10 to 8 in all bioreactors but activated sludge still were classified to the first quality class characterized as a very well colonized and stable sludge with excellent biological activity and very good performance (Madoni 1994).

The statistically lower rate of reduction of total nitrogen and total phosphorus were noted in oxygen-deficient bioreactors after 24 hours when aeration and mixing was stopped (Figure 5.3 A, B and Table 5.3). Simultaneously in none bioreactors level of reduction of measured parameters decrease below 90%

(Figure 5.2). Differences between COD and NH4-N reduction were not observed between control and treatment bioreactors (Figure 5.2 C, D and Table 5.3 and 5.4). Because assumption of homogeneity of variance for NH4-N reduction data was not met, non-parametric Mann-Whitney U test was used.

79 Figure 5.3. Mean (± SD) value of reduction rate of Ntotal (A), Ptotal (B), COD (C), NH4-N (D) in control and treatment bioreactors all over the time. Mean ± SD.

80 Table 5.3. T- test results of differences in Ntotal, Ptotal and COD reduction between control and treatment bioreactors.

Parameter t df p

Ntotal 3.39 4 0.028

Ptotal 3.89 4 0.018

COD 0.60 4 0.581

Table 5.4. Mann-Whitney U test results of differences in NH4-N reduction between experimental groups.

Parameter Z p

NH4-N 1.75 0.081

On the other hand, the abundance of nitrifying bacteria AOB and NOB as well as polyphosphates accumulating organisms (PAOs) did not differ between treatment and control group (Table 5.4 and 5.5).

Glycogen accumulating organisms (GAOs) has not been detected in analyzed samples of activated sludge.

Obtained results made it possible to reject the second and third hypotheses that oxygen shortage will have the negative impact on the AOB, NOB and PAOs colonies.

Table 5.4. Nitrifying bacteria, PAOs and GAOs abundance in control and treatment bioreactors 24 hours after aeration and mixing stopped. Mean ± SD.

Group AOB NOB PAO GAO

Control 2.1 ± 0.3 1.2 ± 0.4 3.2 ± 0.4 - Treatment 1.8 ± 0.6 1.0 ± 0.4 3.2 ± 0.3 -

Table 5.5. T- test results of AOB and NOB bacteria abundance in experimental groups.

Parameter t df p

AOB 0.75 4 0.50

NOB 0.57 4 0.60

81 Discussion

Protozoa

Oxygen concentration and conditions of the aeration play a crucial role in development of activated sludge protozoa communities (Dubber & Gray 2011b). Additionally, ciliated protozoa inhabiting activated sludge reveal different tolerances to anoxic and anaerobic conditions (Dubber &

Gray 2011b). Results from our experiment suggest, that protozoa community acclimatized to the set of cyclic aerobic/anaerobic conditions during SBR working scheme were quite resistant to relatively short lasting 24 hours oxygen deficiency. Parameters of SBR phases and length of start-up period are crucial for protozoa community development but on the other hand we have to take into consideration the fact that protozoa and metazoa species abundance considerably fluctuated during operation of laboratory scale SBR and steady–state was not observed.

The results of the study show that ciliates and generally protozoa are not good bioindicators of dissolved oxygen conditions in activated sludge reactors. Also in literature exist many not repetitive and inconsistent results about correlation between protozoa species and DO concentration (Table 5.6).

According to the data set only Opercularia spp., Vorticella microstoma and testacea amoebas seem to be reliable bio-indicators of oxygen concentration in activated sludge. But in our study Opercularia spp.

and testacea amoebas were observed in high density in bioreactors even with oxygen shortage.

82 Table 5.6. Value of correlation coefficients between ciliated protozoa species and dissolved oxygen concentration and between functional groups of protozoa and dissolved oxygen concentration in activated sludge investigated by others authors.

Species r Reference

-0.82 Dubber 2011a Aspidisca cicada 0.79 dos Santos 2014

-0.16 Lee 2004 Aspidisca lynceus -0.68 Dubber 2011a Acineria uncinata 0.03 Dubber 2011a

-0.01 Lee 2004 Tritigmostoma cucullulus 0.08 Dubber 2011

0.12 Lee 2004

Opercularia -0.26 Lee 2004

-0.15 Esteban 1991 -0.23 dos Santos 2014 -0.75 Madoni 2011 Vorticella convallaria -0.18 Dubber 2011a

-0.15 Lee 2004 Attached ciliates 0.31 Hu 2012

0.34 Madoni 2011 0.62 Madoni 2011 Crawling ciliates 0.62 dos Santos 2014

0.19 Hu 2012 0.28 Hu 2012 -0.65 Madoni 2011 Swimming ciliates 0.04 dos Santos 2014

0.06 Hu 2012 0.5 Hu 2012 0.65 Madoni 2011

Flagellates 0.24 dos Santos 2014

-0.63 Hu 2012 -0.14 Hu 2012

0.73 Madoni 2011 -0.11 Lee 2004 Testate amoebae 0.43 dos Santos 2014

-0.16 Hu 2012

0.22 Hu 2012

p < 0.05 p < 0.01 p < 0.001

The Sludge Biotic Index seems to be not a good tool to assess DO concentration in activated sludge. We obtained similar Sludge Biotic Index values equal 8 which respond to sludge class I) for

83 control and treatment bioreactors as Rodriguez-Perez and Fermoso (2016) where final SBI values equal 9 (sludge class I) in the three anoxic time exposures studied were similar to oxic control conditions.

Drzewicki and Kulikowska (2011) paid attention to limitation of SBI method and pointed out that shock load discharged periodically from septic tanks did not reduce the value of the SBI below 8. Moreover, in their study the SBI values were fluctuating between 8 and 10 during whole analyzed period and were not good associated with effluent quality.

Additionally, in our study flagellates never reached the density of more than 100 individuals along the diagonal in the Fuchs-Rosenthal chamber but after calculations it turned out that they were the dominant group of protozoa in investigated bioreactors. Although flagellates dominated in bioreactors, effluents had good quality. This showed, that calculation based on counting along the diagonal in the Fuchs-Rosenthal chamber sometimes is not appropriate to specified key group in SBI method.

It is very hard to determine exact species or ecological protozoa group which could be treat like general DO concentration indicator. The best way seems to be work out an indicator species for each treatment plant separately based on regular and systematic microscopic observation combined with on-line DO probes reading.

Lack of differences between treatments can be explained by too short duration of oxygen shortage. The studied activated sludge "worked" in an 8-hour cycle, divided into a two hours anaerobic phase, five hours aerobic phase and one hour of sedimentation phase during which the oxygen concentration also decreased. The microorganism community of the tested sludge was periodically exposure to anaerobic conditions for more than 6 hours a day. Before planned break, microbial community acclimatized to bioreactors working scheme during 57 cycles (3 cycles/day * 19 days).

Probably the time preceding the planned break in the supply of air was enough long to ensure that the protozoa and bacteria community in studied activated sludge were well acclimatized to repeated low oxygen levels. In addition, the activated sludge sample used in experiment was taken from treatment plant adapted for effective biological nutrient removal. In this process bioreactors configuration favors the selection of microorganisms with were high resistant to alternating aerobic and anaerobic conditions.

Rodrigues and co-workers (2001) claimed that cyclic but short absence of oxygen (cyclic aerobic–

anoxic conditions) was not detrimental to the protozoan population. Moreover, Rodrigues and co-workers (2001) paid attention to fact that protozoa community observations could be valuable tool for evaluating SBR bioreactor working status only under long reaction times within each cycle. Within short time frames, changes in protozoa community could not be evident. This opinion is partly consistent with observation of Rodriguez-Perez and Fermoso (2016) of high influence of anoxic time exposures in sequence 12/12 hours and 6/6 hours (oxic/anoxic) on crawling ciliate (Euplotes spp., Chilodonella spp.) and carnivorous ciliate (Tokophrya spp.). The authors suggested simplification of the microbiological population in this conditions based on Shannon biodiversity index which decreased in this anoxic time exposures in comparison to control with constant oxic conditions. Interestingly Shannon biodiversity

84 index in the anoxic time exposure 4/4 was similar to control. Similarly, in our study Shannon biodiversity index did not change significantly in bioreactors exposure to oxygen shortage in comparison to control bioreactors. Shannon biodiversity index decrease in all bioreactors with time and this fact could be explained by the fact that artificial sewage instead of raw sewage was used and that laboratory SBRs were better prevented from microorganisms invasion from surrounding environment than full scale facilities. Rodriguez-Perez and Fermoso (2016) showed also that cycle 6/6 with four periods: two anoxic periods of 6 hours and two oxic periods of 6 hours reduced in the highest rate both protozoa density and abundance of filamentous bacteria in comparison to the constant 24 hours oxic control conditions. Babko and co-workers (2014) noticed distinct ciliates density decrease after 48 hours in bioreactors without air supply, but protozoa species structure was stable during four days after air switch off.

Rodrigues and co-workers (2001) observed that free-swimming ciliates (Uronema sp., Colpidium sp.) and flagellates (Peranema sp.) replaced crawling (Aspidisca sp., Chilodonella sp.) and attached ciliates (Vorticella sp., Epistylis sp., Vaginicola sp.) during cyclic aerobic-anoxic phases in laboratory bioreactor. During over 100 days considerably fluctuation in protozoa and metazoan community and no steady–state in laboratory SBR was also noticed by Cybis and Horan (1997). On the other hand, Cybis and Horan (1997) claimed that cyclical variations in protozoa species composition and individual’s density were valuable as indicators of SBR performance and effluent quality.

In our study protozoa community in different SBR phases in the same cycle was not investigated because this task is very time consuming (more than 1 hour for 1 sample). Personally I am skeptical that it is possible to detect differences in protozoa community composition and density between SBR phases in the same cycle.

Rodrigues and co-authors (2001) and Babko and co-workers (2014) suggested that the cryptobiosis (encystment) might be the reason for the fluctuations in active protozoan numbers in unfavorable condition in activated sludge such like oxygen deficits. I inclined to opinion of Stout (1955) which after Thomas (1942) presented that Tetrahymena pyrifirmis, Colpoda cucullus, Colpoda inflata and Colpoda steinii were able to survive anaerobic conditions for several days. Additionally, Stout experiments (1955) showed that resting cyst formation by C. cucullus, C. inflata and C. steinii was independent of the oxygen tension. Depletion of food and desiccation are two mayor factors induced encystation (Gutiérrez et al. 2001). Moreover, during microscopic observation resting cysts were not observed and statistical analysis did not show significant differences in ciliates and protozoa density between control and treatment bioreactors.

Nitrifiers

No difference in AOB and NOB bacteria abundance between bioreactors with oxygen shortage and control bioreactors could be explained by the fact that some nitrifiers could survive under anaerobic conditions for example: in fish-pond sediments (Diab et al. 1992) and in the anaerobic hypolimnion of

85 wastewater reservoirs (Abeliovich 1987). Diab et al. (1992) suggested that nitrifying bacteria survive anaerobic conditions either by switching their metabolism to a very low rate or by switching from a nitrifying to a denitrifying activity. Nitrosomonas eutropha combined anaerobic ammonia oxidation with cell growth (Schmidt & Bock 1997). The AOB can start ammonia oxidation within very short time at high rates after substrate or oxygen depletion because they possess several enzymological and molecular mechanisms that allow them to maintain the metabolic state of their cells under non-favorable conditions (Geets et al. 2006).

Polyphosphate accumulating organisms

In our study experimental groups differ in effectiveness of enhanced biological phosphorous removal process (EBPR), but Ptotal reduction never decrease below 90%. Simultaneously experimental groups did not differ in abundance of PAOs and none of GAOs colonies was observed. Carvalheira and co-authors (2014) showed that PAOs have a much higher affinity for oxygen and clear kinetic advantage over GAOs at low DO levels. In our experiment mean DO level in aerobic phase was maintained at relatively low level - 1.1mgO2/l. The maximum specific aerobic metabolic rates of PHA consumption, P uptake and glycogen production for PAOs were relatively stable over a wide DO concentration range (0.6−8.0 mg O2/l), decreasing more considerably in the lower DO values (0.1−0.6 mg O2/l) (Carvalheira et al. 2014). On the other hand the results from Brdjanovic an co-workers (1998) experiments indicated that excessive aeration phase (more than 20 hours) in laboratory SBR cycle can cause deterioration in EBPR efficiency because phosphorus uptake was stopped.

Zeng et al. (2004) showed that simultaneous nitrification, denitrification and phosphorus removal (SNDPR) was possible in laboratory SBR with alternating anaerobic and low DO aerobic stages (0.5 mg O2/l). During this simulation no nitrite or nitrate was accumulated in the aerobic period but N2O rather than N2 was the major denitrification end-product. PAOs and GAOs were found to coexist in this particular process and phosphorus uptake was likely accomplished by PAOs utilizing oxygen as the electron acceptor for its PHA oxidation (Zeng et al. 2004).

Oxygen shortage was likely to affected the metabolic rate/efficiency of reduction of nitrogen and phosphorous compounds by the above-mentioned bacteria rather than their quantity.

Wilén and co-authors (2010) observed only small differences in activated sludge properties in full scale WWTP when DO set-point was changed every 3 weeks between 2 and 4 mg/l for a few months.

The settling properties were not influenced significantly whereas slight negative effects of reduced DO concentration on effluent quality could be seen. The microscopic investigation showed a tendency towards poorer sludge properties at a DO concentration of 2 mg/l compared to at 4 mg/l.

Based on mentioned above observation we could assume that activated sludge community has high resilience and is very resistant to fluctuations in DO concentration. Wilén and co-workers (2010) commented that activated sludge plants are very dynamic systems where many parameters change

86 simultaneously and it is very hard to assess effects of a certain parameters such as the DO concentration on sludge properties.

Contrary, in our study most variable parameters presented in full scale systems: flow rate, sludge load, temperature, sewage composition and pH were strictly controlled and visible differences in sludge properties and microorganisms community between experimental bioreactors were not detected. At the same time, we are aware of the potential impact of the scale and type of artificial sewage on the results of our experiment.

In my opinion the scale of experiment had a big impact on the level of observed changes in microbial community during oxygen shortage. Oxygen shortage could differently affect microbial community in laboratory bioreactors with 2.0 l working volume than in bigger bioreactors chambers and in full scale treatment plant bioreactors with thousands of cubic meters volume.

From physical and chemical point of view dissolved oxygen concentration in activated sludge bioreactors is issue of the gas-liquid mass transfer topic. The gas–liquid mass transfer strongly depends on hydrodynamic conditions in the bioreactors. The hydrodynamics conditions depend on the operational conditions, the physicochemical properties of the culture and the geometrical parameters of the bioreactor (e.g. size of bioreactor, design and number of stirrers) (Novak & Klekner, 1988, Garcia-Ochoa & Gomez 2009).

Researchers which used bioreactors with bigger working volume 6.0 l (Cybis & Horan 1997) and 8.0 l (Babko et al. 2014) noticed more pronounced difference in protozoa community composition between aerobic and anaerobic conditions than Rodrigues and co-authors (2001) which used bioreactors with smaller working volume (2.9 l and 1.7 l). In contrary Dubber and Gray (2011b) used bioreactors with similar working volume (3.4 l) and observed more evident differences in protozoa community between bioreactors worked in different aerobic/anaerobic conditions. Muszyński and co-authors (2013) suggest that operational strategies based on the results from laboratory scale bioreactors rarely works in full scale facilities.

Taking under consideration results of existing studies I have to conclude that the topic of experiments scale in laboratory SBR on activated sludge is still open and require new research in near future.

87

CHAPTER VI

The protozoa and metazoa community composition over one-year