Abstract

Filamentous bacteria in addition to wastewater treatment are responsible for the shape of flocs and sedimentation properties of activated sludge. Their dynamics in activated sludge influences the performance of the whole sewage treatment plant. Therefore the composition of activated sludge biocenosis and its dynamics in the nitrification process were investigated. Four laboratory-scale activated sludge membrane bioreactors fed with wastewater highly concentrated with ammonium (synthetic wastewater imitating landfill leachate) were operated to obtain a high rate of nitrification. The sludge age was 8, 12, 24 and 32 days. An additional fifth reactor was conventionally ammonium loaded at 12-day sludge age and served as the reference. A shift in filamentous bacteria population was observed in all operated reactors. There was no influence of sludge age on composition or abundance of filamentous biocenosis. In high ammonium loaded activated sludge Nostocoida limicola, Haliscomenobacter hydrossis and also Type 021N were the most abundant filamentous bacteria. In the reference reactor Type 021N and Sphaerotilus natans dominated the activated sludge.

INTRODUCTION

Biological nutrient removal, covering nitrogen and phosphorus removal processes, is one of the main tasks in wastewater treatment. Among the biological nutrient removal processes nitrification seems to be the most vulnerable and troublesome process in operation (Grunditz & Dalhammar 2001). The problems appear especially when wastewater is characterized by high ammonia concentration, such as landfill leachate. In order to follow changes in bacteria which nitrify a high concentration of ammonia nitrogen, the dynamics of the activated sludge biocenosis was investigated. The results have been described by Raszka et al. (2011). In the mentioned work, the nitrifiers and higher organisms were the object of interest, but during the microscopic analysis of activated sludge we found the filamentous bacteria group and its dynamics worthy of attention.

Filamentous bacteria are mainly considered to be a cause of activated sludge bulking and foaming. Occurrence of such phenomena makes the gravity separation of solids very difficult. The main factors of the activated sludge process favoring the occurrence of filamentous bacteria are: low dissolved oxygen concentration, low food to microorganisms ratio, configuration of aeration chamber, source of organic compounds and a presence of pretreatment in anoxic or anaerobic conditions (Kampfer 1997). Most of the research focused on filamentous bacteria in membrane bioreactors concerns the membrane fouling (Meng et al. 2006; Li et al. 2008; Parada-Albarracín et al. 2011). Parada-Albarracín et al. (2011) found that the density of filamentous bacteria was higher in systems with sludge retention time (SRT) higher than 20 and 35 days. There is little knowledge about the influence of SRT on abundance and structure of filamentous biocenosis. Moreover, there is still a gap of knowledge concerning the characterization of filamentous bacteria, mainly due to the problems of cultivation and maintenance of cultures (Martins et al. 2004). The classical techniques of identification, based on microscopic analyses of fresh and stained samples, are applicable on the assumption that many environmental factors can change the morphological features of particular species (Snaidr et al. 2002; Nielsen et al. 2009). This gives more certainty that the culture conditions described refer to specific species.

The aim of this study was to present the dynamics of filamentous bacteria biocenosis during adaptation of activated sludge and treatment of highly concentrated ammonia wastewater in aerobic conditions. The SRT (or sludge age) was the main factor taken under consideration of filamentous biocenosis fluctuation. The results of filamentous bacteria abundance and type were compared with other data such as adaptation time or nutrient removal efficiency.

METHODS

Reactor system and operation

A nitrification process was performed in five laboratory-scale bioreactors with a submerged membrane module separating wastewater from activated sludge. Four reactors were operated with high ammonia concentrations and a low C:N ratio. The reactors differed mainly in sludge age. One reactor (named M12_ref) served as a reference, because its ammonia load was much lower than the other reactors and similar to that in municipal wastewater treatment plants (WWTPs). The reactors were fed continuously with a synthetic wastewater consisting of 2.4 g or 0.48 g (reference reactor) NH4Cl, 0.125 g Na2HPO4, 0.25 g of beef broth extract (Winiary) and 0.5 g CH3COONa per litre. Table 1 shows the parameters of the reactors.

Table 1

Parameters of membrane bioreactors

Reactor name HRT SRT OLR NLR C:N Volume Flow rate 
M8 3.0 0.079 0.207 0.4 36 12 
M12 3.3 12 0.129 0.379 0.3 36 11 
M12_ref 2.5 12 0.071 0.046 1.5 30 12 
M24 2.8 24 0.052 0.151 0.3 25 
M32 2.8 32 0.071 0.245 0.3 25 
Reactor name HRT SRT OLR NLR C:N Volume Flow rate 
M8 3.0 0.079 0.207 0.4 36 12 
M12 3.3 12 0.129 0.379 0.3 36 11 
M12_ref 2.5 12 0.071 0.046 1.5 30 12 
M24 2.8 24 0.052 0.151 0.3 25 
M32 2.8 32 0.071 0.245 0.3 25 

HRT, hydraulic retention time, in days; SRT, sludge retention time (sludge age), in days; OLR, organic loading rate in mg TOC/gVSS·d (TOC, total organic carbon; VSS: volatile suspended solids), NLR, ammonia loading rate, in mg N-NH4+/gVSS·d; C:N, carbon:nitrogen ratio; volume in litres; flow rate in L/d.

The reactors did not operate in parallel. First, M12 and M32 were investigated (around 1 year), then the other reactors (consecutive 1 year). The reactors were inoculated with activated sludge taken from the nitrification stage of the municipal wastewater treatment plant in Gliwice, Poland. The activated sludge was continuously aerated and the oxygen concentration in the reactors was kept at the level of 4 mg O2/L. The pH level of 7–8 was adjusted on-line by sodium carbonate addition. The temperature of the reactors was approximately 20 °C.

The adaptation of activated sludge was provided in two stages: adaptation to high ammonia concentration and adaptation to particular sludge age. The performance of the reactors was monitored by influent and effluent analyses for ammonia, nitrite, nitrate and organic compounds (as described in Raszka et al. (2011)). SS and sludge volume index were measured according to Standard Methods (APHA 1998).

Identification of filamentous bacteria

Microscopic analyses of activated sludge were performed every 2 weeks of the experiment. The standard approach for the identification of filamentous bacteria was drawn up by Eikelboom (2000) and Jenkins et al. (2003). Native samples were microscopically subjected to phase contrast (magnification 1,000×) while dry, stained samples (Gram and Neisser staining), to direct light (magnification 1,000×). Microscope Motic BA400T was used for the analysis. Abundance of filamentous microorganisms, called filament index (FI), was rated on a subjective scale from 0 (none) to 6 (excessive) (Jenkins et al. 2003). Filament occurrence was distinguished as dominant when the individual abundance was the highest among the detected filamentous bacteria. Filament occurrence was considered to be secondary for the species almost as excessive as dominant.

Molecular methods were used for confirmation of the standard method identification. Fluorescence in situ hybridization was performed according to Daims et al. (2005). In order to detect the Nostocoida limicola group probes NlimI91, NlimII, Nlim192 and NlimIII301 were used (Liu & Seviour 2001) and Type 021N was detected by probes G1B, G2M and G3M (Kanagawa et al. 2000). The probes enabling detection of a particular strain were mixed prior to general identification.

RESULTS AND DISCUSSION

Nitrification of high ammonia synthetic wastewater

The performance of high ammonia nitrification lasted in each reactor about 200–300 days. The adaptation to particular ammonia load and sludge age took from 40 to 200 days, in general around five times the particular sludge age. The detailed data about the reactors' performance and operation can be found elsewhere (Raszka et al. 2011), but some results for organic and ammonia removal are provided in Table 2.

Table 2

Wastewater treatment efficiency in membrane biological reactors of the study

  M8 M12 M12_ref M24 M32 
Organic compounds removal, % 64.3 89.2 54.5 76.9 93.1 
Ammonia removal, % 98.8 99.6 99.8 99.6 99.8 
  M8 M12 M12_ref M24 M32 
Organic compounds removal, % 64.3 89.2 54.5 76.9 93.1 
Ammonia removal, % 98.8 99.6 99.8 99.6 99.8 

The reactors did not operate simultaneously. Therefore the seeding sludge in each reactor differed slightly. The activated sludge taken for seeding the reactors was not free from filamentous bacteria. The sludge seeding reactor M8, M24, and M12_ref was taken from a municipal WWTP in the autumn time and the FI of about 4 was caused by presence of Type 0041 (domination), Type 0092 (subdomination), Haliscomenobacter hydrossis and Nostocoida limicola. Reactor M12 and M32 were inoculated with activated sludge taken from a WWTP in spring (FI 4). The seeding sludge contained Microthrix parvicella (domination), Type 0092 (subdomination) and Nostocoida limicola.

In each reactor, regardless the load of ammonia or sludge age, a shift in a filamentous bacteria biocenosis was observed. Table 3 shows an example of the dynamics of filaments in the activated sludge from the reactor operated with sludge age of 24 days (M24). In each reactor the shift occurred in the steady-state condition and the biocenosis change did not influence the nitrification efficiency. However, in each reactor some problems with the nitrification process have occurred but it was rather connected with a change in nitrifier population (see Raszka et al. 2011). The nitrification collapse and nitrifier population shift happened at another time to the shift in filament population. Moreover the time of changes in filament domination (for example in reactor M24 the period from 71 to 105 days of experiment) was characterized by lower FI, in the range 1–2.

Table 3

Filamentous occurrence in the reactor M24 (as example of the tendency)

Day of experiment Stage of experiment FI Filamentous bacteria occurrence
 
0041 0092 Nl Hh 021N 
4.0  
16 4.0  
31 4.0  
46 4.0  
57 SS 3.0  
71 SS 1.5 
85 SS 1.0  
105 SS 2.0  
120 SS 3.0   
133 SS 3.0   
148 SS 3.5   
161 SS 3.5   
176 SS 4.0   
198 SS 4.0   
211 SS 4.0   
Day of experiment Stage of experiment FI Filamentous bacteria occurrence
 
0041 0092 Nl Hh 021N 
4.0  
16 4.0  
31 4.0  
46 4.0  
57 SS 3.0  
71 SS 1.5 
85 SS 1.0  
105 SS 2.0  
120 SS 3.0   
133 SS 3.0   
148 SS 3.5   
161 SS 3.5   
176 SS 4.0   
198 SS 4.0   
211 SS 4.0   

A, adaptation; SS, steady-state conditions; D, domination; S, subdomination, I, insignificant occurrence; FI, filament index; Nl, Nostocoida limicola; 021N, Type 021N; Hh, Haliscomenobacter hydrossis; 0092, Type 0092; 0041, Type 0041.

In the reactors highly loaded with ammonia, the filamentous population changed to three species: Nostocoida limicola, Type 021N and Haliscomenobacter hydrossis, but the domination was by Nostocoida limicola or Haliscomenobacter hydrossis. In the reactor with higher sludge age (32 days) the shift of filamentous bacteria had two steps: at first, the domination was reached by Haliscomenobacter hydrossis, but after 30 consecutive days previously subdominating Nostocoida limicola replaced Haliscomenobacter hydrossis in the dominant position. In the other reactors the shift had a one-step nature, i.e. the new species displaced the previous, gaining definite hierarchy of domination which stayed constant to the end of the experiment.

In each case Microthrix parvicella remained in the systems around 150–200 days but, after the shift, disappeared completely, while the Type 0041 and Type 0092 stayed in the activated sludge for the whole experimental time but in insignificant amount. The shift time and filamentous bacteria population characteristic observed after the change is presented in Table 4.

Table 4

Occurrence of filamentous bacteria in the examined nitrifying activated sludge after the shift of filamentous bacteria population

Reactor name Shift time a/b Average FI Range FI Filamentous bacteria occurrence after the shift
 
Nl 021N Hh Sn Mp 0092 0041 
M8 46/4 3.5 2.5–4.5 – – 
M12 168/82 3.5 0.5–4.5 – – – – 
M24 105/60 3.5 2.0–4.0 – – – 
M32 223/25 2.5 0.5–3.0 – – – 
M12_ref 71/0 4.0 1.0–5.0 – – – 
Reactor name Shift time a/b Average FI Range FI Filamentous bacteria occurrence after the shift
 
Nl 021N Hh Sn Mp 0092 0041 
M8 46/4 3.5 2.5–4.5 – – 
M12 168/82 3.5 0.5–4.5 – – – – 
M24 105/60 3.5 2.0–4.0 – – – 
M32 223/25 2.5 0.5–3.0 – – – 
M12_ref 71/0 4.0 1.0–5.0 – – – 

Shift time – period when the shift in biocenosis diversity has directly changed: a – counted from the first day of experiment, before adaptation to particular sludge age and ammonia load, b – counted from the end of adaptation; FI, filament index, average FI is calculated from the results achieved in the time after shift; Nl, Nostocoida limicola; 021N, Type 021N; Hh, Haliscomenobacter hydrossis; Sn, Sphaerotilus natans; Mp, Microthrix parvicella; 0092, Type 0092; 0041, Type 0041; D, domination; S, subdomination; I, insignificant occurrence.

Nostocoida limicola, Haliscomenobacter hydrossis and Type 021N are known to be able to use nitrogen compounds for other purposes than assimilation. There was no direct investigation on the conversion of nitrogen compounds by Nostocoida limicola but Lemmer (2000) predicted that the bacteria prefer an environment with high N:C ratio. Type 021N can use ammonia and also nitrate when the ammonia is also present (Williams & Unz 1985). Haliscomenobacter hydrossis had the same abilities (Mulder & Deinema 1981). Many of the filamentous bacteria may perform denitrification (Ramothokang et al. 2006).

At the period of efficient nitrification the amount of nitrogen in influent and effluent was not in balance – total nitrogen concentrations were much lower in effluent than that expected for consumption for biomass incorporation. One of the possible reasons could be an excessive occurrence of the abovementioned filamentous microorganisms and their activity. It is not evidenced so far that the identified bacteria have the ability of nitrate respiration. Some of the filamentous bacteria, such as filamentous sulfur bacteria of the genus Thiotrix, have the capability to denitrify in anaerobic conditions (Trubitsyn et al. 2013, 2014). However, the oxygen condition in all tested reactors was continuously aerobic; thus the loss of nitrogen in the system can be explained only by embedding into bacterial cells during the growth or by simultaneous nitrification–denitrification in large flocs containing anoxic zones.

Another composition of filament biocenosis was reached in the reference reactor (M12_ref). The Type 021N that subdominated in highly ammonia loaded systems here had the dominant position. Only in the reference reactor did Sphaerotilus natans occur and probably that species was responsible for excretion of viscous substances. The viscosity was not measured but it could be seen by eye that the sludge looked like a weak jelly and small, bright pellets were tangled in the activated sludge flocs (Figure 1). The excess growth of Sphaerotilus natans was observed as well by other researchers in low substrate gradients (Guo et al. 2012) and in fully aerated systems with different organic carbon sources (Guo et al. 2014).

Figure 1

Macroscopic (left) and microscopic (middle and right; magnification 1,000×) view of activated sludge from the reference reactor (M12_ref).

Figure 1

Macroscopic (left) and microscopic (middle and right; magnification 1,000×) view of activated sludge from the reference reactor (M12_ref).

Finally, it can be seen that there is another pattern in filament domination in highly ammonia fed systems and in low ammonia loaded. Table 5 summarizes the obtained results.

Table 5

Domination in filamentous bacteria population in nitrifying activated sludge

AL SA Before adaptation (seeding sludge) After adaptation to high ammonium load (lasting several months of operation) 
Type 0041 Nostocoida limicola
Type 021N 
12 Microthrix parvicella
Type 0092 
Haliscomenobacter hydrossis
Nostocoida limicola 
24 Type 0041 Nostocoida limicola
Type 021N 
32 Microthrix parvicella
Type 0092 
Nostocoida limicola
Haliscomenobacter hydrossis 
12 Type 0041 Type 021N
Sphaerotilus natans 
AL SA Before adaptation (seeding sludge) After adaptation to high ammonium load (lasting several months of operation) 
Type 0041 Nostocoida limicola
Type 021N 
12 Microthrix parvicella
Type 0092 
Haliscomenobacter hydrossis
Nostocoida limicola 
24 Type 0041 Nostocoida limicola
Type 021N 
32 Microthrix parvicella
Type 0092 
Nostocoida limicola
Haliscomenobacter hydrossis 
12 Type 0041 Type 021N
Sphaerotilus natans 

AL, ammonium loading (H – high; L – low (conventional)); SA, sludge age (days).

It has to be also mentioned that the shift in filamentous bacteria biocenosis was not referenced to changes in nitrifying bacteria population (see Raszka et al. 2011).

CONCLUSIONS

Filamentous microorganisms Nostocoida limicola, Haliscomenobacter hydrossis and Type 021N occurred in aerated, highly ammonia loaded systems. No influence of different sludge ages was observed on filamentous bacteria. Shift in composition of filamentous bacteria biocenosis was observed with a tendency that Nostocoida limicola or Haliscomenobacter hydrossis dominated high ammonia loaded reactors accompanied by Type 021N. In the low ammonia loaded reactor, Type 021N and Sphaerotilus natans dominated. These observations confirm that Nostocoida limicola and Haliscomenobacter hydrossis prefer nitrogen rich conditions with high N:C ratio.

ACKNOWLEDGEMENTS

The research (study design, sampling and analysis) was partly financed by the Polish Ministry of Science and Higher Education within the projects: 1 T09D 086 30, 3 T09D 074 26 and 08/080/BK_18/0054.

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