1. Introduction
Due to the improvement of socioeconomic levels resulting from rapid urbanization and industrial development, the generation of municipal and industrial wastes has increased considerably worldwide over the years. Moreover, despite the guidelines of the European Union Waste Framework Directive (2008/98/EC), landfilling still constitutes one of the most common methods of waste disposal in the middle-income countries. The major concern associated with landfilling is the generation of large amounts of leachate, which originates from the water percolated through the refuse. The leachate may contain a wide range of toxic, hazardous, and carcinogenic chemical contaminants. Moreover, its composition often varies significantly, both temporally and spatially [
1]. Therefore, the arbitrary disposal of industrial and municipal waste in locations that are unprepared for landfilling may result in the uncontrolled migration of the leachate into the soil, sediments, surface water, and even groundwater.
The application of biological methods in the cotreatment of landfill leachate and domestic wastewater in urban sewage plants has become popular over the last decade [
2,
3,
4,
5]. However, there is scarce information on the impact of this combined treatment on the operation of the wastewater treatment process and, in particular, on the physiological and functional state of the activated sludge microorganisms.
Depending on their source, industrial and municipal waste are characterized by a variable composition, and may contain large quantities of organic and inorganic pollutants, such as phenols, polycyclic aromatic hydrocarbons, chloroform, sulfur compounds, cyanides, heavy metals, chloride, humic acids, and ammonia [
6,
7]. It is also well documented that the pollutant composition of the leachate may be much more complex than the landfilled waste itself due to the occurrence of the compounds that are formed during the processes of waste degradation [
8,
9]. Therefore, the difficulties in the biological treatment of the leachate originated from municipal and industrial waste are primarily related to its chemical load and changes in the important environmental parameters (e.g., pH, temperature, or salinity) during wastewater treatment.
Currently, the activated sludge process (ASP) has become dominant in biological wastewater treatment systems. In this technology, the contaminants are utilized by various microorganisms embedded with organic and inorganic particles within the extracellular polymeric substance matrix (EPS) [
10]. Despite the fact that the activated sludge during wastewater treatment is often exposed to high concentrations of diverse chemicals, including inhibitory and recalcitrant ones, its autochthonous microorganisms may not be able to use them as carbon and energy sources. As a result, the abundance and activity of indigenous bacteria decrease [
11,
12]. Further reduction of the microbial capacity and diversity may weaken the biological wastewater treatment, or even be the cause of its complete breakdown [
13,
14,
15,
16].
The objective of this study was to assess the impact of the KPL that are discharged into the wastewater treatment plant (WWTP) “Klimzowiec” along with the municipal effluents on the activated sludge microorganisms and the laboratory-scale SBR operation. Within the context of the combined treatment of the KPL and synthetic wastewater in activated sludge, the specific goals of this research were to (1) characterize and determine the acute toxicity of the KPL to the activated sludge microorganisms, (2) evaluate the changes in the operational parameters of the SBRs, (3) analyze the functional capacity and structural diversity of the microorganisms, (4) assess the structure of the activated sludge flocs, and (5) establish the relationships between the biological and physicochemical factors being studied. The special attention will be focused on the use of marker fatty acids to assess alterations in the structure of microbial communities under the KPL exposure because such studies have not been performed yet. The analyses that will be carried out enable any effects of the mixtures of the toxic contaminants on the activated sludge microorganisms and wastewater treatment process to be predicted in advance, and thus enable solutions that ensure the stable operation of the WWTP “Klimzowiec” and similar wastewater treatment units in the nearest future to be implemented.
4. Discussion
AS technology is considered to be the most efficient and economical approach for achieving the desired quality of wastewater mixed with landfill leachate [
16]. Although the cotreatment of the landfill leachate in the WWTP may be appropriate under some circumstances [
53], the inherent variability in the leachate composition necessitates a conservative strategy due to the increasingly stringent water quality emission limits that have been placed on the biological treatment units by the Urban Wastewater Directive (91/271/EEC; EEC, 1991) and the Water Framework Directive (2000/60/EC; EU, 2000). Despite the abundance of studies that have been performed on the cotreatment of the landfill leachate in sewage plants, knowledge about the long-term effect of its occurrence in the effluent on activated sludge ecosystem remains limited.
Among many parameters that enable the alternations in the leachate quality to be evaluated, the pH value is considered to be an indicator of its aggressiveness. The alkaline KPL used in this study can be classified according to El-Fadel et al. [
54] as an old and stabilized leachate. It was also characterized by a moderately high strength of recalcitrant phenolic compounds, which had a concentration that surpassed the allowable limits [
29] by more than three orders of magnitude. Moreover, the low BOD
st level of the KPL compared with its COD value confirmed a large amount of non-biodegradable organic matter. In addition, the LPI value of the KPL was more than 5-fold higher than the standard LPI value for the treated leachate disposal limit for inland surface water (7.378), which revealed its high pollution potential [
55]. It was reported that a high concentration of N-NH
4+ in the leachate can disrupt its biological treatment due to the toxicity of this compound [
56,
57]. Although the concentration of ammonium in the tested KPL was almost 40-fold higher than the limits [
29], its content was near the lower limit reported for an old leachate (360 to 2150 mg/L) [
54]. The performed analysis of specific microbial groups indicated that the investigated landfill leachate may be a sanitary and epidemiological hazard. The abundance of total coliforms as well as fecal indicator bacteria in the KPL could have been the result of the significant content of phosphates, which augmented their survival in the leachate [
58]. Coliform bacteria have recently been recognized as one of the most problematic pollutants in an old leachate [
59].
The AS process is often indicated as being a good solution for leachate treatment [
53]. However, the information about the leachate toxicity towards the sludge microbial communities is very scarce. It is known that individual xenobiotics are toxic to microorganisms to a different extent [
60]. However, the leachate toxicity is a result of the synergistic or antagonistic effects of various contaminants [
61]. Therefore, the volumetric ratios of the leachate that is added to domestic wastewater usually do not exceed 10% [
2,
61,
62]. This study showed that the KPL had an extremely detrimental effect on the AS microorganisms, even in the presence of 6.69%. The long-term presence of the KPL in the effluent significantly disrupted the biological treatment of the wastewater in the SBR by affecting its operational parameters. It was found that the poor quality of the leachate resulted in a gradual decrease in CODr in the SBR
KPL from 95% to 69% and 41% during the cotreatment with 3.5% and 5.5% of the leachate, respectively. A similar phenomenon has been observed in the decrease in CODr from 90% to 70% and 51% in a bench scale reactor cotreated with 2% and 5% of the landfill leachate, respectively [
4].
The appropriate pH is a crucial factor for the stable operation of a sewage plant because it affects the strength and surface charge of the AS flocs [
63]. Extreme pH conditions can lead to an inhibition of the growth of the AS microorganisms [
64]. In addition, the removal of xenobiotics, such as phenol and its derivatives, from the wastewater by the AS may also decrease with an increase in the effluent pH [
65,
66]. It can be concluded that a gradual increase in the pH value of more than 9.0 in the SBR
KPL negatively affected the microbial biodegradation of the contaminants in the sewage. Two-fold and 3-fold decreases in the ability of AS to settle were observed with an increase in pH from 7.6 to 8.1 and 9.3 in the SBR
KPL32d and SBR
KPL64d, respectively. Similarly, Ghanizadeh and Sarrafpour [
67] found that an increase in pH from 5.7 to 9.0 resulted in a significant decrease in the SVI from 96 to 44 mL/g. A considerable drop in the SVI from 90 and 80 mL/g to 30 and 20 mL/g was also observed with an increase in the concentration of leachate from a municipal solid waste dumpsite in wastewater from 2.5 to 15% and 40% in India and Poland, respectively [
16,
68]. An SVI below 70 mL/g may result in the turbid effluent associated with the dispersed growth of microorganisms and the formation of small pin flocs [
69].
The next critical factor for ensuring the biological wastewater treatment process and thus the correct operational behavior in the sewage plants is AS concentration. In this study, the MVLSS in the SBR
KPL64d constituted 63% of the MLSS concentration and was below the appropriate level for pollutant removal in the SBR system [
70]. It can be concluded that the compounds present in the KPL may not be used for the maintenance and growth of the AS microorganisms. Moreover, their occurrence in the SBR may inhibit or slow down the utilization of the readily biodegradable components present in the sewage [
71]. In this study, the inhibition of the utilization of 10 and 12 substrates in an EcoPlate
TM was observed for SBR
KPL after cotreatment with 3.5% and 5.5% KPL compared to SBR
CTL, respectively. The microorganisms in the SBR
CTL were able to use the ingested biodegradable matter in the wastewater to synthesize the new biomass, which resulted in an increase of the MVLSS and MLSS. The increase of suspended matter in the SBR
C may be attributed to the increase in the amounts of the extracellular polymeric substances as a result of the presence of
Zooglea sp. in the wastewater. A decrease in the microbial biomass in the SBR
KPL directly affected the structure and quality of the AS flocs, which was manifested in a change in the size and shape of the aggregates, which became smaller, looser and more irregular than those in the SBR
C. Similarly, Pieczykolan et al. [
16] observed a gradual increase in the size of the small flocs when the concentration of the leachate exceeded 15%. The cotreatment with KPL also resulted in a decrease in the abundance of protozoan in the AS. The decrease in the number of Arcella, which was the most common genera in the AS, as well as the appearance of individuals from the Vorticella genus and a significant number of filamentous bacteria in the SBR
KPL may be connected with the presence of leachate in the wastewater and may be correlated with a decrease in the effectiveness of the cotreatment. In the research by Czapluk et al. [
72], more diverse protozoan and metazoan communities were observed in the WWTP in which domestic effluent predominated, while ciliates and metazoans did not occur or were found at very low densities in the AS that had primarily received industrial wastewater.
Because of their quick reaction to the changes in the ambient environment, the AS microorganisms are considered to be a sensitive bioindicator of the response of biological wastewater treatment units to various stress factors and perturbations [
73]. The results presented in this study indicate that the addition of the KPL into the wastewater significantly reduced the enzymatic activity of the dehydrogenases and esterases in the AS, which could not recover after long-term exposure to the leachate. Previous literature indicated that the activity of esterases and dehydrogenases may decrease in the AS in the presence of the more complex contaminants occurred in the leachate [
74,
75,
76]. Although the activity of the AS microorganisms is extremely important for appropriate functioning of this ecosystem, this parameter is hardly ever considered during the evaluation of the effect of a leachate on the WWTP functionality. The impact of different concentrations of the leachate from the WENT landfill (China) on dehydrogenase activity in an AS reactor was only described by Li and Zhao [
56], who observed a decrease in the respiratory activity by 12, 16, 17, 32, and 47% in the presence of 1, 2, 4, 9, and 20% of the leachate, respectively.
The high metabolic capacity of the AS microbial communities is considered to be a pivotal factor for efficient wastewater treatment in the biological units [
77]. The microorganisms in the SBR
KPL were less metabolically active than the bacteria in the SBR
C. Although the leachate affected the microbial growth in the AS, thus contributing to an extension of the duration of the adaptation phase, the t
50 did not change significantly. It can be assumed that the presence of the KPL in the wastewater had a toxic effect on the core populations in the AS. It was also shown that the low AS metabolic activity after contamination was correlated with a lack of the ability of its microbial communities to utilize specific compounds, in particular, the phosphorus sources. The changes in the CLPP patterns for the SBR
KPL may suggest that the occurrence of the leachate in the wastewater led to the metabolic specialization of the AS microorganisms. The functional diversity of the AS microorganisms exposed to a landfill leachate is poorly documented in the literature. The research by Röling et al. [
78] indicated that the microbial communities in an aquifer contaminated with the leachate from Banisveld landfill (Boxtel, The Netherlands) favored the aromatic substrates among the other carbon sources in an EcoPlate
TM. Moreover, Taş et al. [
79] showed a significant increase in the abundance of the genes that belong to the carbohydrate and xenobiotic metabolism categories in groundwater polluted with the same leachate. Similarly, in this study the microorganisms from the SBR
KPL increased their metabolism in relation to the carbohydrates and phenolic compounds. The significant decrease in the microbial metabolic activity and the increase in the inequality of the bacterial abundance that was observed in the SBR
KPL strongly correlated with a decrease in the functional capacity. This may indicate a loss of the functionally significant microorganisms such as phosphate-solubilizing bacteria in the AS during the leachate cotreatment. By contrast, the microbial populations in the SBR
KPL metabolized the compounds containing nitrogen more intensively than the microorganisms in the SBR
C. It may be suggested that the loss of phosphorus utilization as well as the increase in nitrogen metabolism in the SBR
KPL may be related to the predominance of bacterial species originated from the leachate.
In this work, very interesting results regarding the alterations in the structural diversity of microorganisms in the AS were obtained. Over the course of the experiment the contribution of the microorganisms can be ordered as follows; BG− > F > BG+ > Pr > Ac and BG- > BG+ > F > Ac > Pr in the SBR
C and SBR
KPL, respectively. The prevalence of BG− over the other microbial groups was observed in both of the tested bioreactors. Due to their highly impermeable outer membrane, BG− have generally been recognized as being more tolerant than BG+ to the various hydrocarbons commonly found in landfill leachate [
80,
81]. Nonetheless, a gradual decrease in the content of the BG− biomarkers accompanied by a significant increase in the percentages of the FAME markers for BG+ was observed during the cotreatment with the KPL. Similar observations were made by Leal et al. [
81], who found that the introduction of petrol into compost resulted in a significant decrease in the content of the FAME markers for BG− along with a simultaneous increase in the content of the biomarkers for BG+. It was reported that BG+ can exhibit a high metabolic capability towards various contaminants, which may result in their predominance in a contaminated environment [
82]. Here, the decrease in the abundance of the BG− biomarkers in the SBR
KPL was followed by a decrease in the distribution of the fungal and protozoan FAME markers. Although the abundance of fungi in the AS is rather low, they are involved in many important processes during wastewater treatment due to a versatile pollutant-degrading capacity [
83]. Since the changes in the occurrence of fungi in the AS are mainly associated with pH fluctuations, it may be suggested that the gradual increase in wastewater pH during the KPL cotreatment may negatively have affected the fungal population in the SBR
KPL, which contributed to the loss of important catabolic activity in this environment. Furthermore, the multiplication of the Actinomycetes in the SBR
KPL may adversely affect the wastewater treatment process and the sludge quality [
84].
The identified changes in the FAME profiles of the AS indicated that the microorganisms were very sensitive to the contamination and exhibited adaptive mechanisms to the presence of the KPL in the wastewater. The essential changes in the bacteria FAME patterns were related to an increase in the content of the 16:0, 3-hydroxy, and 15:0
anteiso-branched fatty acids, which were responsible for rigidification of the cytoplasmic membrane. Furthermore, a gradual increase in the saturation of fatty acids in bacterial membrane along with an increase of pollutant concentration could be associated with the stimulation of cyclopropane fatty acid formation by some bacterial strains [
85]. Interestingly, there was also a significant increase in the percentages of the
anteiso-branched fatty acids in the FAME profiles under the KPL contamination. The branched fatty acids play an important role in the microbial tolerance to pH stress [
86]. Therefore, it can be suggested that an increase in the yield of the
anteiso/
iso-branched fatty acids in the FAME patterns, which was observed in this study, may be a result of the microbial response to the pH stress resulted from the presence of the KPL in the wastewater.
Multivariate analysis in this work allowed establishing of important relationships between operational parameters of wastewater treatment process and factors affecting activated sludge quality. It was found that the presence of the KPL in the wastewater was the main factor responsible for variations in the activated sludge. There was a close relationship between the microbial activity of the sludge, ability of the AS microorganisms to utilize carbon sources and important operational parameters of wastewater treatment process. The operation parameters of the reactor were positively correlated with enzymatic activity, metabolic capacity and structural diversity of microorganisms in the AS and negatively correlated with content of selected fatty acids. A significant decrease in the ability of microorganisms to utilize carbon, phosphorus and nitrogen sources may be a good indicator of weakening their metabolic functions. Interestingly, a multivariate analysis showed that the selected fatty acids can be used as indicators of structural diversity of microbial communities and xenobiotic degradation. A significant increase in the content of 3-hydroxy fatty acids and cyclopropane fatty acids in the presence of the KPL can be considered as useful biomarkers of stress condition [
87]. Combining physicochemical and biological analyzes seems necessary to accurately identify the major factors affecting activated sludge operation and quality, especially when they achieve critical levels.