Soil Microbial Functional and Fungal Diversity as Influenced by Municipal Sewage Sludge Accumulation

Safe disposal of municipal sewage sludge is a challenging global environmental concern. The aim of this study was to assess the response of soil microbial functional diversity to the accumulation of municipal sewage sludge during landfill storage. Soil samples of a municipal sewage sludge (SS) and from a sewage sludge landfill that was 3 m from a SS landfill (SS3) were analyzed relative to an undisturbed reference soil. Biolog EcoPlatesTM were inoculated with a soil suspension, and the Average Well Color Development (AWCD), Richness (R) and Shannon-Weaver index (H) were calculated to interpret the results. The fungi isolated from the sewage sludge were identified using comparative rDNA sequencing of the LSU D2 region. The MicroSEQ® ID software was used to assess the raw sequence files, perform sequence matching to the MicroSEQ® ID-validated reference database and create Neighbor-Joining trees. Moreover, the genera of fungi isolated from the soil were identified using microscopic methods. Municipal sewage sludge can serve as a habitat for plant pathogens and as a source of pathogen strains for biotechnological applications.

significance. Sludge is increasingly being used to fertilize agricultural and forest areas and to reclaim devastated terrains. Thus, it is important to recognize the distribution of pathogenic fungal species in activated sludge [11]. However, only limited research is available in this area due to regulations in most countries regarding the permissible contents of heavy metals and the compulsory stabilization and disinfection of municipal sludge by treating with Lime, thermophilic digestion and pasteurization prior to land application [26]. Landfilling and the land application of sewage sludge are the most economical sludge disposal methods [2,27], and are necessary for monitoring the environmental state, including the soil microbial parameters. The aim of the study was to evaluate the influences of sewage sludge storage on the functional diversity of the soil microbial population and on the fungal diversity in sewage sludge.

Site Description and Sample Collection
The research site is located on Haplic Luvisols that were formed on heavy loamy sand with a 59% sand, 23% silt and 18% clay in the Eastern region of Poland (N51°17'16", E22°11'35"). The sewage sludge was sourced from a nearby municipal treatment plant at Nałęczów, Lubelskie, Poland. The municipal treatment plant is located in the protected zone of Kazimierski Landscape Park. This plant is surrounded by a health resort. Consequently, it is important to monitor the environmental state and study the influences of the waste on the soil microbial diversity to protect public health. All analyses were performed in 2011. The basic soil and sludge characteristics are given in Table 1. Notes: EC-electric conductivity, TOC-total organic carbon.
The study included three sites near the municipal treatment plant area. The following soils were subjected to analysis (Figure 1):  a soil under the sewage sludge landfill (SS)-the landfill plot was a quadrat of 4 m by 4 m with 40 cm sludge layer on the soil surface,  a soil located 3 m from a sewage sludge landfill (SS3), and  a control soil (C) consisting of an undisturbed soil located 200 m from the sewage sludge landfill.
The soil samples were collected (0-20 cm depth) randomly, mixed, sieved through a 2-mm-mesh screen and stored at 4 °C until analysis. Municipal sewage sludge probes were collected from the plots where the sludge was stored.

Chemical Analysis
The soil total organic carbon (Corg.) content was determined using the Tiurin method. The pH was measured from a soil aqueous extract using the electrometric method. The total N content was measured by using the Kjeldahl method and the total P content was measured by using the flow spectrophotometric method (SKALAR San System, Lilburn, GA, USA). The total K content was measured using the flame emission spectrometry method (AAS-3 Carl Zeiss Jena, Jena, Germany) after wet digesting the samples with sulfuric acid. The heavy metals were extracted using aqua regia and were measured using atomic absorption spectroscopy (AAS). The same methods were used for the soil and the sewage sludge.

Community Level Physiological Profiling (CLPP) Analysis
Community level physiological profiling was assessed using the Biolog EcoPlate TM system (Biolog Inc., Hayward, CA, USA). Each 96-well plate consists of three replicates, with 31 sole carbon sources and a water blank. EcoPlates TM were used to determine the carbon source use patterns of the SS, SS3 and C soil samples. The soils (1 g) were shaken in 99 cm 3 of sterile water for 30 min before incubating for 20 min at 4 °C. After shaking, 120 µL of the sample suspension was inoculated into each well of the Biolog EcoPlates TM and incubated at 26 °C. The utilization rate was indicated by the reduction of tetrazolium violet, a redox indicator dye that changes from colorless to purple [28]. The data were recorded at 590 nm every 24 h for up to 96 h. The microbial activity, which was expressed as the average well color development (AWCD), was determined for each microplate [20]. The data collected at 48 h were used to calculate the AWCD, the Shannon-Weaver (H) diversity indexes and the substrate richness (R), as shown by Gomez et al. [20]. In addition, the percentages of the carbon sources that were used by groups were evaluated.

Fungal Enumeration and Morphotypic Identification of Fungal Strains
The total number of fungi was determined by using the plate method with Bengal rose medium (Biocorp, Warsaw, Poland) and the serial dilution method. Soil (10 g wet weight) was treated with 90 cm 3 of saline peptone water (Biocorp). Aliquots of the suspensions were transferred to petri dishes with the Bengal rose medium using pour plates. The plates were cultivated for 7 days at 26 °C. Fungal strains were isolated from the municipal sewage sludge and soil by using the same medium. The macroscopic variables that were analyzed included the colony diameter, conidia colors, obverse and reverse mycelium colors, degree of sporulation and the presence or absence of exudates [20]. Microscopic characteristics of the mycelia were examined using an optical microscope. Strains were identified at the genus level using microculturing growth on different media-Czapek agar and PDA (Biocorp). Cultures were incubated at 26 °C for 14 days. Genus identification was performed using the systematic classification methods of Domsch et al. [29], Burnett and Hunter [30] and Watanabe [31].

Molecular Identification of Fungi Isolated from Sewage Sludge
The most dominant fungi that were isolated from the sludge were identified using a molecular method. The method is based on rDNA-extraction and the subsequent amplification of the D2 large subunit region of the fungal rDNA [32] by using the Fast MicroSeq D2 LSU rDNA fungal sequencing kit (Applied Biosystems, Foster City, CA, USA). The fungal strains were grown on PDA medium (Biocorp). Preparation of the fungal DNA was performed with the PrepMan Ultra reagent (Applied Biosystems). A very small amount of one fungal colony was collected using tweezers and suspended in 200 µL of the PrepMan Ultra reagent. After incubation for 10 min at 100 °C , the suspension was mixed and centrifuged for 10 s allow the cell debris to settle. The supernatant solution was transferred to a new microcentrifuge tube and diluted at a ratio of 1:100 in deionized nuclease-free water [33]. A fast MicroSeq D2 LSU rDNA fungal PCR kit (Applied Biosystems) was used to amplify the D2 LSU rDNA region. The PCR was performed using the conditions listed in Table 2. The ExoSAP-IT® PCR Products Purification Kit for ABI (Affymetrix Inc., Santa Clara, CA, USA) was used to clean the PCR products before sequencing to eliminate any remaining labelled ddNTPs and for desalting. Next, 5 µL of the PCR product was mixed with 2 µL of the ExoSap reagent and incubated in a thermal cycling profile as follows: 37 °C for 15 min and 80 °C for an additional 15 min. The next step after PCR purification was to prepare the cycle sequencing. For this purpose, 13 µL of the forward/reverse mix from the MicroSeq D2 Fungal ID Sequencing Kit was used with 7 µL of the purified product. The cycling program is listed as follows: 96 °C for 1 min; (96 °C for 10 s; 50 °C for 5 s; 60 °C for 1 min 15 s) × 25. Next, the Performa Purification System (EdgeBio-Performa Gel Filtration Cartridges) was used and 20 µL of the reaction samples eluate was mixed with 20 µL of deionized formamide. Electrophoresis was run through an ABI 3130 ×l capillary sequencer (Applied Biosystems) with a 50 cm capillary array and polymer POP6_1. The MicroSEQ® ID software was used to assess the raw sequence files and to perform sequence matching to the MicroSEQ® ID (Applied Biosystems) to validated the reference database and to create Neighbor-Joining trees.

Statistical Analysis
The average well-color development, richness and the Shannon-Weaver index were statistically analyzed using an analysis of variance (ANOVA). The significance differences were determined using the Tukey test at p = 0.05. A principle component analysis was conducted on the optical density (OD) data from the 31 carbon sources following 48 h of incubation. Cluster analysis, including grouping of the treatments and features, was performed on the standardized data from the average absorbance values at 120 h. A dendrogram was prepared to present the similarity of the carbon utilization patterns of the substrates that were located on the Biolog EcoPlate TM between the soil samples with scaled bond distances on the axis (Ward's method) and boundaries marked according to Sneath`s criteria (33% and 66%). The data were standardized according to AWCD in each microplate to remove the inoculum density effects [19]. All statistical analyses were performed using the Statistica 10.0 software (StatSoft Inc., Tulsa, OK, USA).

Microbial Activity
The results showed that the AWCD in the control soil was the lowest and increased under the SS3 and SS by 30 and 1000%, respectively (Figure 2A). In addition, the corresponding increases in R were 10 and 8000%. The AWCD and R were significantly greater for the SS when compared with the SS3 and C ( Figure 2B). The lack of a significant difference (i.e., the similar response) between the SS3 and C implies that the influential region of the organic inputs from the landfill regarding increases in microbial activity is characterized by the AWCD and the R is below 3 m. The impacts of sewage sludge on the catabolic potential of microorganisms only occur where the sewage sludge is stored. The average utilization (i.e., AWCD) of the C sources increased with incubation time for all treatments (data not shown). The Shannon-Weaver index was the highest in the control soil, but was not significantly different from the other treatments ( Figure 2C). This result indicates that the microbial populations were stable in the soils.
To determine the extent of treatments differentiation with regard to the metabolism of the carbon sources, a principal components analysis was performed. The first and the second principal components (PC1 and PC2) explained 51.41% and 18.71% of the data variance, respectively. The control soil and the soil that was 3 m from the sewage sludge landfill were clustered together (C and SS3) and were different from the soils under sewage sludge accumulation (Figure 3). The carbon sources were significantly correlated with PC1 and PC2 (R > 0.7) ( Table 3). Several substrates, such as pyruvic acid methyl ester, tween 80, α-D-lactose, D-mannitol, N-acetyl-D-glucosamine, DL-α-glycerol phosphate, D-galactonic acid γ-lactone, 4-hydroxybenzoic acid, D-malic acid and L-arginine were intensively metabolized by the microbial communities from the soil under the sewage sludge accumulation. The microbial communities from the control and the soils that were collected 3 m from the sewage sludge landfill intensively used tween 40 and N-acetyl-D-glucosamine. The microbial communities from the SS3 soil used i-erythritol and control soil used D-cellobiose. Optical densities of less than 0.25 (threshold for positive response) were recorded for glycogen and β-methyl-D-glucoside for all samples.     A similar response occurred between the microbial communities under the SS3 and the control (group 1) towards carbon substrate utilization. This findings were confirmed by the carbon substrate utilization intensity diagram ( Figure 5). Group 1 (C and SS3) and group 2 (SS) differed regarding their use of particular carbon sources. A better aptitude to substrate utilization was revealed by the SS than by the C and SS3. The most intensively used substrates by the SS community included the following: β-methyl-D-glucoside, cyclodextrin, L-phenylalanine, D-glucose-1-phosphate, L-threonine, and L-asparagine.
Significant differences were observed in the categorized substrate utilization patterns among the microbial communities of the three soil samples (Figure 6). The carboxylic acids, carbohydrates and miscellaneous groups were the most intensively metabolized, especially in the soil under sewage sludge accumulation.

Pathogenic Fungi and Yeast
The effects of sewage sludge accumulation on the total number of fungi are presented in Figure 7. Mean values of colony forming units were greater in the soil under sewage sludge accumulation (80.43 × 10 6 CFU kg −1 ) relative to in the control (71.20 × 10 6 CFU kg −1 ) and the SS3 soil (55.54 × 10 6 CFU kg −1 ).  In the control soil, different species from the Penicillium and Mucor genera were isolated frequently. We observed a higher fungal diversity in the control soil than in the soil under sewage sludge accumulation.
Molecular identification of the dominant fungi that were isolated from the sewage sludge and the phylogenetic analysis of the DNA sequences showed that Mucor circinelloides and Geotrichum  citri-aurantii were present in settled sludge as a fungal monoculture (Table 4, Figure 9). Phylogenetic analyses of the LSU D2 rDNA nucleotide sequences demonstrated that the fungal monoculture was created by molds from the Mucor and Geotrichum genus. The first group includes molds, and the second group includes yeast species. An analysis of the phylogenetic tree indicated a 98.13% phylogenetic relationship with M. circinelloides. In addition, a very close relationship with M. ramosissimus (96.26%) was observed. Furthermore, M. racemosus and M. plumbeus were closely related to the fungal isolates from the sewage sludge. The genetic distances between the above-mentioned species and the environmental fungi were 6.54% and 8.41%, respectively. The analysis of the second group indicated that the closest phylogenetic relationships were observed between the isolates from the sewage sludge and G. citri-aurantii (93.46%). A higher genetic distance was observed for the different yeast genera of Candida and Pichia with 16.81% (83.19% similarity) and 23.35% (76.65% similarity), respectively ( Figure 9).

Microbial Activity
The preliminary results indicated that municipal sewage sludge containing organic matter and macronutrients (N, P and K) and the permissible values of heavy metals could be useful for improving microbial activity and soil fertility. A stimulating effect of the sludge on the microbial activity and the oxidative capacity developing in the Biolog EcoPlates TM was clearly shown based on the higher AWCD and Richness in the SS soils relative to the SS3 and C soils. Frąc et al. [23] suggested that in the increased diversity indices after applying organic matter potentially resulted from the development of different microbiota. In agreement with our results, previous studies have demonstrated microbial communities with a high functional potential for carbon utilization in dairy sewage sludge [23], vermicompost, manures [20], and straw [34].
As shown in previous studies, the application of sewage sludge improves the soil structure [35] increases the resistance of the soil to erosion [36], or plays a significant role in the natural remediation of metals and aromatic compounds [37]. Besides stimulating microbial activity, these effects can be helpful for controlling erosion from the erodible loess soils that were used in this study [38].
Changes in the categorized substrate utilization patterns among the microbial communities in the studied treatments suggest that the incorporation of sewage sludge may stimulate microbial carbon use and metabolism in the soil. Similar results after the application of organic matter to the soil were observed [28]. The principal components analysis was used to define the different carbon substrate uses that were metabolized the most intensively. According to Gomez et al. [22], the carbon sources in the Biolog TM assay provide a wide set of compounds that can be used to estimate the relative potential metabolic versatility.

Pathogenic Fungi and Yeast
The number of microscopic fungi in sewage sludge and soil under sewage sludge accumulation is usually relatively high. As reported previously, mean values of colony forming units in the same experimental site ranged from 55.54 × 10 6 to 80.43 × 10 6 CFU kg −1 of soil dry matter ( Figure 5). Microscopic fungi play an important role in the decomposition of sludge organic matter [39]. In the present study, the fungal population increased (but not significantly) in the soil under sewage sludge accumulation. A similar effect was observed by Brendecke et al. [40]. The short-term increase in the fungal population that occurred as a function of the addition of sewage sludge may be explained by the presence of easily degradable organic components in the waste.
A molecular analysis of the fungi that were isolated from the municipal sewage sludge indicated that this sludge could be dangerous for plants due to the presence of M. circinelloides and G. citri-aurantii. These fungal species are common plant pathogens [41,42]. Worldwide citrus sour rot, which is caused by Geotrichum citri-aurantii, has been reported as an important postharvest disease in citrus fruit [42]. In addition, M. circinelloides causes disease in fruit [41] and tomatoes [43]. However, fungi such as M. circinelloides are also of biotechnological interest as a source of carotenes, proteins and lipids because they accumulate high levels of these compounds in their mycelium [44,45]. Moreover, M. circinelloides lipids have gained attention because they can be easily converted into biodiesel, suggesting that the biomass of this fungi could be used as an alternative to plant oils as a feedstock for biodiesel production [46,47].
Microscopic identification of the fungi indicated that the control soil had the highest fungal diversity. However, the soil that was collected from the plots with sewage sludge accumulation were nearly -fungal monocultures‖ (Figure 6), as in the sewage sludge. This result suggests that the fungal species from the sludge won the competition for the soil fungi niche. All of the fungi that were isolated from the soil under sewage sludge accumulation or at 3 m from the sewage sludge landfill were closely related to Mucor or Geotrichum. Detecting the plant pathogenic fungi (Mucor) and yeast (Geotrichum) application limits of the sludge without thermal treatment and such use should be avoided, and the fungal and yeast compositions should be monitored before applying sludge in the soils and during the storage of this waste. Pascual et al. [1] reported that the impacts of sanitized (Autothermal Thermophilic Aerobic Digested, ATAD) and non-sanitized (Anaerobic Mesophile Digested, AMD) sewage sludge on the activities and functional diversities of the soil microbial communities and plant growth were similar. However, sanitation can be beneficial under conditions in which the pathogens migrate to the aquatic environment (the plants do not migrate). The changes in the fungal composition potentially resulted from the improved conditions that favored fungal growth. Because the application of non-stabilized sludge on the land or its storage may result in serious hygienic problems, our knowledge regarding sludge borne-fungal compositions is significant from a practical point of view. Fungi are opportunistic organisms that possess potential pathogenic properties for humans, animals and plants. However, some fungi have higher pathogenic potentials than others. To minimize human infection, it is necessary to monitor the mycological quality of municipal sewage sludge. From a biotechnological point of view, sewage sludge can be one source of enzymes or microorganisms that produce enzymes and different metabolites. Enzymes that are secreted or associated with these microorganisms are vital for the rate-limiting step of anaerobic digestion, hydrolysis [48]. The activities of these hydrolases are important for establishing the overall limiting rate of the processes. This rate limiting step involves the breakdown of large polymeric substrates, such as carbohydrates (cellulose, starch, proteins), into small and low molecular weight compounds (glucose, amino acids, etc.) that are capable of entering the microbial cells and participating in its cellular metabolism.

Conclusions
1. This study showed that community-level physiological profiling using Biolog EcoPlates TM can be used as a sensitive and effective indicator for evaluating microbial functional diversity and microbial communities under sewage sludge accumulation. These parameters can be useful for monitoring the soil microbial state following sewage sludge application. 2. Municipal sewage sludge is rich in molds and yeasts, which are known as fungal plant pathogens. Most fungi that were recovered in this investigation can be considered as potential pathogens. Therefore, all sewage sludge research and accumulation workers should be careful to avoid mycotic infections, and their production must be adapted to control the spread of pathogenic fungi in the environment. 3. Fungi in sewage sludges are not only pathogens but also have practical uses for the development of biopreparations for environmental technologies and agricultural biotechnology, especially for the disposal and recycling of organic wastes. Future research studies are needed to assess the properties and use of fungal strains that are isolated from sludges and soils during organic waste degradation.