Feasible and effective reuse of municipal sludge for vegetation restoration: physiochemical characteristics and microbial diversity

The large volume of municipal sludge causes environmental problems in cities worldwide. In this study, municipal sludge, mixed with construction waste residue, was used as substrate to plant Ailanthus altissima. The growth of A. altissima, the substrate characteristics, and substrate microbial diversity were measured to investigate potential recycling and reusing pathways of municipal sludge. The obtained results showed that compared to garden soil, the mixed substrate was weakly alkaline, and had higher nutrient contents, which is beneficial for A. altissima, and results in better growth in mixed substrate. Although the contents of the main heavy metals in the mixed substrate were significantly higher than in garden soil, the values met the criterion of Class II soil in the Environmental Quality Standard for the Soils of China (GB15618-1995). Different substrates showed a variety of microbial diversities. Proteobacteria was the top microbial phylum in all samples, and higher relative abundances were found in samples containing municipal sludge. After growing A. altissima, the relative abundances of Acidobacteria and Gemmatimonadetes increased in the mixed substrate. Therefore, addition of construction waste residue and growth of A. altissima caused a difference. The microbial communities in the mixed substrate with A. altissima are both plant friendly and environmentally friendly. These results suggest this mixed substrate as a potentially feasible and effective pathway for the reuse and recycling of municipal sludge for vegetation restoration.

1 Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing University of Agriculture, Beijing, 102206, P.R. China. 2 College of Landscape Architecture, Beijing University of Agriculture, Beijing, 102206, P.R. China. 3 Beijing Collaborative Innovation Center for Eco-environmental Improvement with Forestry and Fruit Trees, Beijing, 102206, P.R. China. 4  sludge composts to the soil improves soil properties, such as bulk density, porosity, and water holding capacity 6 . A significant enrichment in mineral nitrate and available phosphorus is detected in soil after municipal sludge application and both the pH value and electrical conductivity also increase 7 . However, municipal sludge also contains different contaminants, including heavy metals, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), adsorbable organohalogens (AOX), pesticides, surfactants, hormones, pharmaceuticals, and nanoparticles 8 . These can create potential pollution to the soil and are hazardous to the health of humans 9,10 . Therefore, the land application of municipal sludge in agriculture and horticulture has security risks, while the application of municipal sludge in forests is a relatively ideal disposal pathway. The advantages are that forests are often deficient in major nutrients that can be found in abundance in municipal sludge, and that forests are not the major contributors to the human food chain. Municipal sludge not only improves forestry soil fertility by increasing soil organic matter and available nitrogen and phosphorus, but also promotes the growth and reproduction of trees, including increases in the height and diameter of trees, which is advantageous for the growth of shrubs and herbs of trees 11 . The application of municipal sludge containing low heavy metal concentrations also improves the structure and the organic matter of the clay loam brown forest soil 12 . Moreover, municipal sludge has also been used as substrate for vegetation restoration and reconstruction 13,14 , which is a disposal route with broad prospect. All relevant previous studies have focused on the effect of municipal sludge on soil properties and plant growth; however, the influence on affected microbes received little attention. On the one hand, the many microbes in municipal sludge are pathogenic species, which may result in the pollution of soil. On the other hand, many microbes also can improve the physical and chemical features of soil to promote the growth of plants. So during the application of municipal sludge, the change of microbe diversity need to be investigated.
The construction waste is a main solid waste in city, which causes huge consumption of limited resources and leads to serious environment problem. In some areas all or part of construction waste is unlawfully deposited on land, or in natural drainages including water, contrary to regulations to protect human health, commerce and the environment. At present, landfill is the main disposal pathway of the construction waste, which increases the burden on landfill loading and operation, and the waste from sources such as solvents or chemicals can result in soil and water pollution. China is the largest producer of construction waste in the world. In 2015, totally 2,400 million tons of construction waste was produced in China, which accounts for 40% of the total amount of municipal waste. It is predicted that the amount of construction waste will arrived at 50 and 75 tons in 2020 and 2030, respectively 15 . But at present, the recycling utilization rate of construction waste is only about 5% in China, so the big city besieged by plenty of waste, which harms the urban safety and ecological development. Thus the effective and harmless method for the disposal of construction waste is in serious demand. Basis on the laboratory study of construction waste in the substrate mix for extensive green roofs 16 , we think that using construction waste as substrate in vegetation restoration is feasible.
In this study, the application of construction waste combined with municipal sludge in vegetation restoration was considered. The seeds of Ailanthus altissima were sowed on the substrate composed of municipal sludge mixed with construction waste residue. Then the growth of A. altissima, the substrate characteristics, and substrate microbial diversity were measured to investigate the potential recycling and reusing pathway of municipal sludge.

Results
The pH values of different substrates were tested (Fig. 1). The pH value of pure sludge (W) was about 7.00, while the pH value of garden soil (TYC) was about 8.00. When the sludge was mixed with construction waste residue (WJ), the pH value significantly increased by 6.90% compared with W (P <0.05). And after planting A. altissima, the pH value of mixed substrate (WJC) rose to 7.80. Table 1 showed the nutrient contents of substrates. We found that in W, the nutrient contents were significantly higher than in TYC (P < 0.05). In W the contents of organic matter, total nitrogen, total phosphorus, and total potassium were 6.68, 11.23, 23.11, and 3.39 folds of those in TYC, respectively. Moreover, mixture with construction waste residue resulted in the significant decreases of these nutrient contents (P < 0.05), and further reduction was found after planting A. altissima. In WJC, the contents of organic matter, total nitrogen, total phosphorus, and total potassium decreased to 53.71%, 34.12%, 27.35%, and 45.86% of those in W, respectively, which was also lower than those in WJ; however, these values were still far higher than those in TYC (P < 0.05). The contents of available nitrogen, available phosphorus, and available potassium were also found to show similar patterns.
The contents of heavy metals in substrates were also examined ( Table 2). It was found that the sludge-containing substrates had significantly higher contents of heavy metals than in TYC (P < 0.05), especially in W. The contents of Cu and Zn in W were 4.53 and 5.95-fold of those in TYC. Similar to the nutrient contents, the contents of heavy metals gradually decreased from W to WJ and to WJC. In WJC, the contents of As, Cd, Cr, Cu, Pb, and Zn were also significantly higher than those in TYC by 14.35%, 100.00%, 22.50%, 175.90%, 40.88%, and 317.88%, respectively (P < 0.05).
After growth for eighteen months, the survival rate, height, and coverage of A. altissima were investigated (Fig. 2). The survival rate of in WJC was about 6.66% lower than that in TYC ( Fig. 2A). However, the height and coverage of in WJC were higher than those in TYC (Fig. 2B,C). The height in WJC almost arrived at 35.00 cm, which was 25.00% higher than that in TYC. Compared to TYC, the coverage in WJC was 11.11% higher.
After high-throughput sequencing using the Illumina HiSeq platform, the data of samples were obtained and analyzed (Supplementary Table S1). The Q30 values of all samples exceeded 98.00%, and the GC values were about 55.00%. Moreover, Venn diagrams of OTUs exhibited the difference of microbial diversity between different samples ( Supplementary Fig. 3). To analyze the diversity among all samples, microbial species and their proportions at phylum levels are shown in Fig. 3. Due to the contained sludge, the dominant microbes showed similar proportions in W, WJ, and WJC, which largely differed from TYC. For these three samples, Proteobacteria was the largest proportion of population, which nearly arrived at 50.00%. In TYC, although Proteobacteria also occupied the highest proportion, it only reached about 35.00%, and Acidobacteria accounted for a further high proportion with about 25.00%. In W, WJ, and WJC, the proportion of Acidobacteria did not exceed 10.00%. Bacteroidetes also accounted for high proportions which was larger than that in TYC. Acidobacteria and Thaumarchaeota were found to gradually increase from W to TYC, while the proportion of Firmicutes presented the opposite pattern. In WJC and TYC Gemmatimonacetes also exhibited higher proportions than in W and WJ, which nearly arrived at 12.00% and 5.50%, respectively.  To demonstrate the different compositions of the microbial community structures in four samples, hierarchical cluster analysis of microbial communities at the genus level in shown in Fig. 4. The relative abundances of these 35 genera varied significantly in different soil samples. However, in W and WJ, the relative abundances showed a similar pattern to some extent. Both in W and WJ, Brevundimonas, Cellvibrio, Lysobacter, Streptomyces, Pseudomonas, Planctomyces, Pedobacter, Bacillus, and Pseudarthrobacter presented relatively high abundances. In W, the abundances of Massilia, Psychrobacter, Acinetobacter, Chryseobacterium, Sporosarcina, and Pseudarthrobacter were higher than those of other genera. In WJ, Dokdonella and Rhodanobacter exhibited the highest abundances. In WJC Filimonas, Steroidobacter, Chryseolinea, Sphingomonas, Haliangium, Neisseria, Bryobacter, Gemmatimonas, unidentified_Gemmatimonadaceae, and Ohtaekwangia were found to have high relative abundances. Ten genera (e.g., Terrimonas, Ohtaekwangia, Flavobacterium, Pirellula, and Ramlibacter) showed high abundances in TYC. In these samples, the microbial genera with high abundances belonged to more than two phyla, which indicated high microbial diversities in these samples.
The beta diversity difference among samples of W, WJ, WJC, and TYC was analyzed in Fig. 5. Significant differences were not observed between W and WJ or between WJC and TYC. However, a significant difference was found between the samples (W and WJ) without plants and the samples (WJC and TYC) with planting A. altissima.
PCoA clearly showed variations of microbial communities among the four soil samples (Fig. 6). W and WJ were not separated and thus possessed similar microbial community compositions. WJC was separated from W and WJ along the second component (PC2), while TYC was separated from W and WJ along the first component (PC1).
A persistent difference in microbial taxa patterns was found via LEfSe analysis (Fig. 7). In W, the relative abundances of Gammaproteobacteria class, Pseudomonadales order, Firmicutes phylum, Moraxellaceae family, Bacillales order, Psychrobacter genus, Psychrobacter species, Planococcaceae family, Sporosarcina genus, Sphingobacteriaceae family, Pedobacter genus, Pseudomonas genus, Pseudomonadacear family, and Acinetobacter genus were higher than three other substrates, among which the Gammaproteobacteria class showed the highest LDA score. WJ had high relative abundances of Xanthomooadaceae family, Xanthomonadeles order, Actinobacteria pylum, uni-dentified_Actinobacteria class, Sphingobacteriia class, Sphingobacteriales order, and Lysobacter genus than other substrates. Compared to W, WJ, and TYC, in WJC Gemmatimonadetes phylum, unidentified_Gemmatimonadetes class, Gemmatimonadaceae family, Gemmatimonadales order, unidentified_Gemmatimonadaceae genus, and Holophagae class showed higher relative abundances. Only the phylum Acidobacteria was found to have higher relative abundance in TYC than the other three substrates.

Discussion
Increasing concern is caused by the substantial generation of municipal sewage sludge and the inadequate development of the wastewater treatment industry, which causes environmental problems. Due to its high nutrient content, municipal sludge is used as soil conditioner and fertilizer to recycle its nutrients through the soil-plant system [17][18][19] . Sewage sludge has many beneficial effects on soil fertility, enriching it with its nitrogen, phosphorus, and micronutrient contents, and improving the physicochemical, microbiological, and enzymatic properties of  soil 11,20,21 , which is an economic and effective method for disposal. However, a high level of contaminants, especially of heavy metals and pathogenic microbes, leads to a long-term environmental risk in soil 18 . Application of municipal sludge for vegetation restoration in a degraded ecosystem can avoid the potential risk that toxic contaminants enter the human food chain. This study investigated the potential application of municipal sludge combined with construction waste for vegetation restoration. The utilized municipal sludge was found to have near neutral in pH, and the mixture with construction waste residue increased the pH value; moreover, after the growth of A. altissima for eighteen months, the pH value further increased. Although the mixed substrate was weakly alkaline, the pH value was evidently lower than that of garden soil. The application of sludge in tree peony-soil ecosystems also decreased the pH value, which was close to neutral soil pH, and was conducive to tree peony 20 . The municipal sludge used in this study had high nutrient contents (Table 1), which was too high for plants, while the construction waste residue contained few nutrients ( Table 3). The addition of construction waste residue not only decreased the nutrient contents to a still high but  acceptable level for plants (Table 1), it also improved the air permeability. A. altissima seedlings in the mixed substrate obtained better nutrient conditions than in garden soil; therefore, we can find that the height and coverage of A. altissima in mixed substrate was better than that in garden soil. The plant height, flower diameter, and flower numbers per plant of tree peony significantly increased after sludge compost amendment 22 . Application of sewage sludge compost as fertilizer in soil with mass ratios of 30.00% also promoted the height, ground diameter, and biomass of Mangifera persiciforma trees 23 . Compared to the application of sludge to soil, the mixed substrate of municipal sludge and construction waste residue also achieved the same beneficial effect of A. altissima plant growth; moreover, both of them were reused. Although the survival rate of A. altissima in mixed substrate was impacted, and was a little lower than in garden soil, it still achieved about 75.00%. Moreover, after mixing with construction waste residue, the contents of main heavy metals in the municipal sludge decreased significantly. In the mixed substrate with A. altissima (with reference to Environmental Quality Standard for Soils of China, GB15618-1995), the contents of As, Cr, and Pb reached the level of first-degree soil, and the contents of Cd, Zn, and Cu met the criterion of second-degree soil. Many previous studies focused on the heavy metals during the land application of municipal sludge, and found that as long as the application amount was controlled within reasonable levels, the pollution and hazard of heavy metals could be avoided 7,23-26 . Based on our data, with regard to heavy metal contents, the mixed substrate belonged to Class II soil, which could be used to grow crops, vegetables, tea trees, fruit trees, and forage grasses, and would not cause pollution or be hazardous for plants and the environment. In addition, the moisture content (Fig. 8) and the activities of soil enzymes including urease, catalase, invertase, and alkaline phosphatase (Table 4) in the mixed substrate were significantly higher than those in garden soil, which was also beneficial for A. altissima growth.    The microbes play an important role for the physicochemical properties of soil and plant growth. Large numbers of microbes can be found in municipal sludge, many of which are species that decompose organic matter, while some of them are pathogenic species that may be environmentally hazardous. Therefore, in the application of municipal sludge, the diversity of microbes is a focal point. In this study, using high-throughput sequencing on the Illumina HiSeq platform, the microbial diversity of mixed substrate was investigated. Our experimental results demonstrated that the microbial community structure observed in the substrate samples containing municipal sludge significantly differed from that of garden soil. Phylum abundance analysis showed that although Proteobacteria was the top microbial phylum in all samples, compared to garden soil, higher values were found in samples that contained municipal sludge. Furthermore, the relative abundance of Proteobacteria in activated sludge of a municipal wastewater treatment plant of Northern China in summer arrived at about 50.50% 27 . Proteobacteria included many important ammonia oxidizing bacteria involving nitrogen fixation, such as Betaproteobacteria and Gammaproteobacteria 28 . Therefore, municipal sludge can provide more nitrogen than garden soil. Bacteroidetes, Fimicutes, and Actinobacteria were the other main phyla in samples containing municipal sludge with higher relative abundance than garden soil. However, in a recent study, the proportions of Fimicutes and Actinobacteria in activated sludge were low 27 . This may result from differences in sludge sources. In garden soil, Acidobacteria and Thaumarchaeota were found to have a higher relative abundance compared to samples containing municipal sludge. Acidobacteria has been reported to be a main bacterial phylum in agricultural or other types of soils, with some variation in their relative abundance 29,30 . Thaumarchaeota was named by Brochier-Armanet et al. 31 , and plays an important role in the ammonia oxidization and carbon fixation in soil.

Nutrient index
No reports exist about Thaumarchaeota in municipal sludge. Therefore, a large difference was found between the substrate samples containing municipal sludge and garden soil.
In addition, after growing A. altissima, the relative abundances of Acidobacteria and Gemmatimonadetes increased in the mixed substrate, especially for Gemmatimonadetes whose relative abundance in WJC was also far higher than that in TYC. Moreover, the relative abundance of Fimicutes decreased after growing A. altissima in the mixed substrate. Therefore, it was demonstrated that the growth of A. altissima resulted in these microbial changes. The planting of apple seedlings was reported to exert a large impact on the bacterial community in the soils of replanted new planting sites 32 .
At the genus level, heat map analyses and PCoA also showed the difference among these four substrates. Similarly, at phylum level, the microbial communities in TYC were significantly different from W, WJ, and WJC, and compared to W and WJ, WJC was somewhat closer to TYC, which was also demonstrated by the analysis of beta diversity. As the dominant microbial communities in WJ, Dokdonella and Rhodanobacter presented low relative abundance in WJC. Rhodanobacter was reported to be likely involved in the denitrifying process, leading to denitrification in low pH soil 33 . In this study, Rhodanobacter was found to be part of the prominent microbial communities in the alkaline substrate. For common soil, Rhodanobacter had a negative effect on nitrogen nutrition supply due to nitrogen losses, while for the substrate containing municipal sludge with high nitrogen content, its denitrification can decrease the nitrogen nutrition content to a suitable level for the growth of A. altissima, which played a positive role. However, in WJC, nine microbial communities, such as Filimonas, Chryseolinea, Sphingomonas, and Gemmatimonas accounted for high relative percentages. It has been reported that Sphingomonas can effectively degrade dibenzo-p-dioxin 34 , hexachlorocyclohexane 35 , and phenazine-1-carboxylic acid 36 in soils. Moreover, Sphingomonas was also capable to degrade fenvalerate, permethrin, fenpropathrin, beta-cypermethrin, cyhalothrin, deltamethrin, bifenthrin, and 3-PBA, and showed multi-resistance to heavy metals and antibiotics 37 . Therefore, Sphingomonas had potential for degrading organic pollutants, especially pesticides in substrate. Furthermore, Gemmatimonas was confirmed to reduce the emission of N 2 O, which is a potent greenhouse gas and ozone depletion agent originating from soil 38 . The high relative abundance of Gemmatimonadaceae suggested that WJC may be beneficial for the atmospheric environment.

Conclusions
This study showed that using municipal sludge mixed with construction waste residue as substrate, increased the growth of A. altissima seedlings and the physiochemical characteristics were suitable for plants, suggesting that the substrates showed potential for vegetation restoration. The different substrates showed various microbial diversities. Proteobacteria was the top microbial phylum in all samples and a higher relative abundance were found in the samples containing municipal sludge. After growing A. altissima, the relative abundances of Acidobacteria and Gemmatimonadetes increased in the mixed substrate. Therefore, the addition of construction waste residue and the growth of A. altissima caused the difference. The microbial communities in WJC was plant-friendly and environment-friendly. The results suggested that using as substrate for vegetation restoration was a potentially feasible and effective pathway for the reuse and recycling of municipal sludge. The heavy metal contents in WJC met the criterion of the Class II soil although the values were significantly higher than in TYC. Therefore, using this mixture as substrate in vegetation restoration was a potentially feasible and effective pathway for the reuse and recycling of municipal sludge and construction waste residue.

Materials and Methods
Experimental Design. The experiment was conducted in the Science and Technology Park of the Beijing University of Agriculture. The municipal sludge used in this study was taken from the Xiaotangshan sewage treatment plant of the Changping district in Beijing, and was naturally dried in the air. The construction waste residue originated from a construction site near the Tingzizhuang village of Changping district in Beijing, and big bricks and stones were removed. The municipal sludge was mixed with the construction waste residue at a volume ratio of 1:1 to obtain the treatment substrate. The garden soil in the nursery was used as control substrate. Both types of experiment plots (5 m × 5 m) were paved with treatment and control substrate to 50 cm thickness, respectively. Three plots were prepared for every type, acting as three replicates. A. altissima was used as plant material. Five hundred A. altissima seeds (China National Tree Seed Corporation, Beijing) were uniformly sowed (100 seeds per square meter) on two experimental plots in April of 2015. The A. altissima seedlings grew under natural condition. Soil Sampling. Pure sludge and mixed substrate without planting A. altissima were sampled. After planting A. altissima, both mixed and control substrates were collected in October of 2016. In each plot, at a depth of 10-15 cm, the substrates close to the roots of 10 A. altissima seedlings with medium size were collected and were divided into two parts. One part of the substrates was used to determine the characteristics, while the other part was frozen in liquid N 2 immediately for subsequent DNA extraction. The samples are listed in Table 5.
Substrate Characteristics Measurement. The pH value was determined with a Delta 320 pH meter (Mettler-Toledo International Inc., Switzerland) at substrate: water = 1:5. The content of organic matter was determined using the potassium dichromate volumetric method. Using the Kjeldahl determination, the content of total nitrogen was investigated in substrate after heating digestion by concentrated sulfuric acid. The contents of total phosphorus, total potassium, and heavy metals were determined by ICP-AES (6,300, Thermo Fisher Scientific Inc., USA) after microwave digestion.
Growth Investigation of A. altissima. In October, the survival rate of A. altissima was calculated. The height of 30 seedlings with medium size were measured with a meter stick. Moreover, the coverage was investigated using a canopy projection method. DNA Extraction. Total genomic DNA from soil samples was extracted using the CTAB/SDS method. DNA concentration and purity were monitored on 1.00% agarose gels. Each sample extraction was conducted three times. DNA concentration and purity were monitored on 1.00% agarose gels. According to the concentration, DNA was diluted to 1 ng·L −1 using sterile water. Then, the DNA extractions were used as template DNA for high-throughput sequencing (HTS) analysis by Novogene Bioinformatics Technology Co., Ltd., Beijing, China.
Microbial 16S rRNA Gene Amplification. The protocol of Caporaso et al. 39 was used to determine the diversity and composition of the microbial communities in soil samples. PCR amplifications were conducted with the 515F-806R primer set amplifying the V4 region of the 16 S rDNA gene. All PCR reactions were conducted in 30 μL reactions with 15 μL of Phusion ® High-Fidelity PCR Master Mix (New England Biolabs); 0.2 μM of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consisted of an initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and elongation at 72 °C for 30 s. Finally, 72 °C for 5 min.
PCR Product Quantification and Qualification. The same volume of 1X loading buffer (contained SYB green) was mixed with PCR products and electrophoresis was conducted on a 2.00% agarose gel for detection. Samples with bright main strip between 400-450 bp were chosen for further experiments.
PCR Products Mixing and Purification. PCR products were mixed at equidensity ratios. Then, mixture PCR products were purified with the GeneJET Gel Extraction Kit (Thermo Scientific).
Library Preparation and Sequencing. Sequencing libraries were generated using the TruSeq DNA PCR-free Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit@2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina HiSeq platform and 250 bp paired-end reads were generated. Data Analysis. Paired-end reads from the original DNA fragments were merged using FLASH 40 , which is a very fast and accurate analysis tool that was designed to merge paired-end reads when there were overlaps between reads1 and reads2. Paired-end reads were assigned to each sample according to unique barcodes. Sequences were analyzed using the QIIME software package (Quantitative Insights Into Microbial Ecology) 39 , and in-house Perl scripts were used to analyze alpha-(within samples) and beta-(among samples) diversity. First, reads were filtered by QIIME quality filters. Then, pick_de_novo_otus.py was used to pick operational taxonomic units (OTUs) by making a OTU table. Sequences with ≥97.00% similarity were assigned to the same OTUs. A representative sequence was picked for each OTU and the RDP classifier was used to annotate taxonomic information for each representative sequence 41 . QIIME calculates unweighted unifrac, which is phylogenetic measure of beta diversity. Unweighted unifrac was used for Principal Coordinate Analysis (PCoA). To further mine the data of microbial diversity of the differences between the samples, LEfSe was conducted.