Molecular Analysis of Bacterial Community Succession During Prolonged Compost Curing

RESEARCH ARTICLE Molecularanalysis of bacterial community succession during prolonged compost curing Michael Danon1, Ingrid H. Franke-Whittle2, Heribert Insam2, Yona Chen1 & Yitzhak Hadar1

1Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel; and 2Institute for Microbiology, University of Innsbruck, Technikerstraße 25d, A-6020 Innsbruck, Austria Downloaded from https://academic.oup.com/femsec/article/65/1/133/620549 by guest on 30 September 2021

Correspondence: Yitzhak Hadar, Abstract Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food The compost environment consists of complex organic materials that form a and Environmental Quality Sciences, The habitat for a rich and diverse microbial community. The aim of this research was to Hebrew University of Jerusalem, PO Box 12, study the dynamics of microbial communities during the compost-curing phase. Rehovot 76100, Israel. Tel.: 1972 8 9489935; Three different methods based on 16S rRNA gene sequence were applied to fax: 1972 8 9468785; e-mail: monitor changes in the microbial communities: (1) denaturing gradient gel [email protected] electrophoresis of PCR-generated rRNA gene fragments; (2) partial rRNA gene clone libraries; and (3) a microarray of oligonucleotide probes targeting rRNA Received 20 December 2007; revised 11 March gene sequences. All three methods indicated distinctive community shifts during 2008; accepted 10 April 2008. First published online 4 June 2008. curing and the dominant species prevailing during the different curing stages were identiﬁed. We found a successional transition of different bacterial phylogenetic DOI:10.1111/j.1574-6941.2008.00506.x groups during compost curing. The Proteobacteria were the most abundant phylum in all cases. The Bacteroidetes and the Gammaproteobacteria were Editor: Alfons Stams ubiquitous. During the midcuring stage, Actinobacteria were dominant. Different members of nitrifying bacteria and cellulose and macromolecule-degrading Keywords bacteria were found throughout the curing process. In contrast, pathogens were biosolids compost; clone library; community not detected. In the cured compost, bacterial population shifts were still observed composition; oligonucleotide microarray; after the compost organic matter and other biochemical properties had seemingly organic matter degradation; PCR-DGGE. stabilized.

toward Sclerotium rolfsii has been demonstrated during Introduction prolonged curing time (Danon et al., 2007). Compost of an Composting is an aerobic process by which organic materi- appropriate age should therefore be used to control disease als are degraded through the activities of successive groups in infested soils before sowing. The successful application of of microorganisms. Soil amendment with composted or- compost is considerably dependent on the selection of an ganic material is an ancient practice that is applied through- appropriate curing period. out the world, and the long-term benefits of compost The biochemistry of the compost-curing process has been application to fields are well documented (Ros et al., 2006). studied extensively. Chen et al. (1989) found that the Previous studies have emphasized the importance of achiev- original cellulose and hemicellulose contents of cattle man- ing compost maturity to ensure balanced plant nutrition ure were reduced by one-third during a 5-month compost- and for the biological control of soil-borne plant disease ing and maturing process. Hemicellulose and cellulose may (Fuchs, 2002; Noble & Coventry, 2005). Compost maturity be the main substrates for microorganisms during the is achieved during the curing process. The duration of maturation process because these components are present curing in the industry varies according to a number of in large quantities in cattle manure and the less complex factors, including source materials, composting process and carbon sources are consumed early on in the process (Chen facility, climate, and planned utilization of the final product. et al., 1989). Recently, Tang et al. (2006) reported that Noncured composts may be phytotoxic, whereas extensively organic matter decomposition during the maturation of cured composts may lose their plant-disease-suppressive cattle-manure compost resulted in decreased C/N ratios, properties. For instance, loss of compost suppressiveness microbial biomass, and microbial diversity. The C/N ratio in

FEMS Microbiol Ecol 65 (2008) 133–144 c 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 134 M. Danon et al. the compost may level off much before the compost vation-independent techniques based on 16S rRNA gene stabilizes (Chefetz et al., 1996) and although the concentra- sequences: PCR-denaturing gradient gel electrophoresis tion of the dissolved organic carbon (DOC) of municipal (DGGE), clone libraries, and an oligonucleotide microarray solid-waste compost did not change at maturation, the (COMPOCHIP). Molecular techniques are becoming in- relative concentrations of different hydrophilic and hydro- creasingly useful for the detection of different microorgan- phobic fractions in the dissolved organic matter continued isms without ﬁrst having to isolate and cultivate them (Dees to change concomitant with the decrease in phytotoxicity & Ghiorse, 2001; Green et al., 2004; Franke-Whittle et al., (Chefetz et al., 1998). Tang et al. (2006) showed that during 2005; Kelly et al., 2005). Although they share the same basis, the maturation period, the proportion of Actinobacteria i.e. exploiting conserved and variable regions in the riboso- increases slightly, and this was associated with the disap- mal small subunit sequence, each of the techniques used in

pearance of phytotoxicity. our study has its inherent advantages and disadvantages. A Downloaded from https://academic.oup.com/femsec/article/65/1/133/620549 by guest on 30 September 2021 Considerable effort and a variety of techniques have been combined analysis was therefore expected to produce more applied to the study of compost microbial populations reliable information on the qualitative and quantitative (Cahyani et al., 2003; Schloss et al., 2003; Ryckeboer et al., succession of bacterial populations during compost curing. 2003b). The initial phase of composting is thought to be the most dynamic part of the process and is characterized by rapid increases in temperature, large changes in pH, and the Materials and methods degradation of simple organic compounds. Schloss et al. Prolonged compost-curing process and compost (2003) reported two significant shifts in the composition of sampling the microbial community: one occurring between 12 and 24 h and the other between 60 and 72 h into the process. Compost samples were obtained from a commercial com- Ryckeboer et al. (2003b) attempted to determine the micro- posting facility (Shacham, Givaat Ada, Dlila Facility, Israel) bial succession of the dominating taxa and functional that prepared compost from a mixture of sewage sludge and groups of microorganisms, as well as the total microbial yard waste (1 : 1, v/v) in windrows (2.5 m wide and 2 m activity during composting of biowaste, using incubation, high). Aeration was achieved by bi-weekly turning for the isolation, and enumeration techniques. They reported that entire 12-week composting process. The biosolids compost, bacteria dominated during the thermophilic phase while considered to be mature by the producers, i.e. appropriate fungi, Streptomycetes, and yeasts were below detection for field application, was sampled and further treated as limits. Different bacterial populations were found in the follows: c. 700 L of bulk compost was collected and placed in thermophilic and mesophilic composting phases. During a cubic, 700-L bin, rewetted and turned every 2 weeks for the the peak-heating phase of fresh wastes, the only bacteria first month, then every month for the next 4 months. The isolated were bacilli; however, during the cooling and resultant compost pile was then left unturned for seven maturation phases, the bacterial diversity of both Gram- additional months (in total, 1 year of prolonged curing). positive and Gram-negative bacteria increased (Ryckeboer Compost temperature in the container increased after et al., 2003b). Using cultivation-independent methods, rewetting to 42 1C in the first week, and then decreased over Cahyani et al. (2003) studied the bacterial communities in the next 2 weeks to 32 1C(c.51C above ambient). Approxi- composting of rice straw. They reported a successional mately 20 L of compost was sampled from the bin at time 0 transition of bacterial community members: Alphaproteo- (beginning of prolonged curing), then after each turning, bacteria in the raw materials, Bacillus and Actinomycetes at and stored at 4 1C until analysis. the thermophilic stage, and Cytophaga and clostridial members at the middle and curing stages. These authors reported DNA extraction, general molecular procedures, that the microbial community remained stable during the and replications curing phase. In contrast, Steger et al. (2007) revealed compositional changes within the Actinobacteria commu- DNA from 0.25 g of compost subsamples was extracted nity in a full-scale composting process of organic household using a ‘PowerSoil’ kit (Mobio, Solana Beach, CA). The waste over a period of 57 weeks. DNA extracts were used as templates for PCR amplification Despite the important role of microbial populations in of bacterial partial 16S rRNA genes for DGGE, clone compost quality, only a few studies have thoroughly exam- libraries, and oligonucleotide microarray analysis. For ined the dynamics of the total microbial populations during DGGE, we used two replicate DNA samples. The DNA was compost curing. The aim of this study was to characterize amplified using the 341-907 primer set with a GC clamp changes in the microbial community structure during (Muyzer et al., 1998) and the products were run in separate prolonged curing of biosolids compost, using molecular lanes. For clone libraries we used three replicate DNA techniques. For this purpose, we used three different culti- samples. The DNA was amplified using the 341-907 primer

c 2008 Federation of European Microbiological Societies FEMS Microbiol Ecol 65 (2008) 133–144 Published by Blackwell Publishing Ltd. All rights reserved Bacterial succession during compost curing 135 set without a GC clamp. The PCR products were pooled and viously in the composting process, as well as plant, animal, mixed before ligation to a vector. and human pathogens, and plant-disease-suppressive bacteria, was applied to DNA extracted from different PCR-DGGE analysis composts. The specificity of all probes was assessed in silico, using the ARB program (Ludwig et al., 2004), and the PCR amplification and DGGE were conducted as described array was tested with pure cultures of microorganisms, previously (Danon et al., 2007). Briefly, DGGE was prepared and was shown to work well with only a low percentage according to Muyzer et al. (1993) using an IngenyPhor-U2 of nonspecific hybridizations (Franke-Whittle et al., 2005). system (Ingeny, Goes, The Netherlands) with 6% (w/v) For most target organisms, at least three probes were polyacrylamide gel [acrylamide/bisacrylamide (37 : 1)] in spotted on the slide, and for a few organisms there were 1 Â Tris acetate–EDTA (TAE) buffer and a 20–60% dena-

only two probes. All the probes included on the COMPO Downloaded from https://academic.oup.com/femsec/article/65/1/133/620549 by guest on 30 September 2021 turing gradient (80% denaturant corresponding to 7 M urea CHIP microarray were designed so as to have similar and 32%, v/v formamide). Dominant DGGE bands were melting temperatures, and probe sequences ranged in excised and reamplified. Reamplified bands that migrated length from 17 to 25 nucleotides. Five or six replicate identically to the excised bands in the DGGE were cloned DNA samples were analyzed. Fluorescence labeling of and sequenced. target DNA, hybridization, scanning of arrays, and image analysis were conducted as described by Franke-Whittle Cloning, sequencing, and phylogenetic analysis et al. (2005). Two clone libraries, for noncured (0 days) and cured (336 days) compost samples, were constructed using the pGEM- Statistical analysis T easy vector system (Promega, Madison, WI). Ligation and Statistical analysis of the DGGE data was conducted using transformation were performed according to the manufac- Dice correlation coefficients and the unweighted-pair-group turer’s directions. Colonies were screened for the presence of method with arithmetic averages (UPGMA) to form a the correctly sized insert, and plasmid DNA was reamplified complete linkage dendrogram (Fingerprinting II Informa- before sequencing. Plasmids from cloned PCR-DGGE bands tix, BioRad Laboratories, Hercules, CA). were extracted and purified using a miniprep DNA purifica- Statistical analysis of clone libraries and coverage deter- tion kit (Genomed, Lohne,¨ Germany). mination were conducted using the procedure developed by Sequencing was performed at the Macrogen Inc. Sequen- Kemp & Aller (2004) to confirm that an asymptotic accu- cing Center (Seoul, Korea). Sequences were examined using mulation curve in both libraries had been reached. Unifrac the CHECK_CHIMERA program located at the Ribosomal Data- (Lozupone et al., 2006) was used to define phylotypes at 3% base Project (Cole et al., 2005), and chimeric sequences were similarity cutoff. removed from phylogenetic analyses. Statistical analyses of microarray data were performed Phylogenetic analysis of sequences derived from clones using the program CANOCO 4.5 (ter Braak & Sˇmilauer, 2002). and from DNA of excised bands was performed by align- Data from the microarray analysis, the physicochemical ment to known bacterial sequences using the ‘greengenes’ analysis, and the cloning of DGGE bands were subjected to 16S rRNA gene database and alignment tool (DeSantis et al., principal component analysis (PCA). As differing numbers 2003) (http://greengenes.lbl.gov/). Aligned sequences and of replicates were used for each set of experiments, an close relatives were imported to the MEGA software package average was used for each set of replicates in the statistical version 3.1 (Kumar et al., 2004). Similarity was tested to analysis. Because a strong correlation between SD and sequences available at the National Center for Biotechnology means of replicate samples was found, a log-transformation Information (NCBI) using BLAST analysis (Altschul et al., of microarray data was conducted to equalize variances. For 1997). The phylogenetic tree was constructed using the MEGA the covariance-based redundancy analysis (RDA), the fol- software with the Kimura two-parameter method for dis- lowing settings were used: inter-sample distance scaling, no tance matrix calculations and the neighbor-joining method post-transformation of scores, log data transformation (no for tree design. Tree topologies were evaluated by perform- offset), and center by species. ing bootstrap analysis of 1000 data sets. The rRNA gene sequences were submitted to the GenBank database under accession numbers EU215227–EU215310. Results

Oligonucleotide microarray analysis PCR-DGGE The COMPOCHIP microarray, spotted with 369 probes The community composition of bacteria in biosolids com- targeting microorganisms that have been reported pre- post samples subjected to different curing times is shown in

40 50 60 70 80 90 100 Compost curing time (days) 0 0 19 19 41 41 67 67 130 167

Fig. 1. PCR-DGGE analysis of the bacterial Downloaded from https://academic.oup.com/femsec/article/65/1/133/620549 by guest on 30 September 2021 205 community in composts from different stages of 167 curing. A UPGMA algorithm was applied to a 205 similarity matrix of Dice and Pearson correlation 130 coefﬁcients generated from the DGGE banding patterns. The numbers correspond to bands 336 identiﬁed by 16S rRNA gene sequence analysis 336 (Table 1).

Table 1. Identification of PCR-DGGE bands by 16S rRNA gene sequence Clone libraries analysisÃ Clone libraries were constructed from cured (0 days) and Band Accession noncured (336 days) composts, each library including a number number Phylum/class Family total of 41 and 43 clones, respectively. The complete 8 EU215311 Bacteroidetes Cryomorphaceae phylogenetic analysis is given in the supplementary infor- 10 EU215312 Bacteroidetes Sphingobacteriaceae mation. Figure 2 illustrates the phylogeny of compost 13 EU215313 Alphaproteobacteria Phyllobacteriaceae bacteria members affiliated with (a) Actinobacteria and (b) 15 EU215314 Alphaproteobacteria Caulobacteraceae 19 EU215315 Gammaproteobacteria Xanthomonadaceae Betaproteobacteria. The clone libraries were found to differ 21 EU215316 Gammaproteobacteria Xanthomonadaceae significantly with respect to phylotype composition (P value 23 EU215317 Gammaproteobacteria Xanthomonadaceae 0.01). Clone rRNA gene sequences were found to belong 26 EU215318 Gammaproteobacteria Xanthomonadaceae to seven different phyla: Actinobacteria, Bacteroidetes, Chlor- 29 EU215319 Bacteroidetes Flavobacteriaceae oflexi, Deinococci, Gemmatimonadetes, Firmicutes, and Pro- 30 EU215320 Actinobacteria Corynebacterineae teobacteria. Most of the clones, derived from both the cured 37 EU215321 Alphaproteobacteria Caulobacteraceae and noncured composts, were classified as belonging to the 38 EU215322 Actinobacteria Promicromonosporaceae Alpha-, Beta-, Gamma-, and Deltaproteobacteria classes. The Ã Dominant DGGE bands were excised, reamplified and cloned. Ampli- Gammaproteobacteria class contained the highest number of cons and clones were compared with the original bands in DGGE. clones: five from the noncured compost and 12 from the Amplicons and clones that did not migrate to the identical positions of cured samples. Eight clones from the noncured compost and the excised bands in the DGGE were not identified. five clones from the cured compost were found to belong to Alphaproteobacteria. Only clones from the noncured compost grouped within the Caulobacteraceae family. Other Fig. 1. UPGMA analysis of the DGGE profiles generated groups found only in the noncured compost were Promicro- from individual PCR products showed that the duplicate monosporaceae (Actinobacteria) (Fig. 2a), Comamonadaceae samples of specific ages clustered together. The noncured (0 (Betaproteobacteria) (Fig. 2b) and members of the Chloro- days) samples were distinctly different from those represent- flexi phylum. Members of the Deinococcus phylum were ing longer curing times. Among the cured compost samples, present only in the cured compost clone library (Supple- the 19- and 41-day samples clustered separately from the mentary Fig. S1). longer curing times. The oldest sample (336 days) was distinctly different from the 67- to 205-day samples. The Microarray bands were numbered (1–44) and identified according to similarity to sequences in the GenBank and the greengenes The COMPOCHIP 16S rRNA gene microarray was applied database. The identifications of DGGE bands are listed in in order to directly determine which bacteria were present in Table 1. the different compost samples analyzed. Because a linear

60 Actinomadura pelletieri AF163119.1

(a) Actinobacteria 99 Actinomadura str. JCM 3308 AF134067.1 Cured compost clone M37 EU215289 91 Cured compost clone M46 EU215297 Thermomonosporaceae Noncured compost clone M3 EU215229 Cured compost clone M41 EU215293 Microbacteriaceae str. Ellin145 AF408987 65 97 89 Leifsonia str. PTX1 DQ901014.1

99 Noncured compost clone M11 EU215237 Noncured compost clone M65 EU215238 Downloaded from https://academic.oup.com/femsec/article/65/1/133/620549 by guest on 30 September 2021 56 Promicromonospora sukumoe AB023375.1 58 Promicromonosporaceae Cellulomonas sp. str. X7 AF060791.1 95 Compost PCR-DGGE band 38 EU215322

89 Noncured compost clone M62 EU215242 Cured compost clone M17 EU215270

81 Cured compost clone M23 EU215276 Cured compost clone M25 EU215278

Compost PCR-DGGE band 30 EU215320 91 Cured compost clone M36 EU215288 79 60 Cured compost clone M52 EU215301 Cured compost clone M42 EU215294 Corynebacterineae 98 Mycobacterium terrae X52925.1 Mycobacterium tokaiense AF480590.1

77 Mycobacterium thermoresistible M29570.1 93 Noncured compost clone M76 EU215252

0.01 Noncured compost clone M86 EU215261 67 (b) Betaproteobacteria Cured compost clone M85 EU215307 Noncured compost clone M87 EU215262 71 Oxalobacteraceae Cured compost clone M32 EU215284 99 Oxalobacter str. HI-D2 DQ196473.1

Oxalobacteraceae isolate D8-13b AM403210 Variovorax paradoxus str. WP1 AF208386.1

Noncured compost clone M2 EU215228

Fungal ascocarp clone AY599736.1 99 Sludge clone H21 AF234687.1 61 Denitrifying reactor clone 81 AJ412678.1

97 Noncured compost clone M6 EU215232 Comamonadaceae Ottowia thiooxydans str. K11 AJ537466.1 91 Acidovorax avenae AY512827.1 55 Acidovorax sp. AHL 5 AY379977.1 Noncured compost clone M9 EU215235 69 Noncured compost clone M75 EU215251

0.01

Fig. 2. 16S rRNA gene phylogeny of bacteria found in composts at different curing stages (a) Actinobacteria and (b) Betaproteobacteria phyla. Neighbor-joining phylogenetic tree drawn to scale, with branch lengths computed using the maximum composite likelihood method. The scale bar represents 0.01 base substitutions per site. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Sequences of PCR-DGGE bands are marked by triangles. Sequences from noncured and cured compost are marked by black and white circles, respectively.

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41-c Downloaded from https://academic.oup.com/femsec/article/65/1/133/620549 by guest on 30 September 2021 130 d 67-e 67-d RDA Axis 2 0.0 67-f 336 d 41-e 41-a 67-a 336-d 336-e 130-e –0.2 67-c 336-b 130-f 130-d 336-a 336-f 130-c 336-c 67 d –0.4 130-a Fig. 3. Two dimensional ordination plot of compost communities from different curing stages –0.6 analyzed by the COMPOCHIP microarray and –1.0 –0.5 0.0 0.5 1.0 canonical analysis (redundancy analysis). Indivi- RDA Axis 1 dual replicates are represented by letters (a–f).

correlation between target concentrations and signal inten- the 336-day samples. These results were supported by strong sities of the various probes has been reported by others hybridization signals for probe 350, specific for A. faecalis. (Taroncher-Oldenburg et al., 2003; Tiquia et al., 2004), Despite its name, A. faecalis is not typical to feces, but is a the results obtained in this study were analyzed semi- common, nonpathogenic, environmental bacterium that quantitatively. has been reported to be present in composts by other Figure 3 shows an ordination graph: the two relevant axes authors (Droffner et al., 1995; Ryckeboer et al., 2003a). explained 34.1% of the variance. Multivariate analysis The 295 and 296 probes, specific for members of the showed that the bacterial-community compositions of the genera Nitrosovibrio and Nitrosospira, indicated the presence composts changed with curing time, namely, the noncured of these bacteria in composts throughout the curing process. and cured composts of different ages clustered separately. However, in the noncured samples, the probes exhibited Bacteria belonging to the low G1C and Alphaproteobacteria higher signal levels. groups were found in high numbers in composts from all curing stages, as well as noncured compost. The probe Discussion targeting the genus Actinomyces also revealed high levels of these organisms in both cured and noncured compost The microbial community in compost during prolonged samples. The microarray included probes specific for mem- curing was studied using three different culture-indepen- bers of the genus Chryseobacterium. Higher levels of this dent techniques. We followed the dynamics of the microbial genus were found in the younger composts than in the more population from the point at which the commercial produ- mature ones. Higher signals for the probes targeting Micro- cer considered the compost appropriately mature for field bacterium and Sphingobacterium were detected in the non- application and at which point it was found to be suppres- cured composts. The EUB 338II probe specific for the sive to plant pathogens (Danon et al., 2007). The results Verrucomicrobia group was found to correlate well with the obtained from analysis using DGGE, clone libraries, and a more cured composts. The Pseudomonas genus-specific microarray revealed that under the conditions tested here, probes gave signals above the threshold values for samples the compost community continued to change during the from days 0, 41, and 336, but not for those from days 67 curing phase. Recognizing the inherent advantages and and 130. disadvantages of each technique, we attempted to integrate High signal-to-noise ratios were obtained for composts the data analysis to produce a comprehensive picture of the from all curing stages with probe 270 specific for Alcaligenes microbial community dynamics. Microarrays allow the faecalis/defragrans. Although the signal intensities were parallel detection of up to several thousand microbial found to decrease with time, Alcaligenes was still present in strains, species, genera, or higher taxonomic groups in a

c 2008 Federation of European Microbiological Societies FEMS Microbiol Ecol 65 (2008) 133–144 Published by Blackwell Publishing Ltd. All rights reserved Bacterial succession during compost curing 139 single experiment, depending on the availability of the gobacterium, and Flavobacterium (Fig. 5). We found mem- probe set used on the microarray. However, because differ- bers of Sphingobacterium, both by DGGE (band 10) and ent probes have different afﬁnities for their targets (Loy & with the microarray (probe 550) in the noncured compost. Bodrossy, 2006), the information on the relative abundance The phylum Bacteroidetes includes a wide variety of of the different microorganisms derived from the micro- bacteria known for their utilization of macromolecules such array analysis needs to be interpreted with caution. We as proteins, starch, cellulose, and chitin (Manz et al., 1996), found higher signals (indicative of better hybridization) for and its members have been detected previously by mole- most probes (both higher taxonomic group level probes as cular methods in various composts (Alfreider et al., 2002; well as genus- and species-speciﬁc probes) upon hybridiza- Michel et al., 2002; Verkhovtseva et al., 2002). Green et al. tion with the noncured compost samples when compared (2004) reported that the most frequently detected

with the cured compost samples. This would indicate that sequences in compost-amended potting mixes belong to Downloaded from https://academic.oup.com/femsec/article/65/1/133/620549 by guest on 30 September 2021 the longer the curing time, the less likely it is that the Chryseobacteria. resulting compost product will have a bacterial community Nitrosovibrio and Nitrosospira were predominantly found that matches the expected typical compost bacteria targeted in the noncured composts in close proximity to the NH4 by the microarray. As a result of this, clone libraries were vector (Fig. 5). These bacteria were detected by the micro- more reliable for the comparison of higher taxonomic array only, indicating that they represent a minor popula- groups comprising the cured compost microbial commu- tion in the compost total microbial community nity (Fig. 4). (Fig. 4). Nevertheless, minor populations may play impor- In a previous report (Danon et al., 2007), we have tant roles in their environment, and ammonium-oxidizing described how biosolids compost undergoes a transition bacteria, including members of the above genera, are often from a highly active to a stabilized biosolid during pro- reported in composts (Kowalchuk et al., 1999; Innerebner longed curing. Samples of noncured compost were charac- et al., 2006). terized by intense activity, with rapid decomposition of We found bacteria belonging to the Betaproteobacteria to organic matter, as reflected by a higher respiration rate, a be more abundant in noncured than cured composts (Fig. faster utilization of glucose, and a higher concentration of 4). Members of the Comamonadaceae family were detected DOC. The solubilized material became depleted in carbohy- only in the noncured compost clone library (Fig. 2b). These drates and enriched in aromatic compounds. As the avail- bacteria may originate from the activated sludge process able carbon was depleted, the microbial activity, indicated during wastewater treatment, as they were related to Acid- by respiration, decreased, the response to added glucose was ovorax sequences isolated from wastewater-treatment plants. 1 slower, and competition for NH4 decreased, allowing an Interestingly, Valle et al. (2004) reported that in a phenol- increase in nitrification. It was proposed that as curing degrading activated sludge system, the population size of progresses, the remaining substrate in the compost is Acidovorax sp. AHL-5 (AY379977.1) changes in association more lignocellulosic in nature (Danon et al., 2007). The with the phenol-degradation rate. Acidovorax species have biochemical data of the previous report were used for also been shown to be the dominant 3-hydroxybutyrate comparison of the same samples with microbial population (PHB)-degrading bacteria in soils, composts, and freshwater analysis conducted in the current study (Fig. 5). Some (Mergaert & Swings, 1996). Other Betaproteobacteria, of the bacteria associated with the changes in the chemical namely members of the Oxalobacteraceae, were found in properties of compost during prolonged curing were then both libraries (Fig. 2b). identified. We found bacteria of the phylum Chloroflexi only in the noncured compost clone library (Fig. S1). Filamentous members of the phylum Chloroflexi have also been found in Noncured compost populations activated sludge, and they have occasionally been associated Differences between the noncured and cured composts in with incidences of bulking (Bjornsson et al., 2002). As the relative abundances of bacterial groups are illustrated in compost used in this study was derived from sewage sludge, Fig. 4. Using both the cloning and the microarray ap- the detection of these bacteria is not surprising. Chloroflexi proaches, we found that the members of the Bacteroidetes filaments appear to be specialized in polysaccharide phylum were more abundant in the noncured composts degradation. than in the cured composts (Fig. 4). With a more detailed Members of the genus Brevundimonas (Alphaproteobac- principal component analysis including DGGE and micro- teria; Caulobacteraceae) were present and dominant only in 1 À array data as well as process parameters (NH4 ,NO3, DOC, the noncured compost clone library (Fig. S1). However, and sugar concentration), we found that the dominant PCA of the DGGE bands (Fig. 5) showed that band 15, population in the noncured compost consisted of a variety identified as Caulobacteraceae, appears to be a dominant of Bacteroidetes species, including Chryseobacterium, Sphin- microorganism in the compost sample with a curing time of

100

Clones in library 90

Proteobacteria 80

Alphaproteobacteria 70

Betaproteobacteria 60

Gammaproteobacteria 50 Proteobacteria

40 Downloaded from https://academic.oup.com/femsec/article/65/1/133/620549 by guest on 30 September 2021 Deltaproteobacteria Gammaproteobacteria 30 Actinobacteria % of cured compost bacteria 20 Actinobacteria Bacteroidetes Alphaproteobacteria 10 other Bacteria Bacteroidetes Betaproteobacteria 010 20 30 40 50 60 70 80 90 100

100 Universal Probes in microarray 90

Proteobacteria 80 Alphaproteobacteria 70 Low G+C Betaproteobacteria 60 Gammaproteobacteria Proteobacteria Epsilonproteobacteria 50 Actinobacteria 40 Actinobacteria Bacteroidetes 30 Alphaproteobacteria Low G+C Gammaproteobacteria Nitrospirae % of cured compost bacteria 20 Planctomycetes Bacteroidetes 10 Universal Betaproteobacteria Other Bacteria 0 0 10 20 30 40 50 60 70 80 90 100 % of noncured compost bacteria

Fig. 4. Percentage contribution of various bacterial phyla to the total bacterial community in cured and noncured compost, as detected by clones in libraries vs. microarray probes. Groups below the diagonal line were more abundant in the noncured compost, and those above it were more abundant in the cured compost.

41 days. Pedro et al. (2001) found Brevundimonas present in Populations at intermediate compost-curing the mesophilic phase of industrial and agricultural waste times composting. These bacteria may play an important role in the composting process, and it is possible that they origi- PCA of microarray data showed that the Actinobacterium nated from the biosolids, as Caulobacteria members have Microbacterium dominated composts with intermediate been isolated previously from sewage and activated sludge curing times of 41–130 days (Fig. 5). Manickam et al. (Baker et al., 1983). (2006) reported the ability of Microbacterium isolated from

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–0.5

–1.0 –1.0 –0.5 0.0 0.5 1.0 1.5

Fig. 5. Principal component analysis of chemical and molecular data. Circles indicate the clustering time of curing in days. The principal DGGE bands and microarray probes are highlighted (in red and blue, respectively). contaminated soil to degrade the persistent and toxic hexa- at the end of the thermophilic stage (Gray et al., 1971). At chlorocyclohexane. Members of the Actinomycetes genus have the end of the composting process, however, the cellulose is been reported to develop more slowly than most other micro- often inaccessible to enzymatic attack because of low water organisms and are comparatively ineffective competitors under content or association with protective substances such as high-nutrient conditions. The proportion of Actinomycetes lignin (Stutzenberger et al., 1970). This results in a decrease relative to other bacteria has been suggested as an indicator of in the number of cellulolytic organisms. It has also been compost maturity (Palmisano & Barlaz, 1996). Thus, it is not reported that in materials with high cellulose content, surprising that microarray analysis revealed high levels of thermotolerant microorganisms with the ability to degrade Actinomycetes in the composts from longer curing times. cellulose can dominate the end stages of the composting DGGE analysis showed the presence of Promicromono- process (Ryckeboer et al., 2003b). Actinobacteria may sur- sporaceae members between 130 and 205 days of curing. We vive the thermophilic phase of composting and become also found Promicromonosporaceae in the noncured com- active during compost maturation. The decomposition of posts clone library (Fig. 2a). Promicromonospora sukumoe cellulose by cellulolytic bacteria such as Cellulomonas occurs and related organisms of the Cellulomonas group have been during the maturing process (Tang et al., 2006). characterized as cellulose-degrading Actinobacteria (Bakali- dou et al., 2002). Cellulomonas has been reported previously Cured compost populations in mature-compost bacterial populations (Ryckeboer et al., 2003b). Cellulolytic bacteria are common in compost Gammaproteobacteria were also abundant in the cured (Herrmann & Shann, 1997) and are known to occur mainly compost, and were detected in the clone library as well as

FEMS Microbiol Ecol 65 (2008) 133–144 c 2008 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 142 M. Danon et al. by microarray (Fig. 4). It is possible that different species of days). A retarded response to added glucose in the cured Pseudomonas were responsible for hybridization with the compost (Fig. 5) indicates a shift from microbial r-strate- genus-level probe, and that certain species were present in gists to K-strategists. The r-strategists are adapted to inter- higher numbers at the start of the curing process, while vals of rapid growth, depending on the availability of their other species were present later in the curing process. substrate, as opposed to the K-strategists, which typically Pseudomonas species are widely distributed in the environ- have slow growth rates, presumably benefiting from poly- ment and have been reported previously in mature-compost merized substrates that have a long residence time (Fontaine bacterial communities (Ryckeboer et al., 2003a). Pseudomo- et al., 2003). nas strains are known to be able to participate in N2 fixation, In conclusion, we found a successional transition of denitrification, and degradation of pollutants or interaction bacterial populations during compost curing. Comparison

with toxic metals (Lalucat et al., 2006), and several strains of the results obtained by the three methods was used for Downloaded from https://academic.oup.com/femsec/article/65/1/133/620549 by guest on 30 September 2021 are known to confer plant-disease suppressiveness (Haas & identification of different bacterial phylogenetic groups that Defago,´ 2005). We found sequences affiliated to the Gam- changed during prolonged compost curing. The Proteobacter- maproteobacteria, Xanthomonadaceae families both in the ia were the most abundant phylum in all cases. The Bacter- cured compost clone library and among the DGGE bands of oidetes and the Gammaproteobacteria were ubiquitous but cured compost samples (Table 1). opposite in their relative dominance. At the beginning of the The dominant Actinobacteria found in the cured compost compost-curing process, Bacteroidetes were dominant. Later, were Corynebacterineae (i.e. Mycobacterium species), as during the midcuring stage, Actinobacteria were dominant. detected in the clone library and by DGGE. Many of the Finally, in the cured compost, the Gammaproteobacteria were Mycobacteria are potential pathogens. Mycobacterium more abundant. Despite using a microarray that targets many avium, an environmental opportunistic pathogen, has been pathogens (Franke-Whittle et al., 2005; Franke-Whittle et al., isolated from environmental water samples. Furthermore, 2005), we did not detect any human, animal, or plant growth of M. avium in natural water was stimulated by the pathogens by this method. This suggests efficient eradication addition of humic and fulvic acids in acidic, microaerobic of pathogens during the composting. Furthermore, this environments (Kirschner et al., 1999). observation indicates that no reinfestation with pathogens We found only a few Firmicutes sequences in the compost occurred during the prolonged curing. clone libraries; we did not detect Firmicutes in DGGE and The initial phase of composting is thought to be the most their detection rate in the microarray was low. This finding dynamic part of the process (Schloss et al., 2003). Never- is surprising considering the dominance previously attrib- theless, we found that the microbial community continued uted to this phylum in compost. Ryckeboer et al. (2003b) to change long after the compost organic matter and other reported Bacillus as the most dominant bacterial taxon biochemical properties had stabilized. It is assumed that recovered from compost feedstock, and it was also the most bacterial populations, playing their specific role in changing abundant group of bacteria recovered from compost during the chemical environment, were replaced in turn by selec- the thermophilic phase and throughout the entire compost- tion pressures driven by these changes in the microhabitat. ing process. Dees & Ghiorse (2001) reported that the two Changes in complex lignocellulose-rich organic matter may most abundant cultivated isolates from hot synthetic com- have been too minute to detect using standard physico- post belong to the genera Aneurinibacillus and Brevibacillus. chemical analysis. However, bacterial community response We suggest that Firmicutes members do not always persist in to these changes was more easily observed. compost after the thermophilic phase has ended. It is apparent that members of different bacterial species Acknowledgements capable of similar functions were found throughout most of the process. For example, at the beginning of the curing We gratefully acknowledge the assistance of Dana Levinson, and thank Sharon Nahum-Zmora for her significant role in process, the NH4 concentration was high (Fig. 5) and supported a population specializing in nitrification (i.e. carrying out the compost-curing process and providing the samples, the chemical data, and critical remarks. This Nitrosovibrio, Nitrosospira); thus, NH4 concentrations de- research was supported by The Negev Foundation and the creased rapidly and NO3 increased until 41 days. From 41 to Israeli Ministry of Agriculture and Rural Development, the 130 days, NO3 levels continued to increase, suggesting that nitrification activity was maintained by more versatile Wastewater Treatment Program. bacteria (Danon et al., 2007). Biodegradation of macromolecules, especially of complex materials such as lignocellulose, is a much slower process. A population specializing in References cellulolytic activity (i.e. Promicromonosporaceae) became Alfreider A, Peters S, Tebbe CC, Rangger A & Insam H (2002) established during the intermediate curing time (130–205 Microbial community dynamics during composting of organic

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Ryckeboer J, Mergaert J, Coosemans J, Deprins K & Swings J Please note: Blackwell Publishing is not responsible for the (2003b) Microbiological aspects of biowaste during content or functionality of any supplementary materials sup- composting in a monitored compost bin. J Appl Microbiol 94: plied by the authors. Any queries (other than missing material) 127–137. should be directed to the corresponding author for the article.