Bioprocess Biosyst Eng DOI 10.1007/s00449-014-1289-z

ORIGINAL PAPER

Microbial community analysis of a full-scale DEMON bioreactor Alejandro Gonzalez-Martinez • Alejandro Rodriguez-Sanchez • Barbara Mun˜oz-Palazon • Maria-Jesus Garcia-Ruiz • Francisco Osorio • Mark C. M. van Loosdrecht • Jesus Gonzalez–Lopez

Received: 15 August 2014 / Accepted: 16 September 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Full-scale applications of autotrophic nitrogen removal technologies for the treatment of digested sludge liquor have proliferated during the last decade. Among these technologies, the aerobic/anoxic deammonification process (DEMON) is one of the major applied processes. This technology achieves nitrogen removal from wastewater through anammox metabolism inside a single bioreactor due to alternating cycles of aeration. To date, microbial community composition of full-scale DEMON bioreactors have never been reported. In this study, bacterial community structure of a full-scale DEMON bioreactor located at the Apeldoorn wastewater treatment plant was analyzed using pyrosequencing. This technique provided a higher-resolution study of the bacterial assemblage of the system compared to other techniques used in lab-scale DEMON bioreactors. Results showed that the DEMON bioreactor was a complex ecosystem where ammonium oxidizing bacteria, anammox bacteria and many other bacterial phylotypes coexist. The potential ecological role of all phylotypes found was discussed. Thus, metagenomic analysis through pyrosequencing offered new perspectives over the functioning of the DEMON bioreactor by exhaustive identification of microorganisms, which play a

A. Gonzalez-Martinez (&)  M.-J. Garcia-Ruiz  F. Osorio Department of Civil Engineering, University of Granada, Campus de Fuentenueva, s/n, 18071 Granada, Spain e-mail: [email protected] A. Rodriguez-Sanchez  B. Mun˜oz-Palazon  J. Gonzalez–Lopez Institute of Water Research, University of Granada, C/Ramo´n y Cajal, 4, 18071 Granada, Spain M. C. M. van Loosdrecht Department of Biotechnology, Technical University of Delft, Julianalaan 67, 2628 BC Delft, The Netherlands

key role in the performance of bioreactors. In this way, pyrosequencing has been proven as a helpful tool for the indepth investigation of the functioning of bioreactors at microbiological scale. Keywords Anammox  Deammonification  DEMON  Pyrosequencing  Wastewater treatment

Introduction In the past decade, full-scale applications of autotrophic nitrogen removal technologies for the treatment of digested sludge liquor have been constructed [1]. The Apeldoorn DEMON is a full-scale bioreactor operating at the wastewater treatment plant of Apeldoorn, The Netherlands. This bioprocess was designed by Grontmij and has been operating since 2009. The Apeldoorn DEMON treats sludge liquor coming from anaerobic co-digestion of urban and industrial sludge. Autotrophic nitrogen removal processes are based on the metabolism of anammox bacteria. Anammox bacteria can utilize ammonium as substrate and nitrite as terminal electron acceptor, yielding molecular nitrogen as a result. Anammox bacteria have been found at many engineered and natural environments such as wastewater treatment plants, marine sediments or agricultural soil [2–4], among others. Also, anammox bacteria have proposed as fundamental players in the nitrogen cycle at marine ecosystems [5]. The DEMON process achieves autotrophic nitrogen elimination from wastewater. Anammox elimination of nitrogen requires ammonium and nitrite in molar fractions of 1:1, so previous oxidation of ammonium to nitrite is required. In this way, attainment of ammonium oxidation to nitrite and anammox reaction in the same environment is

123

Bioprocess Biosyst Eng

not an easy task given the different requirements for the two bioprocesses. The DEMON technology, developed at the University of Innsbruck, makes possible the elimination of nitrogen through anammox pathway inside a single bioreactor by cycles of aeration-no aeration [6]. Molecular biology techniques have been utilized for the investigation of microbial community structure of many different environments. Among these, one of the most powerful is the high-throughput sequencing process. This PCR-based next-level sequencing technique has been successfully applied for the analysis of microbial community structure of many ecosystems such as aquifers, arctic seas, greywater biofilters, hot springs or partial nitritation biofilters [7–11]. Also, identification of microbial populations of autotrophic nitrogen removal bioreactors through nextgeneration sequencing has been achieved. Analysis of labscale DEMON bioreactor communities has been done by DGGE/TGGE techniques, but this has been focused only to Planctomycetes microorganisms and ammonium oxidizing bacteria (AOB) [6, 12]. In this way, phylotypes not related to these bacterial groups have never been investigated in these systems. Moreover, studies conducted over autotrophic nitrogen removal bioreactors have aimed that the presence of bacterial communities not related to AOB or anammox bacteria might have an effect over the performance of these systems [13, 14]. In this way, an accurate analysis of all bacterial communities thriving in the DEMON bioreactor and the identification of their ecological roles is needed for a deeper understanding of the performance of the system. It has been defended that pyrosequencing offers a deeper and more accurate characterization of microbial communities in environmental samples in comparison to DGGE/ TGGE techniques [15, 16], leading to higher-level resolution on diversity and abundance of microbial communities in a given environment [17]. In this sense, pyrosequencing identification of bacterial communities of a full-scale DEMON bioreactor will provide a more accurate insight into the bacterial diversity and abundance of these systems than those achieved previously by other procedures. In addition, study of microbial communities in a full-scale DEMON bioreactor through pyrosequencing has never been attempted, thus this work is the first approach for this task. Here the microbial community structure of the full-scale DEMON bioreactor at Apeldoorn wastewater treatment plant was studied by means of pyrosequencing, which poses the first pyrosequencing study of full-scale DEMON bioreactors. Investigation of all microbial communities thriving in this system was conducted. The effect that the growth of these communities could have over the performance of the full-scale DEMON bioreactor is unknown to date, but potential ecological roles of the most important phylotypes is given. Identification of these bacterial groups

123

Table 1 Apeldoorn DEMON bioreactor operational conditions given by the plant operation personnel at the moment of sampling Apeldoorn DEMON operational conditions Flow (m3 day-1) ?

-1

1,020

NH4 (mg-N L )

1,208

COD (mg L-1)

2,472.5

BOD (mg L-1)

176.5

pH

6.71–6.69

Oxygen demand (D.O) (mg O-1 2 L)

0–0.3

Temperature Celsius (°C) Efficiency of N-removal (%)

30 85

is the first step towards a more accurate understanding of the functioning of DEMON bioreactors. The results obtained from the analysis will stand as important information and, combined with other techniques such as metatranscriptomics, will provide new perspectives for the future design, operation, maintenance and control of these bioprocesses.

Materials and methods The Apeldoorn DEMON bioreactor The Apeldoorn DEMON bioreactor has been operating since its construction in 2009. Digested sludge liquor was treated in cycles of 8 h, divided in 6 h of filling and mixing and 2 h of biomass settling and treated wastewater discharge. At the start of the filling and mixing step, the reactor is intermittently aerated. Ammonium will be oxidized to nitrite in the presence of oxygen, and therefore pH of the system drops. Once pH has dropped a certain amount, taken as 0.01, aeration stops and autotrophic nitrogen removal can take place, raising the pH at the same time. When pH raises a certain amount, taken as 0.01, aeration starts again. Therefore, autotrophic nitrogen removal happens in the DEMON bioreactor by cycles of aeration-no aeration controlled by changes in pH. Operational conditions of the Apeldoorn DEMON bioreactor can be seen in Table 1, as reported by the plant operation personnel. Sampling The DEMON bioreactors are not subjected to environmental changes, given the technological configuration of the system (e.g.: DEMON bioreactors are not exposed to changes in climate conditions). Therefore, stability of the system depends only on the influent wastewater composition and operational procedures. Prior to the date of

Bioprocess Biosyst Eng

sampling, the system was found to operate under stable conditions, as reported by plant operation personnel. Therefore, the investigation of the main bacterial assemblage structure of the bioreactor was conducted. Sampling was conducted for the investigation of the bacterial communities of the bioreactor. An amount of 200 mL of wastewater containing suspended and granular biomass was collected from five different points distributed evenly among the bioreactor volume. Samples were immediately submerged into saline solution (0.9 % NaCl) for the preservation of microbial cells. Biomass collection was accomplished by centrifugation of samples at 3,500 r.p.m. for 10 min at room temperature. Biomass collected was stored at -20 °C for further molecular biology analysis.

AnRareReadme.html). Hill diversity indeces were calculated using ‘‘vegan’’ package v.2.0 implemented on the R-Project v.2.15.1 statistical software [19].

DNA extraction and 454-pyrosequencing process

Results and discussion

350 mg pellet was taken from each sample of biomass collected for the purpose of DNA extraction. FastDNA SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA) was used according to the manufacturer’s instructions. DNA extracts from the five samples were merged prior to pyrosequencing process. Primer pair 530F (50 -GTGCCAG CMGCNGCGG) and 1100R (50 -GGGTTNCGNTCGTTG) [18] were chosen for the amplification of the V4-V5-V6 regions of 16S rRNA gene of Bacteria. Pyrosequencing procedure followed Dowd et al. [18] and was done by the Research and Testing Laboratory (Lubbock, TX, USA). PCR conditions for the pyrosequencing process were the following: preheating for 3 min at 94 °C; 32 cycles of: 30 s at 94 °C, 40 s at 45 °C, and 1 min at 72 °C; 5 min of final elongation.

Species richness and diversity

Pyrosequencing post-process Raw data generated through pyrosequencing was further treated for removal of reads with poor end quality, detection and elimination of chimeras, denoising, screening for quality selection of reads and phylogeny-based clustering using specific software such as USEARCH, UCHIIME or KrakenBLAST. Sequences that failed to meet quality criteria required at any of these processes were discarded from the analysis. Sequences were affiliated phylogenetically at the following thresholds: up to 3 % for OTU level, up to 5 % for genus level, up to 10 % for family level, up to 15 % for order level, up to 20 % for class level and up to 23 % for phylum level. Species richness and diversity calculations Species richness of the samples was calculated through rarefaction analysis developed by aRarefactWin software (University of Georgia, Athens; http://www.uga.edu/strata/

Phylogenetic tree assemblage A phylogenetic tree representing the OTUs with [0.5 % relative abundance in the Apeldoorn DEMON was constructed using the MEGA 6.0 software. Genetic sequences were aligned using CLUSTALW algorithm. Calculation of the tree was developed by the neighbor-joining method with 1,000 bootstraps replications for test of phylogeny utilizing a Jukes-Cantor substitution model [7].

Species richness of the DEMON bioreactor was evaluated by the conformation of a rarefaction curve, which is shown in Fig. 1. In a total of 9,924 reads, a number of 256 different species were identified. For the pyrosequencing sample, Hill diversity index of order one was 3.5618 and Hill diversity index of order two was 0.9399. The evenness of the sample based on the Hill diversity index of order one was of 0.6423. Relative abundance and diversity of the DEMON bioreactor At phylum level (Fig. 2), the dominant phylotypes belong to Proteobacteria with almost a 34 %. Chlorobi with a 17.88 % relative abundance and Bacteroidetes with a 16.67 % follow in importance. A remarkable presence of Firmicutes, Verrucomicrobia and Planctomycetes accounting for 9.24, 7.54 and 7.50 %, respectively, has to be taken into account. Acidobacteria, Chloroflexi and Fibrobacteres have relative abundance of around 2.14 and 1.22 %. All other phyla together only explain the 2.41 % relative abundance of the system. A representation of the bacterial community at OTU level can be seen in Fig. 3. At OTU level, the dominance of the system belongs to OTU 41459 with a 22.87 % relative abundance. The three closest sequences identified at species level to OTU 41459 were GU556309.1 and GU556323.1, with 89 % similarity and affiliated with Pedomicrobium sp., and JX114411.1, affiliated to Ignavibacterium sp. clone at 86 % similarity. This similarity may signify an affiliation at order level with these two species. Nevertheless, sequence of OTU 41459 shares a 99 % similarity with the uncultured planctomycete clone 5GA PLA HPK 17 with accession number GQ356164.1 [20].

123

Bioprocess Biosyst Eng Fig. 1 Rarefaction curve of the DEMON bioreactor

Fig. 2 Relative abundance and diversity of the Apeldoorn DEMON bioreactor at phylum level

Fig. 3 Relative abundance and diversity at OTU level of the Apeldoorn DEMON bioreactor

123

Bioprocess Biosyst Eng

Park et al. [20] identified clone 5GA PLA HPK 17 as a strain of the phylum Planctomycetes not related with anaerobic ammonium oxidation thriving in a steady-state lab-scale DEMON bioreactor. It has been suggested that strains of the Planctomycetes phylum not related with anammox bacteria could develop important roles in the functioning of these autotrophic nitrogen removal technologies, such as granular biomass conformation [20]. In this way, OTU 41459 could be of ecological relevance in the Apeldoorn DEMON bioreactor given that it can achieve the formation of granular biomass inside the system. Identification of OTU 41459 is still unknown and further research should be done to fulfill this purpose. The second OTU in importance inside the Apeldoorn DEMON bioreactor was the OTU 11256, with a 7.15 %. OTU 11256 showed a 99 % similarity with the Pedosphaera sp. clone KF956764.1. Also, it has a 98 % similarity with the uncultured Verrucomicrobia clone JN038977.1, and a 97 % similarity with the Verrucomicrobium sp. clone DQ263384.1. Pedosphaera strains were affiliated with Verrucomicrobia subdivision three species [21]. Pedosphaera parvulla has been described as an obligate aerobic microorganism [22]. Nevertheless, Verrucomicrobia phylum is poorly characterized to date [23]. It can be said that OTU 11256 could grow within the bioreactor during aeration steps of operation cycles by BOD degradation. Also, species of Verrucomicrobia phylum have been described in municipal anaerobic digestion reactors [24]. In this sense, presence of OTU 11256 can be derived from its entrance with the influent in addition to the growth of its communities. The following OTU in terms of dominance was OTU 71090, accounting for a 6.46 % relative abundance. OTU 71090 had a 98 % similarity with GU245918.1 and a 97 % similarity with GU245920.1, which stand for Clostridium sp. [25] clones. Some Clostridium species are human pathogens related with tetanus or gas gangrene diseases, among others [26, 27]. The main energy source of Clostridium species has been found to be the anaerobic fermentation [26]. It has been reported that Clostridium acetobutylicum is capable of nitrogen fixation and nitrate reduction [28]. Thus, OTU 84104 can grow inside the bioreactor by fermentation of organic carbon compounds and it can also develop a metabolism based on nitrate reduction, which could help to reduce nitrate accumulation inside the Apeldoorn DEMON bioreactor. OTU 71090 also share a 97 % similarity with the Anaerolinea sp. clone JQ861772.1. Anaerolinea species have been described as strictly anaerobic, thermophilic, chemoorganotrophic, filamentous bacteria. Species of the Anaerolinea genus have the ability to oxidize N-acetylglucosamine with nitrite as terminal electron acceptor or with nitrate as terminal electron acceptor [29]. Species of Clostridium and

Anaerolinea have been reported in anaerobic digester biomass [30], and though their presence in the Apeldoorn DEMON bioreactor could be related with the influent composition. OTU 8488 represented a 6.10 % relative abundance within the Apeldoorn DEMON. This OTU shares a 99 % similarity with sequences AM285341.1, GQ356182.1 found at a lab scale DEMON reactor [20], KF606756.1 and KF606759.1 found at partial nitritation-anammox movingbed biofilm reactor. This sequences stand for Candidatus Brocadia sp. clones. Candidatus Brocadia sp. and other anammox bacteria perform the anammox reaction, which refers to the anaerobic, autotrophic oxidation of ammonium with nitrite as terminal electron acceptor, yielding molecular nitrogen as result [31]. In this sense, OTU 8488 was the main contributor to autotrophic nitrogen removal in the Apeldoorn DEMON bioreactor. OTU 20719 dominates the 4.65 % relative abundance in the Apeldoorn DEMON. This OTU has a 98 % similarity with Microbulbifer sp. AM936202.1. The earliest description of Microbulbifer genus reported these microorganisms as strictly aerobic, heterotrophic bacteria [32]. Nevertheless, strains of Microbulbifer have been identified for facultatively anaerobic metabolism either by fermentation or by respiration with nitrate [33]. Growth on N-acetyl-Dglucosamine for species of Microbulbifer has been described [34]. In this sense, metabolic capabilities described for Microbulbifer make OTU 20719 to be wellfitted for the environment that the Apeldoorn DEMON is, growing on organic carbon and with possibilities for cell biomass degradation and aerobic and anaerobic growth utilizing oxygen or nitrate. OTU 10180 has a 4.61 % relative abundance. This OTU has a 98 % similarity with Flavobacterium sp. FR772064.1 and 97 % similarity with HM776706.1 and KF214259.1. Flavobacterium are aerobic, heterotrophic bacteria, with the ability to generate EPS of hydrophobic nature, which is thought to trigger granulation of biomass [35]. According to this, Flavobacteriales order species are subject to an increase in relative abundance with formation of aerobic granular biomass from aerobic bioflocs [36]. In this way, ecological role of OTU 10180 was of importance for the performance of the DEMON system given that it helps in the formation of the granular biomass. Also, chitinolytic activity of Flavobacterium has been reported, which implies that OTU 10180 could thrive in the bacterial biomass available in the system when aerobic conditions are met in the bioreactor. OTU 17125 was found to have a 4.41 % relative abundance inside the DEMON bioreactor. This OTU was affiliated with sequence FN394322.1 at a 99 % similarity, and with sequences AJ224410.1 and GQ891780.1 at 97 % similarity. These three sequences are related to

123

Bioprocess Biosyst Eng

Nitrosomonas sp. clones. Nitrosomonas species have been widely reported as ammonium oxidizers in wastewater treatment bioreactors, achieving partial nitritation in autotrophic nitrogen removal systems [7, 36]. Therefore, OTU 17125 stands as the main ammonium oxidizer inside the DEMON bioreactor. This ecological role is of great importance for the autotrophic ammonium removal process, given that partial nitritation is needed for anammox reaction to take place. OTU 12738 represents a 3.62 % relative abundance of the Apeldoorn DEMON. OTU 12738 shares 97 % with Sphingobacterium sp. clone JX535192.1 and 96 % with Sphingobacterium sp. clone FN668067.2. Strains of Sphingobacterium genus have been described as strictly aerobic bacterium with uncapability for chitin utilization [37]. Also, Sphingobacterium sp. can develop anaerobic/ anoxic removal of phosphate with nitrite as terminal electron acceptor [38]. In this sense, OTU 12738 could oxidize organic matter inside the DEMON during aeration cycles and eliminate phosphorous and nitrogen during anaerobic cycles. OTU 35059 has a 3.36 % relative abundance in the system. Its sequence was closely related with the uncultured Rubrivivax sp. clone JN802224.1 at 99 % similarity and with Rubrivivax clones FN995214.1 and EF664279.1 at 97 % similarity. Complete genome of Rubrivivax gelatinosus IL144 strain showed a facultative anaerobic bacterium with structural genes for nitrogenase activity, which implied that this strain was capable of nitrite but not nitrate reduction [39]. Photoheterotrophic growth of Rubrivivax gelatinosus has been reported [39]. Phototrophic growth seems not possible inside the Apeldoorn DEMON due to technical reasons. In this way, it can be hypothesized that OTU 35059 grows in the system by heterotrophic metabolism utilizing oxygen and nitrite as terminal electron acceptor. OTU 46260 represented a 3.28 % relative abundance of the DEMON bioreactor. Its sequence shared a 99 % similarity with Nitrosococcus sp. clone GU271755.1 and Steroidobacter sp. clone JX576027.1 and also shared a 98 % with Nitrosococcus sp. clones GU271302.1 and GU245896.1. Nitrosococcus strains have been widely reported as ammonium-oxidizing microorganisms in autotrophic nitrogen removal bioreactors [40]. It has been previously reported that Nitrosococcus species closely cooperate with anammox bacteria in the autotrophic denitrification process [41]. On the other hand, type strain of Steroidobacter genus, Steroidobacter denitrificans, is capable of aerobic respiration and anaerobic reduction of nitrate and nitrite in presence of organic carbon compounds [42]. Although both classifications could be possible in this ecosystem, OTU 46260 finds more affiliation with Nitrosococcus sp. clones than with Steroidobacter sp. clones. In

123

this sense, OTU 46260 develops autotrophic, aerobic oxidation of ammonium inside the bioreactor, which is the necessary first step for autotrophic nitrogen removal by anammox bacteria. The OTU 47200 has a relative abundance of 3.25 %, and shares a 96 % similarity with a Chitinophagaceae bacterium clone sequence JX493631.1 and a 95 % similarity with a Chitinophaga sp. clone sequence FJ946530.1. Therefore, OTU 47200 was related to Chitinophaga sp. bacterium at genus level. The genus Chitinophaga was created to cover strains of aerobic bacteria capable of chitin biodegradation. Ability for N-acetylglucosamine utilization was demonstrated through analysis of complete genome of Chitinophaga pinensis [43]. In this way, OTU 47200 thrives in the DEMON system by predation and/or scavenging of bacterial biomass. OTU 2598 possess the 2.00 % of the bacterial community structure of the system. This OTU has been affiliated to Acidobacteria bacterium clones AB291529.1, CU925586.1 and CU927180.1 with a 99 % similarity. Acidobacterium sp. has been reported as a species driving BOD removal in activated sludge systems [44]. It is then possible that OTU 2598 thrives in the organic matter present in the system during aeration steps. OTU 6335 represents a 2.00 % relative abundance in the DEMON bioreactor. OTU 6335 has a 98 % similarity with sequences KF287743.1, HM769664.1 and JN125283.1. Denitratisoma genus type strain, Denitratisoma oestradiolicum, has been reported for heterotrophic growth with reduction of oxygen, nitrate and nitrite [42]. In this way, OTU 6335 can grow in the system by aerobic heterotrophic metabolism during aeration steps and by nitrate or nitrite reduction during no-aeration steps. Therefore, OTU 6335 is of importance for the system, given its ability to mend nitrite accumulation. OTU 26344 showed a 1.23 % relative abundance. This OTU was affiliated at 95 % similarity with GU323642.1 and JQ937375.1, which are Fibrobacter sp. clone sequences. Thus, OTU 26344 is related to Fibrobacter sp. at genus level. Study of the complete genome of Fibrobacter succinogenes demonstrated its capability of polysaccharide hydrolysis [45]. Therefore, fermentative oxidation of organic matter in the DEMON bioreactor could support the development of OTU 26344. OTU 71279 accounted for a 1.10 % relative abundance in the DEMON bioreactor. This strain showed a 97 % similarity with Fluviicola sp. clones GU257757.1 and GU257755.1 and a 96 % similarity with Fluviicola sp. clone GU257758.1. Analysis of complete genome of Fluviicola taffensis showed that this strain develops an aerobic, heterotrophic metabolism and that it is not able to biodegrade N-acetylglucosamine or to reduce nitrate or nitrate [46]. In this sense, OTU 71279 could grow in the

Bioprocess Biosyst Eng

Fig. 4 Metabolic capabilities and ecological roles of OTUs [1 % in the Apeldoorn DEMON bioreactor

Apeldoorn DEMON by uptake of organic carbon coming with the influent or released by activity of chitinolytic bacteria in the system. OTUs individually accounting for \1 % relative abundance in the DEMON bioreactor summed up to 23.85 % relative abundance. Interestingly, development of autotrophic elimination of nitrogen in the Apeldoorn DEMON bioreactor was done by a small percentage of bacteria. Based on the ecological analysis of OTUs with [1 % relative abundance, oxidation of ammonium was done by OTUs 17125 and 46260, which sum up a 7.70 % total relative abundance, while anammox bioprocess is done by OTU 8488, which accounts for a 6.09 % relative abundance (Fig. 4). In this sense, performance of the Apeldoorn DEMON bioreactor is related to microbial groups with a small representation with respect to the total microbial community structure. Nevertheless, relative abundance of OTUs thriving in the bioreactor might be affected by the amount of bacterial species that enters with the influent. The activity of these microorganisms needs to be measured for a more accurate characterization of the ecological role each of the OTUs have for the performance of the DEMON bioreactor. The use of pyrosequencing for the analysis of microbial assemblage of DEMON bioreactors has several advantages over other molecular techniques used in previous studies, such as DGGE [5, 19]. Studies conducted before over the bacterial community structure of DEMON bioreactors have focused over the diversity of AOB and anammox bacteria [5, 19] and have disregarded other bacteria that might be important for the performance of the system, as has been reported before [12, 13]. However, this pyrosequencing analysis was able to capture a richer diversity of microorganisms, which leads to a better understanding of the functioning of the system. Also, relative abundance of bacterial phylotypes in DEMON bioreactors has never been

explored through DGGE techniques. On the other hand, pyrosequencing analysis provided a measure of the relative abundance of bacterial phylotypes within the system, which also deepens the understanding of the microbiological processes that drive the performance of the system. In this way, as previously reported, pyrosequencing offers a more accurate analysis of microbial assemblage of an ecosystem [14–16]. Phylogenetic analysis The phylogenetic affiliation of OTUs with [0.5 % relative abundance in the Apeldoorn DEMON bioreactor is shown in Fig. 5. At phylum level, the highest diversity belongs to the Proteobacteria. OTUs related to b-Proteobacteria resemble species from genera such as Ralstonia/Burkholderia/Derxia/Azoarcus, Rubrivivax, Nitrosospira, Denitratisoma and Nitrosomonas. Among the c-Proteobacteria OTUs related to Nitrosococcus/Steroidobacter, Haliea, Pseudomonas and Microbulbifer genera can be found. The phyla Acidobacteria and Fibrobacteres account for only one OTU, which in both cases are affiliated with the type genus of the phylum, Acidobacterium and Fibrobacter, respectively. The phylum Planctomycetes has two representatives, standing for Candiadtus Brocadia and uncultured clone 5GA PLA HPK 17. In spite of the low diversity, Planctomycetes phylum accounts for the most important phylotypes with respect to the Apeldoorn DEMON bioreactor performance. A sole OTU representative for the phyla Spirochaetes, Verrucomicrobia and Cyanobacteria were found, being affiliated with Turneriella, Pedosphaera/Verrucomicrobium and Prochlorococcus genera, respectively. Firmicutes phylum accounts for two OTUs, which are related to Clostridium and Pelotomaculum genera. The phylum Bacteriodetes has a high diversity in the Apeldoorn DEMON bioreactor. Several

123

Bioprocess Biosyst Eng Fig. 5 Phylogenetic tree of OTUs with [0.5 % relative abundance in the Apeldoorn DEMON bioreactor

123

Bioprocess Biosyst Eng

OTUs belonging to this phylum were affiliated with Flavobacterium, Anaerophaga, Chitinophaga, Sphingobacterium, Bacteriodes/Parabacteroides/Macellibacteroides, Flexibacter and Fluviicola genera. A single OTU of the Bacteriodetes phylum could not be identified up to genus level. As happened with the 5GA PLA HPK 17 clone, this species requires more investigation for its phylogenetic identification.

Conclusions Analysis of the bacterial community structure of the Apeldoorn full-scale DEMON bioreactor has been developed using pyrosequencing for the first time. A higherlevel resolution study of the bacterial assemblage of this system has been achieved in comparison to previous studies using other techniques such as DGGE. A high diversity of OTUs was found, with many of them not related to AOB or anammox bacteria. The potential ecological roles that these OTUs can play in the functioning of the system are discussed. The most relevant of these OTUs for the purpose of the performance of the system showed close similarity with Candidatus Brocadia, Nitrosomonas and Nitrosococcus genera. Relative abundance of these bacteria could be reduced by the amount of biomass entering with the influent, which might also increase the relative abundance of OTUs affiliated with Clostridium/ Anaerolinea or Verrucomicrobium/Pedosphaera genera, among others. Also, a high relative abundance of the uncultured planctomycete clone 5GA PLA HPK 17 was observed, which is thought to help in the formation of biomass inside the DEMON systems. Overall, metagenomic analysis through pyrosequencing is a promising approach for the investigation of microbial assemblages of bioreactors and their linkage to the performance of these systems. A further study into the activity of microbial communities is proposed as the next step for the in-depth understanding of the functioning of this full-scale DEMON bioreactor at microbiological scale. This task can be successfully accomplished by available techniques, such as metatrasncriptomics.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

References 1. Rodriguez-Sanchez A, Gonzalez-Martinez A, Martinez-Toledo MV, Garcia-Ruiz MJ, Osorio F, Gonzalez-Lopez J (2014) The effect of influent characteristics and operational conditions over the performance and microbial community structure of partial nitritation reactors. Water 6:1905–1924 2. Kotay SM, Mansell BL, Hogsett M, Pei H, Goel R (2013) Anaerobic ammonia oxidation (ANAMMOX) for side-stream

17.

18.

treatment of anaerobic digester filtrate process performance and microbiology. Biotechnol Bioeng 110:1180–1192 Shen LD, Liu S, Lou LP, Liu WP, Xu XY, Zheng P, Hu BI (2013) Broad distribution of diverse anaerobic ammonium-oxidizing bacteria in chinese agricultural soils. Appl Environ Microbiol 79:6167–6172 Ganesh S, Parris DJ, DeLong EF, Stewart FJ (2014) Metagenomic analysis of size-fractionated picoplankton in a marine oxygen minimum zone. ISMEJ 8(1):187–211 Thamdrup B, Jensen MM, Ulloa O, Faria L, Escribano R (2006) Anaerobic ammonium oxidation in the oxygen-deficient waters off northern Chile. Limnol Oceanogr 51(5):2145–2156 Innerebner G, Insam H, Franke-Whittle IH, Wett B (2007) Identification of anammox bacteria in a full-scale deammonification plant making use of anaerobic ammonia oxidation. Syst Appl Microbiol 30(5):408–412 Gonzalez-Martinez A, Rodriguez-Sanchez A, Martinez-Toledo MV, Garcia-Ruiz MJ, Hontoria E, Osorio-Robles F, GonzalezLopez J (2014) Effect of ciprofloxacin antibiotic on the partialnitritation process and bacterial community structure of a submerged biofilter. Sci Total Environ 476–477:276–287 Dalahmeh SS, Jo¨nson H, Hylander LD, Hui N, Yu D, Pell M (2014) Dynamics and functions of bacterial communities in bark, charcoal and sand filters treating greywater. Water Res 54:21–32 Han D, Kang I, Ha HK, Kim HC, Kim OS, Lee BY, Cho JC, Hur HG, Lee YH (2014) Bacterial communities of surface mixed layer in the Pacific sector of the Western Arctic Ocean during sea-ice melting. PLoS One 9(1):e86887 Huang Q, Jiang H, Briggs BR, Wang S, Hou W, Li G, Wu G, Solis R, Arcilla CA, Abrajano T, Dong H (2013) Archaeal and bacterial diversity in acidic circumneutral hot springs in Philippines. FEMS Microbiol 85:452–464 Livermore JA, Jin YO, Arnseth RW, LePuil M, Mattes TE (2013) Microbial community dynamics during acetate biostimulation of RDX-contaminated groundwater. Environ Sci Technol 47:7672– 7678 Zekker I, Rikmann E, Tenno T, Saluste A, Tomingas M, Menert A, Loorits L, Lemmiksoo V, Tenno T (2012) Achieving nitritation and anammox enrichment in a single moving-bed biofilm reactor treating reject water. Environ Technol 33(6):703–710 Mozumder MSI, Picioreanu C, van Loosdrecht MCM, Volcke EIP (2014) Effect of heterotrophic growth on autotrophic nitrogen removal in a granular sludge reactor. Environ Technol 35(8):1027–1037 Sorokin DY, Lu¨cker S, Vejmelkova D, Kostrikina NA, Kleerebezem R, Rijpstra WIC, Damste´ JSS, Le Paslier D, Muyzer G, Wagner M, van Loosdrecht MCM, Daims H (2012) Nitrification expanded: discovery, physiology and genomics of a nitriteoxidizing bacterium from the phylum Chloroflexi. ISMEJ 6:2245–2256 Dowd SE, Sun Y, Secor PR, Rhoads DD, Wolcott BM, James GA, Wolcott RD (2008) Survey of bacterial diversity in chronic wounds using pyrosequencing, DGGE, and full ribosome shotgun sequencing. BMC Microbiol 8:43 Nakayama J (2010) Pyrosequence-based 16S rRNA profiling of gastro-intestinal microbiota. Biosci Microflora 29(2):83–96 Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM, Neal PR, Arrieta JM, Herndl GJ (2006) Microbial diversity in the deep sea and the underexplored ‘‘rare biosphere’’. PNAS 103(32): 12115–12120 Dowd SE, Callaway TR, Wolcott RD, Sun Y, McKeehan T, Hagevoort RG, Edrington TS (2008) Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tagencoded FLX amplicon pyrosequencing (bTEFAP). BMC Microbiol 8:125

123

Bioprocess Biosyst Eng 19. R Development Core Team (2008) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna 20. Park H, Rosenthal A, Ramalingam K, Fillos J, Chandran K (2010) Linking community profiles, gene expression and N-removal in anammox bioreactors treating municipal anaerobic digestion reject water. Environ Sci Technol 44(16):6110–6116 21. Kant R, van Passel MWJ, Sangwan P, Palva A, Lucas S, Copeland A et al (2011) Genome sequence of ‘‘Pedosphaera parvula’’ Ellin514, an aerobic Verrucomicrobial isolate from pasture soil. J Bacteriol 193:2900–2901 22. Sangwan P, Kovac S, Davis KER, Sait M, Janssen PH (2005) Detection and Cultivation of Soil Verrucomicrobia. Appl Environ Microbiol 71(12):8402–8410 23. Galperin MY (2008) New feel for new phyla. Environ Microbiol 10:1927–1933 24. Chouari R, Le Paslier D, Dauga C, Daegelen P, Weissenbach J, Sghir A (2005) Novel major bacteria candidate division within a municipal anaerobic sludge digester. Appl Environ Microbiol 71(4):2145–2153 25. Haldar S, Choudhury SR, Sengupta S (2011) Genetic and functional diversities of bacterial communities in the rhizosphere of Arachis hypogaea. Antonie Van Leeuwenhoek 100:161–170 26. Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, Shiba T et al (2002) Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci USA 99:996–1001 27. Bruggemann H, Baumer S, Fricke WF, Wiezer A, Liesegang H, Decker I et al (2003) The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc Natl Acad Sci USA 100:1316–1321 28. No¨lling J, Breton G, Omelchenko MV, Kira S, Zeng Q, Gibson R et al (2001) Genome sequence and comparative analysis of the solvent-producing Bacterium Clostridium acetobutylicum. J Bacteriol 183(16):4823–4838 29. Miura Y, Watanabe Y, Okabe S (2007) Significance of Chloroflexi in performance of submerged membrane bioreactors (MBR) treating municipal wastewater. Environ Sci Technol 41(22): 7787–7794 30. Nelson MC, Morrison M, Yu Z (2011) A meta-analysis of the microbial diversity observed in anaerobic digesters. Biores Technol 102:3730–3739 31. Strous M, Fuerst JA, Kramer EHM, Logemann S et al (1999) Missing lithotroph identified as new planctomycete. Nature 400:446–449 32. Yoon JH, Kim IG, Oh TK, Park YH (2004) Microbulbifer maritimus sp. nov., isolated from an intertidal sediment from the Yellow Sea, Korea. Int J Syst Evol Microbiol 54(4):1111–1116 33. Miyazaki M, Nogi Y, Ohta Y, Hatada Y, Fujiwara Y, Ito S, Horikoshi K (2008) Microbulbifer agarilyticus sp. nov. and Microbulbifer thermotolerans sp. nov., agar-degrading bacteria isolated from deep-sea sediment. Int J Syst Evol Microbiol 58(5):1128–1133

123

34. Gonzalez JM, Mayer F, Moran MA, Hodson RE, Whitman WB (1997) Microbulbifer hydrolyticus gen. nov., sp. nov., and Marinobacterium georgiense gen. nov., sp. nov., two marine bacteria from a lignin-rich pulp mill waste enrichment community. Int J Syst Bacteriol 47:369–376 35. Guo F, Zhang S-H, Yu X, Wei B (2011) Variations of both bacterial community and extracellular polymers: the inducements of increase of cell hydrophobicity from biofloc to aerobic granule sludge. Biores Technol 102(11):6421–6428 36. Gonzalez-Martinez A, Calderon K, Albuquerque A, Hontorio E, Gonzalez-Lopez J, Guisado IM, Osorio F (2013) Biological and technical study of a partial-SHARON reactor at laboratory scale: effect of hydraulic retention time. Bioprocess Biosyst Eng 36:173–184 37. Yoo SH, Weon HY, Jang HB, Kim BY, Kwon SW, Go SJ, Stackebrandt E (2007) Sphingobacterium composti sp. nov., isolated from cotton-waste composts. Int J Syst Evol Microbiol 57(7):1590–1593 38. Bao HX, Ma XP, Wang J, Jing K, Shen ML, Wang AJ (2012) Performance of denitrifying phosphorous removal process employing nitrite as electron acceptor and the characterization of dominant functional populations. Adv Mater Res 455–456: 1019–1024 39. Nagashima S, Kamimura A, Shimizu T, Nakamura-Isaki S, Aono E, Sakamoto K et al (2012) Complete genome sequence of phototrophic betaproteobacterium Rubrivivax gelatinosus IL144. J Bacteriol 194(13):3541–3542 40. Xie B, Lv Z, Hu C, Yang X, Li X (2013) Nitrogen removal through different pathways in an aged refuse bioreactor treating mature landfill leachate. Appl Microbiol Biotechnol 97(20):9225–9234 41. Lam P, Jensen MM, Lavik G, McGinnis DF, Mu¨ller B, Schubert CJ, Amann R, Thamdrup B, Kuypers MMM (2007) Linking crenarchaeal and bacterial nitrification to anammox in the Black Sea. Proc Natl Acad Sci USA 104(17):7104–7109 42. Fahrbach M, Kuever J, Remesch M, Huber BE, Ka¨mpfer P, Dott W, Hollender J (2008) Steroidobacter denitrificans gen. nov., sp. nov., a steroidal hormone-degrading gammaproteobacterium. Int J Syst Evol Microbiol 58(Pt 9):2215–2223 43. Glavina Del Rio T, Abt B, Spring S, Lapidus A, Nolan M, Tice H et al (2010) Complete genome sequence of Chitinophaga pinensis type strain (UQM 2034). Stand Gen Sci 2(1):87–95 44. Chen HJ, Lin YZ, Fanjiang JM, Fan C (2013) Microbial community and treatment ability investigation in AOAO process for the optoelectronic wastewater treatment using PCR-DGGE biotechnology. Biodegradation 24(2):227–243 45. Suen G, Weimer PJ, Stevenson DM, Aylward FO, Boyum J, Deneke J et al (2011) The complete genome sequence of Fibrobacter succinogenes S85 reveals a cellulolytic and metabolic specialist. PloS One 6(4):e18814 46. Woyke T, Chertkov O, Lapidus A, Nolan M, Lucas S, Del Rio TG et al (2011) Complete genome sequence of the gliding freshwater bacterium Fluviicola taffensis type strain (RW262). Stand Gen Sci 5(1):21–29