A Unique Autothermal Thermophilic Aerobic DigestionProcess Showing a Dynamic Transition of Physicochemicaland Bacterial Characteristics from the Mesophilic to theThermophilic Phase

Yukihiro Tashiro,a,b Kosuke Kanda,a Yuya Asakura,a Toshihiko Kii,a Huijun Cheng,a Pramod Poudel,a Yuki Okugawa,a

Kosuke Tashiro,c Kenji Sakaia,b

aLaboratory of Soil and Environmental Microbiology, Division of Systems Bioengineering, Department ofBioscience and Biotechnology, Faculty of Agriculture, Graduate School of Bioresources and BioenvironmentalSciences, Kyushu University, Fukuoka, Japan

bLaboratory of Microbial Environmental Protection, Tropical Microbiology Unit, Center for InternationalEducation and Research of Agriculture, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

cLaboratory of Molecular Gene Technology, Division of Systems Bioengineering, Department of Bioscienceand Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan

ABSTRACT A unique autothermal thermophilic aerobic digestion (ATAD) processhas been used to convert human excreta to liquid fertilizer in Japan. This study in-vestigated the changes in physicochemical and bacterial community characteristicsduring the full-scale ATAD process operated for approximately 3 weeks in 2 differentyears. After initiating simultaneous aeration and mixing using an air-inducing circula-tor (aerator), the temperature autothermally increased rapidly in the first 1 to 2 dayswith exhaustive oxygen consumption, leading to a drastic decrease and gradual in-crease in oxidation-reduction potential in the first 2 days, reached �50°C in the mid-dle 4 to 6 days, and remained steady in the final phase. Volatile fatty acids wererapidly consumed and diminished in the first 2 days, whereas the ammonia nitrogenconcentration was relatively stable during the process, despite a gradual pH increaseto 9.3. Principal-coordinate analysis of 16S rRNA gene amplicons using next-generationsequencing divided the bacterial community structures into distinct clusters cor-responding to three phases, and they were similar in the final phase in bothyears despite different transitions in the middle phase. The predominant phyla(closest species, dominancy) in the initial, middle, and final phases were Proteo-bacteria (Arcobacter trophiarum, 19 to 43%; Acinetobacter towneri, 6.3 to 30%),Bacteroidetes (Moheibacter sediminis, 43 to 54%), and Firmicutes (Thermaerobactercomposti, 11 to 28%; Heliorestis baculata, 2.1 to 16%), respectively. Two predomi-nant operational taxonomic units (OTUs) in the final phase showed very low sim-ilarities to the closest species, indicating that the process is unique comparedwith previously published ones. This unique process with three distinctive phases wouldbe caused by the aerator with complete aeration.

IMPORTANCE Although the autothermal thermophilic aerobic digestion (ATAD) pro-cess has several advantages, such as a high degradation capacity, a short treatmentperiod, and inactivation of pathogens, one of the factors limiting its broad applica-tion is the high electric power consumption for aerators with a full-scale bioreactor.We elucidated the dynamics of the bacterial community structures, as well as thephysicochemical characteristics, in the ATAD process with a full-scale bioreactor fromhuman excreta for 3 weeks. Our results indicated that this unique process can be di-vided into three distinguishable phases by an aerator with complete aeration andshowed a possibility of shortening the digestion period to approximately 10 days.

Received 14 November 2017 Accepted 20December 2017

Accepted manuscript posted online 5January 2018

Citation Tashiro Y, Kanda K, Asakura Y, Kii T,Cheng H, Poudel P, Okugawa Y, Tashiro K, SakaiK. 2018. A unique autothermal thermophilicaerobic digestion process showing a dynamictransition of physicochemical and bacterialcharacteristics from the mesophilic to thethermophilic phase. Appl Environ Microbiol84:e02537-17. https://doi.org/10.1128/AEM.02537-17.

Editor Volker Müller, Goethe UniversityFrankfurt am Main

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Kenji Sakai,kensak@agr.kyushu-u.ac.jp.

BIODEGRADATION

crossm

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This research not only helps to identify which bacteria play significant roles and howthe process can be improved and controlled but also demonstrates an efficientATAD process with less electric power consumption for worldwide application.

KEYWORDS autothermal thermophilic aerobic digestion, bacterial communitystructure, human excreta, physicochemical properties, three phases

Several biological processes have been utilized to treat various types of generatedorganic solid and liquid wastes, including human excreta, kitchen garbage, munic-

ipal wastewater, and livestock manure. The processes are grouped into two categories:anaerobic and aerobic processes. Methane fermentation is the most popular among theanaerobic processes, and it has been carried out worldwide to treat several types ofwastes (1, 2). On the other hand, several types of aerobic processes, including theactivated-sludge process (3, 4), composting (5, 6), and autothermal thermophilic aer-obic digestion (ATAD) (7–9), have also been applied to organic waste treatmentprocesses. It has been reported that unique bacterial community structures are formedin each process, which can be attributed to their distinctive physicochemical propertiessuch as temperature and aerobic or anaerobic conditions (6, 10).

The ATAD process was first introduced in the 1970s, and it has been adopted for usein relatively small- and medium-size full-scale reactors over the activated-sludge pro-cess (11). The maximum temperatures differ between the ATAD process and theactivated-sludge process, as the ATAD process is operated under thermophilic condi-tions higher than 45°C, whereas the activated-sludge process is operated under roomtemperature or mesophilic conditions below 45°C (12). The ATAD process using batchmode has been considered to be divided into two distinct phases: mesophilic andthermophilic (13). The temperature increases to approximately 45°C at the mesophilicphase after the initiation of digestion, following which it is maintained at �45°C duringthe thermophilic phase. The ATAD process has several advantages, such as a highdegradation capacity, a short treatment period, and inactivation of pathogens (11). TheATAD process is currently applied mainly for the treatment of animal waste, sewagesludge, food processing wastes, and wastewater for release to the environment andlandfills after reducing their amounts and volumes (9, 12). Nevertheless, the mostsignificant bottleneck that prevents the application of the ATAD process worldwide isattributed to the high energy cost to supply oxygen compared with that observed inthe anaerobic digestion process. Therefore, detailed monitoring of its physicochemicaland microbial properties would be required to better understand the ATAD process andto improve its efficiency with low energy costs.

In Chikujo Town, Japan, human excreta have been treated with the ATAD processand then utilized as a useful organic liquid fertilizer for the agricultural cultivation ofcrops such as rice, wheat, and others. During the digestion, the temperature increasesto above 50°C without any additional heating. To date, there has been only one studythat performed a thermal balance analysis of this process using a full-scale bioreactor,which reported that the microbial heat generated from microbial activity, mechanicalheat derived from the aerator, and the jacket to prevent heat loss from the bioreactorparticularly contributed to the increase in the digestion temperature (14). We hypoth-esized that the adoption of an air-inducing circulator apparatus (aerator) for simulta-neous aeration and mixing would have two favorable effects on this digestion process:the direct transfer of mechanical heat to the sludge and the unique supply/circulationof oxygen.

Although many studies have reported on bacterial community structures of full-scale and lab-scale ATAD processes for several types of sludge (7, 13, 15–20), most ofthem have focused on analyzing samples in the thermophilic phase. To our knowledge,only one paper reported the dynamics of bacterial community structures by usingdenaturing gradient gel electrophoresis (DGGE) profiles from mesophilic to thermo-philic phases (20). Nevertheless, since the authors did not analyze the sequences of theDGGE bands, little is known of the phylogenetic dynamics of the bacterial community

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structures and the relationship between physicochemical properties and bacterialcommunity structures throughout the ATAD process. This study aimed to investigatethe dynamics of the physicochemical characteristics as well as bacterial communitystructure using high-throughput sequencing during the whole ATAD process forhuman excreta in a full-scale bioreactor. Here, we report the unique behaviors, groupedinto three phases, of the bacterial community structure corresponding to the dynamicchanges in temperature, dissolved oxygen (DO), oxidation-reduction potential (ORP),and other physicochemical characteristics during the full-scale ATAD process.

RESULTSPhysicochemical parameters during the ATAD process. The changes in the

physicochemical parameters of the ATAD process for the 2 years are presented in Fig.1. The temperature immediately started to increase after the initiation of the digestion,from atmospheric temperature to 52°C and higher after 5 days of digestion (Fig. 1a).Different increasing rates for temperature were observed during 0 to 2 days (0.4°C/h),2 to 5 days (0.2°C/h), and 5 to 22 days (no increase) (Fig. 1a). DO exhibited differentbehaviors during these three phases: low levels at �1 mg/liter (0 to 2 days), increaseswith drastic fluctuations of 1 to 3 mg/liter (2 to 5 days), and high levels with slightfluctuations at �3 mg/liter (5 to 22 days) (Fig. 1b). Corresponding to these results, ORP

FIG 1 Changes of physicochemical parameters in the ATAD process with human excreta in 2013 (blue) and 2014 (red). (a)temperature; (b) DO; (c) ORP; (d) pH; (e) acetic acid (closed symbols) and propionic acid (open symbols) in supernatant; (f) totalcarbon in supernatant; (g) ammonia nitrogen (closed symbols) and nitric acid nitrogen (open symbols) in supernatant; (h) totalnitrogen; (i) phosphate in supernatant.

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showed similar changes of �500 to �100 mV (0 to 2 days), �100 to 0 mV (2 to 5 days),and 0 mV (5 to 22 days) (Fig. 1c).

The pH increased gradually during the ATAD process from approximately 7.5 to 9.0or higher after 8 days, and it then became stable (Fig. 1d). Our high-pressure liquidchromatography (HPLC) system detected approximately 2 to 3 g/liter acetic acid,approximately 1 g/liter propionic acid, and a small amount of butyric acid in thesupernatants of the initial digestion broths. These organic acids were consumedpromptly and completely within 2 days (Fig. 1e), corresponding to the decreases intotal carbon (Fig. 1f), and they were not detected thereafter. The rapid increase in pHat the initial phase likely resulted from the consumption of these organic acids. On theother hand, ammonia nitrogen concentrations in the supernatant fluctuated untilapproximately 8 days and decreased slightly from approximately 1.6 g N/liter at 0 daysto approximately 1.3 g N/liter after 22 days for both years (Fig. 1g), whereas the nitrateconcentrations were negligible (approximately 0.0001 g N/liter) throughout the ATADprocess, much lower than those for ammonia nitrogen (Fig. 1g). These results indicatedthat the nitrification reaction did not occur. Total nitrogen concentrations also varieduntil 9 days and then clearly exhibited a slight decrease to approximately 1.7 g N/literafter 22 days (Fig. 1h). As a result, the main nitrogenous component in the final liquidfertilizer was ammonium ion, which is known as a soil adhesive and an effectivenitrogen fertilizer component. Concentrations of phosphate, another fertilizer compo-nent, were almost constant (Fig. 1i).

Thus, several physicochemical properties changed during the initial phase just afterstarting the digestion, and thereafter, most of the properties were stable.

Change in the total numbers of viable and dead cells. In regard to foodbornebacteria, Enterococcus faecalis and Clostridium perfringens were detected at 6.9 � 103

cells/ml and 3.3 � 102 cells/ml, respectively, in the original excreta samples (beforeinitialization of the ATAD process). These pathogenic bacteria diminished to thedetection limit of 1� 101 cells/ml after 6 days of digestion, when the temperaturereached higher than 50°C. Other pathogenic bacteria analyzed were not detectedthroughout the ATAD process. Therefore, the ATAD process effectively inactivatedseveral pathogenic bacteria in human excreta, which would result in the production ofa more stable and sanitary liquid fertilizer.

Figure 2 shows the changes in the total cell number and percentage of viable cellsduring the ATAD process. Total numbers greater than 1 � 109 cells/ml were foundthroughout the process. The values increased gradually from 1.6 � 109 cells/ml at 0days to 7.5 � 109 cells/ml at 4 days and then slightly decreased to 1.8 � 109 cells/mluntil the end of the ATAD process, although there were no statistically significantdifferences. This trend was caused by the drastic increase in viable cells during 0 to 4days. As a result, the percentages of viable cells increased drastically from 15% at 0 daysto 55% at 2 days and then remained at high levels of approximately 70% until 4 days.Viable cells also showed high levels, ranging from 40 to 60%, until 22 days. These resultssuggested that certain cells remained active because of the aeration and agitation afterthe initiation of the ATAD process until the end of the ATAD process.

Changes in the bacterial community structures during the ATAD process. Atotal of 454,122 reads derived from 20 ATAD samples were generated by 454 sequenc-ers (see Table S1 in the supplemental material). After the removal of reads with barcodeand primer mismatches, homopolymers, ambiguous bases, chimeras, and contami-nants, 184,798 reads were clustered to operational taxonomic units (OTUs) at a 97%and higher similarity, and then alpha and beta diversities were analyzed with QIIMEsoftware (6).

Figure 3 shows the distinguishable beta-diversity structures of each digestion phaseobtained by principal-coordinate analysis (PCoA). The diversities of the original humanexcreta before the digestion (0 days) and in the initial phase (1 to 2 days) for both yearswere clearly distinguishable and grouped into individual clusters. After 3 days and 4days, diverse clusters were formed between the 2013 and 2014 ATAD processes. These

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different bacterial structures converged upon the same cluster group after 6 days, 8days, 9 days, 14 days, and 22 days for both years. In addition, denaturing gradient gelelectrophoresis (DGGE) analysis (see Fig. S1 in the supplemental material) also showeddistinguishable patterns among the original human excreta (0 days) and the corre-sponding three phases (1 to 2 days, 3 to 4 days, and 6 to 22 days), and DGGE bandsshifted from low to high denaturing gradients. This suggested that bacteria with a low

FIG 2 Changes in total number of bacteria and percentage of viable cells during the ATAD processoperated in 2014. Circles and triangles indicate total cell number and percentage of viable cells,respectively.

FIG 3 Beta diversity of bacterial community structures in the ATAD process with human excreta at 3%OTU distance. Blue and red symbols, diversities in 2013 and 2014, respectively.

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G�C content were abundant in the initial phase, and then bacteria with a high G�Ccontent became predominant during the final phase. As suggested by the physico-chemical changes, the ATAD process for treating human excreta formed three distinc-tive bacterial community structures depending on operation time: the initial (1 to 2days), middle (3 to 4 days), and final (6 to 22 days) phases. Alpha-diversity analysis alsoshowed different values for several indexes among the phases (Table 1). In particular,the values of all the indexes, including observed OTU, Chao 1, Shannon, and phyloge-netic diversity (PD) whole, of the original human excreta were significantly (P � 0.01)higher than those in the initial, middle, and final phases.

Based on both the physicochemical properties (temperature increase rate, ORP, DO,organic acids, and total carbon) and bacterial community structure (alpha and betadiversities) findings, the full-scale ATAD process could be clearly divided into threephases: initial (0 to 2 days), middle (2 to 6 days), and final (6 to 22 days) (Table 2).

Bacterial community structure at the phylum and species levels. Figure 4 showsthat five phyla, i.e., Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Spiro-chaetes, accounted for more than approximately 94% of the members throughout theATAD process. In the 0-day samples before the initiation of the ATAD process, Bacte-roidetes (32% and 52%) and Firmicutes (46% and 18%) were the shared predominantphyla in both years. In the initial phase, Proteobacteria drastically increased from 2.4 to8.6% at 0 days to 52 to 70% in both years, with a decrease in Bacteroidetes andFirmicutes. In the middle phase (3 days and 4 days), the structure changes were differentin 2013 and 2014: Proteobacteria slightly decreased and there were small increases inFirmicutes and Bacteroidetes in 2013, whereas in 2014, Bacteroidetes drastically increasedto 80% and there was a decrease in Proteobacteria. These differences in the predom-inant phyla in the middle phase between 2013 and 2014 corresponded to the resultsof the beta-diversity analysis (Fig. 3). Thereafter, Actinobacteria grew actively, increasingfrom 1.2 to 2.6% after 4 days to 30 to 39% after 6 days in both years, and then showedstable high abundances of 30 to 69% in the final phase (until 22 days). Moreover,Firmicutes became a major phylum, with 18 to 41% abundance, in the final phase.

To analyze the predominant bacteria in more detail, we assigned representative OTUsequences in terms of closely related bacterial species. Figure 5 shows the phylogenetictree and heat map of the major OTUs of samples with abundances greater than 2%.Before the initiation of the ATAD process (day 0), no common OTUs were sharedbetween the samples from 2013 and 2014. On the other hand, OTU 535, related toArcobacter trophiarum (each relative abundance, 19 to 43%; pairwise similarity, 99.2%),and OTUs 9776, 11054, and 13623, related to Acinetobacter towneri (10 to 33%; 95.4 to98.8%) were abundantly present in the initial phase in both years. In the middle phaseduring the 3- to 4-day phase of the ATAD process, OTUs 9776, 11054, and 13623,related to Ac. towneri (13 to 14%; 95.4 to 98.8%), were still predominant in 2013,whereas different OTUs, 7006, 9145, 10799, and 14158, related to Moheibacter sediminis(43 to 54%; 91.4 to 94.6%), were predominant in 2014. After this phase, OTU 9470,related to Thermaerobacter composti (10 to 28%; 84.7%), and OTUs 3393 and 4762,related to Heliorestis baculata (2.1 to 16%; 84.1 to 84.4%), appeared as the predominantspecies in the final phase in both years. Note that these three OTUs showed very low

TABLE 1 Summary of alpha diversity at each phase in the ATAD process in 2 years by16S rRNA amplicon analysis using 454 pyrosequencing

Phase (sampling day[s])

Alpha diversitya by index:

Observed OTU Chao 1 Shannon PD whole

Original (0) 319 � 56 A 1,283 � 482 A 6.75 � 0.38 A 30.4 � 3.9 AInitial (1, 2) 234 � 24 B 818 � 188 C 5.38 � 0.48 C 22.5 � 2.2 BMiddle (3, 4) 247 � 57 B 1,031 � 310 B 5.80 � 1.10 BC 24.3 � 5.0 BFinal (6, 8, 9, 14, 22) 235 � 39 B 741 � 198 C 5.85 � 0.53 B 24.0 � 3.2 BaThe values represent the averages and standard deviations of the 10 trial values per one sample at eachphase in 2013 and 2014. Different letters indicate statistically significant different parameters among phases(P � 0.01).

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similarities, of approximately 84%, to the closest types of assigned culture strains. Theseresults indicated that the major bacterial species included bacteria that have not beenisolated or identified.

In summary, the bacterial community structures in the original human excreta werealtered by decreasing the alpha diversities and altering predominant phyla (species)through the ATAD process: original material, Bacteroidetes and Firmicutes; initial phase(0 to 2 days), Proteobacteria (Ar. trophiarum- and Ac. towneri-related strains); middlephase (2 to 4 days), Bacteroidetes (M. sediminis); and final phase (4 to 22 days), Firmicutes(T. composti and H. baculata).

DISCUSSION

In this report, we present the specific features of an ATAD process for human excretawhich was specifically modified via a novel aeration system to reduce mixing andenergy costs. The ATAD process has been performed under thermophilic conditions at�45°C to degrade various types of organic waste and wastewater and to inactivatepathogens for the production of sanitary fertilizer (8). The heat energy is mainlyproduced autothermally; that is, it comes from the oxidative degradation of organicmatter in the waste. Nevertheless, relatively little is known about the ATAD processcompared with traditional treatments, including the activated-sludge process, com-posting process, and methane fermentation process (12). In particular, most reportsfocused on the analysis of bacterial community structures during the thermophilicphase of the ATAD process in either full-scale or lab-scale bioreactors (7, 13, 15–20). Inthis study, we dynamically monitored bacterial community structures from the meso-philic to thermophilic phases using a high-throughput sequencer with higher resolu-tion than the PCR-DGGE method (6), and we elucidated the unique behaviors regardingthe physicochemical parameters and bacterial community structures of the ATADprocess for treating human excreta. Especially, it was revealed that the bacterialcommunity structure of the ATAD process in this study was distinct from those of theother ATAD processes reported in the literature, which was specifically caused bycomplete aeration.

The DO and ORP in the fermentation broth are important physicochemical param-

FIG 4 Change in bacterial community structure during the ATAD process at the phylum level.

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FIG 5 Heat map of major OTUs with abundance higher than 2% for any samples and phylogenetic tree including major OTUs and their most relatedtype strains.

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eters for evaluating the levels of oxygen consumption by bacteria in a given fermen-tation state. Although previous studies on the ATAD process using lab-scale bioreactorshave reported variations in the characteristics depending on temperature profiles andcompounds in the substrates (11, 21–23), to our knowledge, our study is the first thatreports drastic changes in DO (Fig. 1b) and ORP (Fig. 1c) as well as temperature (Fig. 1a).These characteristics changed according to the digestion phase: initial phase, drasticincreases in temperature and low DO and ORP values; middle phase, slight increases intemperature and slight recovery of DO and ORP with fluctuations; and final phase,constant temperatures and DO and ORP at maximum levels (Fig. 1a to c). A modelreported by Wang et al. (14) indirectly predicted that the heat generated by themicrobial oxidation of organic materials would be primarily responsible for the increasein digestion temperature during the treatment of human excreta with the ATADprocess. Our on-site measurements of DO and ORP directly confirmed the contributionsof oxygen consumption to the increase in digestion temperature. This study investi-gated the relationship between the increases in temperature and oxygen consumptionduring the ATAD process in full-scale bioreactors.

Many studies have reported the initial production and subsequent consumption ofvolatile fatty acids, such as acetic acid and propionic acid, during ATAD processes (21,22, 24). However, in the process described here, all acetic acid and propionic acid wererapidly consumed in the initial phase, after which no volatile fatty acids were detected(Fig. 1e). The major OTUs 9776, 11054, and 13623, related to Ac. towneri, in the initialphase contributed to the rapid consumption of organic acids in this study, becauseAcinetobacter spp. are reported to utilize organic acids, including acetic acid and citricacid (25). We successfully isolated Ac. towneri strains from the ATAD samples using asystematic feedback isolation technique (26) (data not shown), and further studies areneeded to elucidate its functions in the ATAD process.

Interestingly, the ATAD process presented here showed a small fluctuation in theammonia nitrogen concentration during the initial and middle phases, followed byconstant values of approximately 1.3 g/liter during the final phase (Fig. 1g). Variation inammonia nitrogen in the ATAD process have been reported in the literature, includinga simple increase (23), an increase and subsequent decrease (22), and a simple decrease(27). Ammonia nitrogen is removed as nitrogen gas via nitrification and denitrificationreactions (28). Nitrification and (aerobic) denitrification reactions are known to beperformed by nitrifying bacteria, such as ammonium-oxidizing bacteria and nitrite-oxidizing bacteria, and (aerobic) denitrifying bacteria (28–31). The bacterial communitystructure analysis performed here revealed no data related to these nitrifying and(aerobic) denitrifying bacteria, even though it is an aerobic process. Although the abilityof Acinetobacter spp. in a coke wastewater plant to remove ammonia nitrogen has beenreported (25), the predominance of Ac. towneri in Fig. 5 indicates a different function.These results suggested that this ATAD process has unique features, with fewernitrification and denitrification reactions. It is notable that the high ammonia nitrogenconcentration (1.3 g/liter) in the final product produced by ATAD of human excreta isadvantageous for its application as a liquid fertilizer (32).

Thermophilic conditions at �56°C have been shown to eliminate pathogenic bac-teria such as Salmonella spp. and enterococci from sludge in full-scale ATAD processes(33). Although the maximum temperatures of approximately 53 to 55°C (Fig. 1a) wereslightly lower in this study than that reported previously (33), this full-scale ATADprocess could inactivate pathogenic E. faecalis and C. perfringens within 6 days, as didanother run in the same full-scale bioreactor (data not shown). Therefore, this processis effective to yield a sanitary liquid fertilizer from human excreta.

We were able to successfully determine drastic changes in the community structuresthrough the mesophilic phase to the thermophilic phase (initial, middle, and finalphases) during the ATAD process for treating human excreta (Fig. 3 to 5; Table 2), whichimparted the respective functions that are necessary when producing liquid fertilizerfrom human excreta. It was also suggested that proliferating bacteria would be selectedby adaptation to the physicochemically changing environments in each phase (Fig. 1)

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or by the cycles of cell growth and lysis being repeated in each phase, and each of themwould play a function during the ATAD process. Previous reports focused on analyzingbacterial community structures not in the mesophilic phase but in the thermophilicphase, after reaching the highest temperatures, and a few reports have analyzed thebacterial community structures only at the thermophilic phase in the ATAD processwith full-scale (13, 15, 16, 19) and lab-scale (7, 17, 18) bioreactors using molecularmethods (Table 3). Our results indicated that several Proteobacteria related to Ar.trophiarum (pairwise similarity, 99.2%) and Ac. towneri (95.4 to 98.8%) became predom-inant in the initial phase with the rapid increase in temperature, whereas the closestrelative of M. sediminis (91.4 to 94.6%), belonging to Bacteroidetes, proliferated in themiddle phase with the gradual increase in temperature. Two distinct members ofFirmicutes related to T. composti (84.7%) and H. baculata (84.1 to 84.4%) were recog-nized as major OTUs during only the final phase in this ATAD process, whereas strainsbelonging to the same phylum of Firmicutes have been reported as the predominantstrains in other ATAD processes (Table 3). In particular, it is notable that the pairwisesimilarities of these predominant OTUs in the final phase were very low, around 84 to85%, which suggested that as-yet-uncultured and unidentified bacteria play roles in theconversion of human excreta to liquid fertilizers. Although isolations of Ac. towneri, thepredominant species in the initial phase, succeeded, we failed to isolate strains relatedto those predominant OTUs at the final phase using several conventional culturingtechniques, probably because growth conditions such as medium compositions havenot been proper (data not shown). These findings suggested that this ATAD process forhuman excreta formed very unique bacterial community structures that underwentdrastic changes during the process, which contributed to the distinct physicochemicalcharacteristics that were observed. We speculate that the adoption of an air-inducingcirculator in the treatment system accounted for these unique characteristics of theATAD process shown here.

In addition, it was novel that several bacteria belonging to the Clostridium andBacillus genera were not predominant in this ATAD process (Table 3); nevertheless,these genera have been considered to be major participants at the thermophilic phasein full-scale bioreactors as reported thus far (13, 15, 16). Piterina et al. suggested thatincomplete aeration and inefficient oxygen supplementation in full-scale bioreactors byan aerator would partially contribute to an anaerobic condition, which resulted inproliferation of those anaerobic bacteria in the ATAD process (15). From the level of DOat �3 mg/liter (Fig. 1b) and ORP at approximately 0 mV (Fig. 1c), on the other hand,aerobic conditions would be established at the thermophilic phase regardless of usageof an aerator in this study. Therefore, the aerator in this ATAD process would providemore efficient aeration and oxygen supplementation with higher strength than thatreported previously, which resulted in different predominant bacteria. Further researchis planned to investigate the function of aerators in the dynamics of bacterial commu-nity structures in the ATAD process.

The energy cost of electric power consumption using an aerator for simultaneousaeration and mixing in an ATAD process would be much higher than that in ananaerobic digestion process, which prevents the application of the ATAD processworldwide. Not only physicochemical properties, including temperature, DO, ORP, pH,etc. (Fig. 1), but also bacterial community structures (Fig. 3 to 5) were mostly stable after6 days during the final phase, with the inactivation of several pathogenic bacteria. Ourresults imply that the operation time would be shortened to around 10 to 14 days toproduce a sanitary liquid fertilizer, which would lead to drastically less electric powerusage. The minimization of the aerator operation energy is worthy of further study toestablish a more efficient ATAD process.

In conclusion, we elucidated the distinctive physicochemical characteristics andbacterial community structures during the ATAD process for human excreta using afull-scale bioreactor. Previously, the ATAD process has been considered to consist oftwo distinct phases: a mesophilic phase and a thermophilic phase (9). Our resultsindicated that the process can be divided into three distinguishable phases (Table 3).

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TAB

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However, little is known about the mechanism of the drastic transitions in the bacterialcommunity structure during the ATAD process. Additional studies are required toinvestigate the functions of each type of bacteria in this ATAD process.

MATERIALS AND METHODSSampling of sludge from the full-scale ATAD bioreactor. The facility for the full-scale ATAD

bioreactor for human excreta is located at 33°39=03.0N, 131°04=36.9E in Chikujo Town, FukuokaPrefecture, Japan. Briefly, collected human excreta were roughly filtered to remove foreign materials suchas stone, plastic, and metal, and then enzymatic powder (Asahi Kasei Clean Chemical Co. Ltd., Shizuoka,Japan) was added to the raw slurry at 300 ppm. Although the enzymatic powder is considered to includehydrolytic enzymes for polysaccharides, proteins, and lipids, according to the manufacturer’s instructions,our preliminary investigations found that the powder had negligible effects on the ATAD process, withsmall numbers of bacterial cells and weak lipase activity (unpublished data). Approximately 150 m3 ofraw slurry was fed to the semiunderground empty bioreactor, which was made of concrete with athickness of 300 mm and a working volume of 180 m3, and covered above ground by soil (14). Thedigestion started with simultaneous mixing and aeration using an aerator apparatus in batch modewithout any control of the temperature, pH, and DO parameters for approximately 3 weeks. After everyATAD process of approximately 3 weeks, the digested slurry was removed from the bioreactor, stockedin a tank, and used as liquid fertilizer, and the next ATAD run was then started using a new raw slurrywithout sterilizing and washing the bioreactor. In this study, two sets of treated human excreta sampleswere collected at days 0, 1, 2, 3, 4, 6, 8, 9, 14, and 22, on 2 to 24 July 2013 and 17 June to 9 July 2014.Samples were stored at �20°C before use. The physicochemical properties of the raw slurries in 2013 and2014, respectively, were as follows: pH, 7.7 and 7.4; mixed-liquor suspended solids (MLSS), 3.5 g/liter and1.1 g/liter; mixed-liquor volatile suspended solids (MLVSS), 2.8 g/liter and 0.90 g/liter; total carbon, 3.5 gC/liter and 2.2 g C/liter; and total nitrogen, 2.7 g N/liter and 2.0 g N/liter.

Physicochemical analysis during the ATAD process. The changes in temperature, DO, electricalconductivity (EC), and ORP in the ATAD process were measured every hour in situ using a portable sensor(WQC-24; DKK-TOA Corp., Tokyo, Japan). The pH levels were measured using a Laqua twin pH meter(Horiba Ltd., Kyoto, Japan) during the sampling. A Gastec GV-100S meter (Gastec, Kanagawa, Japan) wasused for on-site measurements of odor (ammonia [3HM glass tube], methyl mercaptan [71 glass tube],and hydrogen sulfide [4HN glass tube]) in the headspace of the ATAD bioreactor. The total carbon andnitrogen contents of the supernatant and pellet were measured with a CN corder (Macro CorderJM1000CN; J-Science Lab, Kyoto, Japan) using hippuric acid as a standard. Prior to the other chemicalanalyses (total organic acids, ammonia, nitrate, and phosphate), 3 ml of each sample was pretreated byultrasonication (UD-200; Tomy, Tokyo, Japan) for 30 s, centrifugation for 10 min at 12,000 rpm at 4°C, andfiltration through a cellulose acetate filter membrane (pore size, 0.45 �m; Advantec, Osaka, Japan) toobtain supernatants. Total organic acid content was analyzed using a specific HPLC system (Organic AcidAnalyzer; Shimadzu, Kyoto, Japan) (34). The indophenol method was adopted for ammonium ionmeasurements (35). NO3

� and PO43� anions in the supernatants were measured with an ion chromato-

graph (IC-2001; Tosoh, Tokyo, Japan) under the following conditions: flow rate of the eluting solutioncontaining 1.9 mM sodium hydrogen carbonate and 3.2 mM sodium carbonate, 0.800 ml/min; column,TSKgel SuperIC-AZ (Tosoh); column temperature, 40°C; and detector, electric conductivity.

Microbial analysis during the ATAD process. (i) Counting viable and dead cells. A BacLight kit(Thermo Fisher Scientific Inc., Kanagawa, Japan) was used to count viable or dead cells according apreviously described protocol (36). One milliliter of each sample was incubated at room temperature inthe dark for 15 min. Five microliters of bacterial cell suspensions stained with the dyes was spotted ona 12-well microscope slide, dried for 20 min at 40°C, and observed under fluorescence microscopy(BZ-9000; Keyence, Osaka, Japan).

(ii) Detection of foodborne pathogenic bacteria. Several foodborne pathogens, such as Escherichiacoli O157 (as well as nonpathogenic E. coli), Salmonella spp., Shigella spp., Enterococcus faecalis,Enterococcus hirae, Clostridium difficile, Bacillus cereus, and Staphylococcus aureus, were enumerated usingselective medium for each (see Table S2 in the supplemental material). One milliliter of each ATADsample (0 days, 6 days, and 22 days) taken in 2014 was aseptically pipetted into 9 ml of sterilephosphate-buffered saline (PBS) (0.2 g potassium dihydrogen phosphate, 0.2 g potassium chloride, 1.15g disodium hydrogen phosphate, and 8.0 g sodium chloride in 1,000 ml of deionized water) and dilutedto 10�4. Each 0.1 ml of diluent was spread onto the respective agar plate and incubated (Table S2). Forfurther identification, DNAs from a few colonies on each type of agar medium were extracted andsubjected to 16S rRNA gene sequence analysis. Amplification of the 16S rRNA gene was performed usingthe universal primer set 8F (5=-AGAGTTTGATCCCTCAG-3=) and 1492R (5=-GGTTACCTTGTTACGACTT-3=)(37) in a total volume of 50 �l containing 1.25 U Ex Taq DNA polymerase (TaKaRa Bio, Shiga, Japan), 0.2mmol liter�1 deoxynucleoside triphosphates (dNTPs), 0.8 �mol liter�1 both primers, and 20 ng �l�1 DNAtemplate. The amplification conditions were as follows: 30 cycles of DNA denaturation at 98°C for 10 s,primer annealing at 55°C for 5 s, and elongation at 72°C for 1 min. The PCR products were purified usinga QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions. The sequencing ofpurified DNA was performed by Fasmac Co. Ltd., (Kanagawa, Japan). The similarity scores of nearlyfull-length sequences were calculated by the EzTaxon-e server (38).

(iii) Bacterial community structure analysis by high-throughput pyrosequencing. Total DNA wasextracted from the centrifugation pellet of 1 ml of the broth for each sample using a PowerSoil DNAisolation kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. The

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partial 16S rRNA gene (V3 to V5 region) (6) was amplified using the extracted DNA samples as templateswith universal primer pair for eubacteria, 357f (5=-CCTACGGGAGGCAGCAG-3=) and 926r (5=-CCGTCAATTCCTTTRAGTTT-3=), with 11-bp barcoded sequences in a total volume of 25 �l containing 0.675 U of ExTaq DNA polymerase (TaKaRa Bio), 0.2 mM dNTPs, 0.5 �M each primer, and 1 �l DNA template. The PCRcycling conditions were as follows: 94°C for 40 s, 50°C for 40 s, and 72°C for 1 min for 30 cycles and afinal extension at 72°C for 5 min (6). PCR products were purified using a QIAquick PCR purification kit. Thepurified PCR products were sequenced using the 454 GS FLX Titanium XL� platform (Roche, Basel,Schwarz) according to the manufacturer’s instructions. After the pyrosequencing was performed, thedata were processed using QIIME software (39) and the Black Box Chimera Check (B2C2) softwarepackage (40). Grouping into operational taxonomic units (OTUs) with a similarity higher than 97%,assignment of representative OTU sequences of closely related bacteria at the phylum and species levels,and analysis of alpha diversity (observed OTU, Chao 1, Shannon index, and PD whole) (Table S3) and betadiversity (principal-coordinate analysis [PCoA]) were performed as described previously (6). Because theindexes of alpha diversity have been considered to be basic and important to monitor the diversity ofmicrobial community structures in treatment process and ecologic environments (6), an analysis ofvariance (ANOVA) was performed to reveal the differences of alpha-diversity indexes between thephases.

Accession number(s). The nucleotide sequences of 37 selected OTUs have been deposited underaccession numbers LC326070 to LC326106 in the DNA Data Bank of Japan (DDBJ).

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02537-17.

SUPPLEMENTAL FILE 1, PDF file, 0.2 MB.

ACKNOWLEDGMENTSThis work was partly supported by grants from Chikujo Town office, Fukuoka

Prefecture, Japan, and from JST/JICA, SATREPS (Science and Technology ResearchPartnership for Sustainable Development).

We acknowledge the staff at the ATAD process facility in Chikujo Town, Fukuoka,Japan, for taking digestion samples.

We have no conflicts of interest to declare.

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