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Accepted Manuscript: Bioresource Technology
Accepted Manuscript: Bioresource Technology
Yu Qin, Atsushi Higashimori, Li-Jie Wu, Toshimasa Hojo, Kengo Kubota, Yu-
You Li
PII: S0960-8524(17)31438-4
DOI: http://dx.doi.org/10.1016/j.biortech.2017.08.124
Reference: BITE 18733
Please cite this article as: Qin, Y., Higashimori, A., Wu, L-J., Hojo, T., Kubota, K., Li, Y-Y., Phase separation and
microbial distribution in the hyperthermophilic-mesophilic-type temperature-phased anaerobic digestion (TPAD)
of waste activated sludge (WAS), Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.
2017.08.124
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1 Phase separation and microbial distribution in the
5 Yu Qina, Atsushi Higashimori a, Li-Jie Wua, Toshimasa Hojoa, Kengo Kubotaa, Yu-You Li
6 *a,b
8 a
Department of Civil and Environmental Engineering, Graduate School of Engineering,
10 b
Department of Frontier Science for Advanced Environment, Graduate School of
12 980-8579, Japan
13
16 E-mail: gyokuyu.ri.a5@tohoku.ac.jp
17
18 Abstract
19 In order to investigate the phase separation and microbial distribution in the TPAD, the
25 was not found in TM-TPAD. The differences on microbial distributions also reflected the
27 abundance in the first stage than the second stage of HM-TPAD but it had similar
28 abundance between the two stages of TM-TPAD. Also, the archaeal communities from the
29 two stages of HM-TPAD shared the least similarity but those from the two stages of
31
34
35 1. Introduction
36 With the expansion of urbanization, the need for proper disposal techniques for the bulk
37 volumes of sewage sludge becomes more and more pronounced. Anaerobic digestion (AD)
38 is the essential link in the sewage sludge disposal: it can recycle bioenergy in the form of
39 methane and achieve the diminution of solid waste. Sewage sludge generally consists of
40 primary sludge and waste activated sludge (WAS). It is known that WAS is less degraded
41 than primary sludge in anaerobic digestions and that hydrolysis of WAS was the
42 rate-limiting step in anaerobic digestion (Gavala et al., 2003; Li & Noike, 1992;
43 Westerholm et al., 2016). Thus, great efforts have been made on enhancing the
44 degradability of WAS.
48 over single-phase mesophilic system (Riau et al., 2010). In recent years, diverse
49 combinations of temperature were applied as new type TPAD, among which the
52 phase, showing a 30~50% enhancement in both solubilization and gas production over
53 single-stage digestion. Other studies also found that the hyperthermophilic condition
54 played an effective role on the hydrolysis and acidogenesis of WAS (Alqaralleh et al.,
55 2016; Ge et al., 2011; Yu et al., 2013). Even as a thermal pretreatment, the 70 ºC harvested
57 degradation of WAS without domesticated sludge as inoculum (Farno et al., 2016; Gavala
58 et al., 2003) As a value of below the boiling point of water, hyperthermophilic condition
60 (Carlsson et al., 2012; Carrere et al., 2010). Assessments on energy production and
63 However, the direct comparison was barely discussed between the conventional TM-TPAD
65 microbial communities in the conventional TM-TPAD has not been yet fully explored in
66 the literature. Previous studies have shown that temperature affected the microbial
67 communities in the pretreatment stage of WAS (Pervin et al., 2013) and that the
69 et al., 2012). For a cascaded process such as TPAD, it is reasonable to assume that the first
70 reactor with different conditions would influence the next stage differently. Yet, current
73 In this case, a lab-scale TPAD process was performed with a single-stage mesophilic
74 anaerobic digestion (MAD) as control. The conditions of TM-TPAD and HM-TPAD were
75 successively applied on the TPAD process. The conversion efficiencies of the four steps in
76 the methane fermentation was calculated to describe the phase separation. The microbial
77 distribution was presented in the form of 16S rRNA gene clone library. Moreover,
78 real-time quantitative polymerase chain reaction (qPCR) was conducted to investigate the
79 changes in the populations of targeted microbial groups, such as Bacteria and Archaea.
82 Figure 1
83 As shown in Figure 1, the digesters were continuous stirring tank reactors (CSTR),
84 equipped with stainless shafts and paddles driven by motors. Each tank had water jacket
85 that allowed warm water to flowing through to maintain its temperature. The slurry of
86 sludge was transported by the fill-and-draw strategy with roller pumps (FURUE SCIENCE,
87 RP-LV2), which were controlled by timers to run 5 times a day. Two processes were
88 operated in parallel: a single-stage MAD system (35 ± 1 ºC) and a TPAD system. The
89 working volume of the first stage and second stage of TPAD were 3 L and 12 L,
91 According to the temperature combination of the first and second stages, TPAD was
92 operated in different conditions in two successive periods: TM period (55±1 ºC & 35±1 ºC,
93 in thermophilic and mesophilic conditions) and the HM period (70±1 ºC & 35±1 ºC, in
96 and the second stage of TPAD are respectively expressed as (TPAD, TM or HM) - (1 or 2).
97 MAD was 35±1 ºC all along the two periods. No pH adjustment was performed and no
99 Table 1
100 WAS was taken monthly from the vacuum belt filters in the Senen Purification Centre in
101 Sendai, Japan. The WAS was preserved below 4 ºC in the substrate tanks to avoid
102 decomposition. Details of the substrate are listed in Table 1. The mesophilic inoculum was
103 also taken from mesophilic digesters in the Senen Purification Centre. The thermophilic
104 inoculum was from thermophilic digesters treating municipal solid waste in the Maihira
105 Cleaning Centre in Niigata, Japan. After inoculation, both the MAD and the TPAD were
106 started up by shortening the HRT from 100 days (for 2 weeks) to 50 days (for 2 weeks),
107 and then 30 days for long-term observations. The HRTs of TPAD-1 and TPAD-2 were 6
110 The volume of the produced biogas was measured with wet-type gas meters
111 (SHINAGAWA, W-NK-0.5) and standardized to standard temperature and pressure (STP,
112 273.15 K and 1 atm). The gas contents (N2, CH4 and CO2) were analyzed using a gas
113 chromatograph (SHIMADZU, GC-8A) equipped with a stainless steel column packed with
114 Porapak-Q and a thermal conductivity detector (TCD). The temperature of the column and
115 the detector were 70 ºC and 100 ºC, respectively. The pH, total solids (TS), volatile solids
116 (VS), suspended solids (SS), volatile suspended solids (VSS) and chemical oxygen
117 demands (COD) were measured according to the Standard Methods (APHA, 2012). The
118 proteins contents were determined by the Lowry method (Lowry et al., 1951). Ammonia
119 concentrations were analyzed using the indophenol method (Bolleter et al., 1961). Volatile
120 fatty acids (VFA) were detected using a gas chromatograph (Agilent 6890) equipped with
121 a 30-meter capillary column (J&W, DB-WAXetr) and a flame ionization detector (FID).
122 For every sample, the column was held at 125 ºC for 5 min, heated linearly from 125 ºC to
123 180 ºC in 22 min and then held at 180 ºC for 10 min. Both the injector and the detector
124 were maintained at 250 ºC. The soluble components were defined as their concentrations
125 in the supernatant after 15000 rpm centrifugation for 20 min. Both the ammonia and VFA
126 were analyzed with the filtered (0.45 μm, hydrophilic PES) supernatant samples.
128 The removal efficiencies η of the systems were calculated based on the influent and
131 Then the contributions to the total removal by each stage, η1/η and η2/η were calculated.
134 where c0 is the concentration in the influent, g/L; c1 and c2 are the concentration in the
135 effluent from the first and the second stage, respectively, g/L. It should be noted that c and
137 In order to express the absolute reacted ratios of the four steps involved in methane
138 fermentation, the average reaction efficiencies were defined as the ratio of the converted
139 mass to the total input mass (on COD basis). The (average) reaction efficiencies of
140 hydrolysis (ηhyd), acidogenensis (ηacid), acetogenesis (ηacet) and methanogenesis (ηmeth)
141 were calculated in accordance with a method outlined in an earlier report (Kobayashi et al.,
142 2014). The efficiencies represented the macroscopic extents of the four steps of the
143 methane fermentation. Especially, a change in the efficiencies between acidogenesis and
147 ηacid = [(cttl,inf – cac,inf) - (cttl,eff – cac,eff)] / c0× 100% Eq. (6)
149 where c0 is the total COD concentration at the entrance of the process/reactor, g/L; cptc,inf
150 and cptc,eff are the particle COD concentration in the influent and effluent, respectively, g/L;
151 cttl,inf and cttl,eff are the total COD concentration in the influent and effluent, respectively,
152 g/L; cvfa,inf and cvfa,eff are the VFA concentration (on COD basis) in the influent and effluent,
153 respectively, g/L; cac,inf and cac,eff are the acetate concentration (on COD basis) in the
156 thermodynamic characteristics at 70 °C. The Gibbs free energy changes and the
157 equilibrium constants of isomerizations were modified with temperature according to the
160 Samples for qPCR were taken on the 113rd day (TM period), and also on the 217th, 229th
161 and 251st day (HM period). A qPCR was conducted according to the method established by
162 Miyashita et al. (2009) and the primers for Bacteria (Eub338f/Eub907r) and Archaea
163 (Arc109f/Arc912r) were selected accordingly. In addition, primers for the members of
164 Coprothermobacter were Cth485f (5’-TAC CCC AGT AGA AAG GGA-3’) (Gagliano et
165 al., 2014) and Cth584r (5’-TGA GAC ACA GAC AAC CAC-3’) (designed in this study).
166 Samples for 16S rRNA gene cloning analysis were taken when reactors were on the steady
167 state. The sludge samples were washed with phosphate buffer solution, extracted for DNA
168 with Ultra Clean® Soil DNA Isolation Kit (MO BIO) and preserved below −20℃. The
169 primer sets for bacterial 16S rRNA genes were Eub8f/Univ1500r and those for archaeal
170 16S rRNA genes were Arc109f /Arc1059r (Amann et al., 1990; Weisburg et al., 1991). The
171 PCR was conducted under TaKaRa ExTaq® Hot Start Version (TaKaRa Bio, Inc). the PCR
172 codnition included an initial denaturation step at 94 °C for 2 min, followed by 24 cycles of
173 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s, with a final extension step of 72 °C for 7
174 min. The PCR products were purified with MinElute PCR Purification Kit (Qiagen) and its
175 ligation and transformation were performed with TOPO TA Cloning Kit (Invitrogen). The
176 clone numbers for bacteria and archaea were 128 and 64, respectively. For each read,
177 about 600 bases starting from 907R (bacteria) or 1059R (archaea), were sequenced by
178 TaKaRa Bio, Inc. The sequencing results were processed according to Kubota et al. (2014).
179 Additionally, principal component analysis (PCA) was performed with the software PAST
180 v3.11 (Øyvind et al., 2001). Calculations for hierarchical clustering were based on the
181 Euclidean distances on the OTU level and the results were displayed in dissimilarities.
184 Figure 2
185 Figure 2 shows the time course of the performance of MAD and TPAD. The results of
186 each reactor during steady states were summarized in Table 2. The MAD system produced
187 biogas at the rate of 0.39 ± 0.04 L-biogas/L-reactor/d (L/L/d), in which 63.01 ± 0.77%
188 were CH4, and the pH maintained 7.34 ± 0.03 with rare VFA accumulation. With respect to
189 the TM-TPAD, biogas production rates in TM-1 were considerably higher than TM-2.
190 Indistinctive differences (p= 0.890>0.05) were found in the pH values between the two
191 stages of the TM-TPAD system. Since no VFA accumulation was observed in either stage,
192 except for temporary appearances in the thermophilic TM-1 at less than 3 g-acetate/L, both
193 digesters of TM-TPAD system were operated stably. The first stage (TM-1) was kept
194 suitable for methanogenesis by maintaining a pH around 7.5: namely, there was no phase
195 separation in the TM-TPAD.
196 After the first stage of TPAD shifted from 55 ºC to 70 ºC on the 134th day, several abrupt
197 changes occurred leading to phase separation. In HM-1, biogas production dropped to 0.40
198 L/L/d as soon as the temperature was shifted, and continued to decrease gradually until the
199 150th day. Also, the CH4 contents fell from 60.8% to 26.0% and then rose to about 40%
200 until the 180th day. The pH also dropped to 6.5 when the VFA concentration was about 8
201 g-acetate/L, indicating that acidification occurred in HM-1. Subsequently, HM-1 reached a
202 steady state with a pH around 6.4 and VFA around 6 g-acetate/L. In HM-2, biogas
203 production ascended slightly to 0.40 L/L/d and the CH4 content in the gaseous phase was
204 stable at 67.59%. No decline in the pH (7.47 ± 0.02) or VFA accumulation was observed in
205 the mesophilic HM-2, indicating the TPAD system was running stably in the HM
206 condition.
207 Based on the data from the steady states, details of the biogas production and organic
209 Table 2
210 The ranking of organic removals in the systems can be summarized as TPAD (HM > TM) >
211 MAD. Within the TM-TPAD system, major organic removals (73% of total VS removal)
212 and major particle solubilizations (83% of total VSS removal) were accomplished in the
213 TM-1, whereas it was different in the HM-TPAD. Even though HM-1 largely shouldered
214 the tasks of solubilizing particles and removing organics, its contribution to the total
215 removal by the whole HM-TPAD system dropped to 53% (of total VS removal) and 70%
216 (of total VSS removal) compared to TM-1. The drop in its contribution implies the
217 application of the hyperthermophilic condition did not substantially accelerate degradation
218 or solubilization within the digester. Under the conditions at 70 ºC, however, degradation
219 was improved in the downstream methanogenic digester. It is considered that the HM-1 in
220 the TPAD played the role of thermal pretreatment rather than microbial hydrolysis when
221 the temperature condition in the first stage changed from thermophilic to
222 hyperthermophilic.
223 Destruction of proteins is regarded as significant item for WAS reduction since protein is
224 the main components instead of carbohydrates and lipids. It should be noted that 88% of
225 the total protein removal in the entire TM-TPAD system was accomplished in the TM-1
226 and that for particle proteins it was as high as 97%, which was higher than the values of
227 VS and VSS removals. Despite the drop from TM-1 to HM-1 mentioned before, 88% of
228 the particle protein solubilization in the whole HM-TPAD system occurred in HM-1. On
229 the other hand, only 9% of the total COD removal occurred in HM-1. It is implicated that
230 in HM-1, major proteins were degraded into smaller molecules but not converted into
231 methane, thus remained in the liquid of HM-1, which was in accordance with the
234 The conversion efficiencies describe the proportions of organics that have undergone a
235 reaction under a given organic loading rate. With COD basis, the tendency of electron
236 flows through each system could be quantified. It should be noted that these percentages
237 represent the capacities of each digester under the steady state.
238 Figure 3
239 As can be seen in Figure 3 (a), 47% of the fed WAS were hydrolyzed in the thermophilic
240 TM-1, higher than the 40% in the single-phase MAD. When considered with the next three
241 steps, the conversion efficiency of the entire TM-TPAD system was about 8% higher than
242 the contemporaneous MAD55. The absence of cumulative VFA detected in TM-1 could be
243 attributed to the rate of VFA consumption being faster than the rate of VFA production
244 from the hydrolysis of WAS. In TM-1, since the following steps were almost equal to the
245 acidogenesis, it appeared to be the rate-limiting step. However, this explanation about
246 rate-limiting step disagrees with the well-accepted opinion that hydrolysis is the rate
247 limiting step in WAS digestion. This was attributed to the method in this study that defined
248 the soluble compounds as those in the centrifuged supernatant. The true soluble COD
249 could be lower than the supernatant COD so that the real hydrolysis efficiency would be
250 the same with the following steps as rate-limiting step. Besides, no difference in the
251 acidogenesis, acetogenesis and methanogenesis was found in either of the stages of the
252 TM-TPAD. Thus, it was concluded that no phase separation occurred in this system.
253 The total HM-TPAD system accomplished removal rates approximately 13-15% higher
254 than that of the contemporaneous MAD70, as indicated in Figure 3 (b). As was mentioned
255 earlier, the hydrolysis efficiencies of the HM-1 were considerably lower than those of the
256 TM-1 and MAD70. Within HM-TPAD, HM-1 was notable for hydrolysis while HM-2 was
258 acid-consuming step), a decline in efficiency left approximately 11% of COD in forms of
259 VFA, which was responsible for lowering the pH. Meanwhile in HM-2, this gradient was
260 reversed and enlarged to 18%. The efficiencies of acetogenesis and methanogenesis even
261 surpassed those of MAD70. The accumulated VFA in the effluent of HM-1 might stimulate
262 the methanogenesis in HM-2. Compared with the control system MAD, it was concluded
263 that HM-TPAD obtained an enhancement of 14%, much higher than the 8% in TM-TPAD.
264 Even though the organic removals (TS, VS and COD) of the thermophilic digester (TM-1)
265 were higher compared to the hyperthermophilic digester (HM-1), the HM-1 improved
266 degradation in the HM-2 by separating the acidogenic phase and methanogenic phase.
268 Figure 4
269 The microbial abundance were quantified with qPCR. The results revealed the relative
271 4.
272 The change in the number of 16S rRNA gene copies in the WAS from the HM period to
273 the TM period was greater by orders of magnitude, with 1.42 ×106 copies/ng and 1.73 ×105
274 copies/ng for Bacteria and 4.17 ×104 copies/ng and 3.63 ×103 copies/ng for Archaea,
275 respectively. This can likely be attributed to seasonal changes in the wastewater treatment
276 plant. For MAD system, whereas, no obvious change in the 16S rRNA gene copies of
277 Bacteria (p= 0.969) or Archaea (p= 0.755) was observed between the TM period and the
278 HM period. Little variance was found in the archaeal and bacterial populations in the
279 MAD system throughout the experiment, as is indicated by the coincidence of the points
281 In TM-TPAD, no significant difference was found between TM-1 and TM-2 with regard to
282 the copies of Bacteria (p= 0.522) and Archaea (p= 0.168), indicating that the bacterial and
283 archaeal populations in these two digesters were similar. It was also found that both
284 reactors were characterized by more Archaea copies than MAD55 systems. In HM-1, the
285 relatively low populations of Bacteria and Archaea might result from the nature of the
286 hyperthermophilic condition. On the other hand, more Archaea were found in HM-2 than
287 in TM-2, which is in agreement with the former conclusion that HM-1 enhanced
290 Coprothermobacter proteolyticus was frequently reported and considered playing key role
291 in the hydrolysis of WAS (Gagliano et al., 2015; Kobayashi et al., 2009).
292 Coprothermobacter spp. were reported to be capable of utilizing protein and producing H2,
293 acetate and small fractions of other VFA. Their syntrophic relation with hydrogenotrophic
294 organisms greatly activates the proteolytic process (Gagliano et al., 2015; Sasaki et al.,
295 2011). The growth of Coprothermobacter spp. reached the maximum at the
296 hyperthermophilic condition of 70 ºC (Kersters et al., 1994), which was the utilized in this
299 A small share of the Bacteria in WAS were occupied by the members of
300 Coprothermobacter, with 0.001% and 0.002% in the TM and HM periods, respectively;
301 their proportions grew to 0.04% and 0.01% in the MAD system. As illustrated in Figure 4
302 (b), their percentages increased to higher levels in both TM-TPAD and HM-TPAD,
305 Coprothermobacter in Bacteria in the first stages, TM-1 and HM-1 were close to each
306 other whereas in the second stages, TM-2 and HM-2 were different. The ratio of
307 Coprothermobacter to Bacteria for TM-2 reached 38.4%, which was 11.6% higher than
308 TM-1. In the case of HM-2, the ratio declined to 5.18% instead. It is implied that after
309 hyperthermophilic fermentation, the lower availability of WAS restricted the members of
313 Table 3
314 The bacterial compositions analyzed with the 16S rRNA gene clone library are shown in
315 Table 3. According to Shannon’s diversity index and Pielou’s evenness index, the ranking
316 of bacterial diversity was substrates (WAS55, WAS70) > mesophilic conditions (MAD >
317 HM-2 > TM-2) > hyperthermophilic condition (HM-1) > thermophilic condition (TM-1).
318 The phyla Proteobacteria, Firmicutes and Bacteroidetes were the major bacterial group in
319 the WAS digesters. At the class level, the bacteria in WAS55 and WAS70 were ranked as
321 (Firmicutes). The MAD system inherited this ranking of bacterial community in class level.
322 In TPAD systems, bacteria affiliated with the class Betaproteobacteria remained at similar
323 share in TM-1 and HM-1 at 24.6% and 26.5%, but decreased differently to 16.7% in TM-2
324 and 1.7% in HM-2. With the elevated temperatures in TM- and HM-TPAD, the class
325 Clostridia outcompeted Betaproteobacteria and became the most abundant group in the
326 digester. Their prevalence is considered responsible for the higher degrading ability of the
327 TPAD.
329 enzymes, was most frequently detected in this study. Their abundances in the results of
330 bacterial communities were in agreement with the results of qPCR stated above. The
331 results also explicated that in WAS55, WAS70, MAD and HM-1, the members related to
332 phosphorus and nitrogen removal were found such as Candidatus Accumulibacter
333 phosphatis, Simplicispira limi and Acidovorax ebreus (Byrne-Bailey et al., 2010; Lu et al.,
334 2007; Tong & Chen, 2007). They are considered to originate from the A2O process, where
335 the WAS was taken. Bacteria related to sulfur or iron metabolism, such as Sideroxydans
336 lithotrophicus, the member of Thioflavicoccus and Rhodoferax ferrireducens, were also
337 widely detected in these samples (Finneran et al., 2003; Imhoff & Pfennig, 2001; Liu et al.,
338 2012). The enrichment of these bacteria was probably due to the addition of iron sulfate as
339 coagulation reagents before the dewatering of WAS. Still, apart from Coprothermobacter
340 proteolyticus, few clones in the bacterial library were related to the hydrolysis of proteins.
341 The results for these bacterial communities suggest that Coprothermobacter proteolyticus
342 was the major protein hydrolyzer and that their dominance was more marked in the TPAD
343 processes.
345 Table 4
346 Archaeal communities, which mainly referred to the methanogens, are shown in Table 4.
347 According to Shannon’s diversity index and Pielou’s evenness index, the archaeal
348 diversities were substrates (WAS55, WAS70) > mesophilic conditions (TM-2 > MAD ≈
349 HM-2) > thermophilic condition (TM-1) > hyperthermophilic condition (HM-1). The
350 general finding was that as the operating temperature of the digester increased, less
352 While the WAS introduced a diversity of methanogens to the digesters, only a few genera
353 thrived and became dominant species. It is well known that aceticlastic methanogens are
354 principal in the archaeal structures since the majority of methane production is harvested
355 via aceticlastic pathway. The results elucidated that the dominant aceticlastic methanogens
356 were Methanosaeta concilii in MAD and Methanosarcina barkeri in TM-1, the latter of
357 which was also the dominant aceticlastic methanogen in TM-2, the following digester in
358 the TM-TPAD system. In HM-1, aceticlastic methanogens lost their dominance to the
360 maladaptation to the hyperthermophilic condition (Demirel & Scherer, 2008). In HM-2,
361 however, Methanosaeta concilii regained dominance in the mesophilic condition, i.e. the
362 influence from the first stage to the second stage for HM-TPAD system was less than for
365 decreasing the hydrogen partial pressure and therefore establishing thermodynamically
366 favorable conditions for VFA oxidization. In the MAD system, the dominant
368 Methanoculleus thermophilus was the dominant methanogen. In the downstream TM-2,
369 the hydrogenotrophilic group remained dominanted by Methanolinea tarda but was
370 characterized by the increased presence of other species of methanogens. In HM-1, of the
373 and accounted for 1 copy out of 64 copies in the 16S rRNA gene clone library of archaea.
374 3.5 The correlations of microbial communities in the digesters with one another
375 Figure 5
376 Hierarchical clustering on bacterial and archaeal OTU describes the similarities of
377 microflora between digesters with more accurate numbers. In the hierarchical clusters
378 shown in Figure 5 (a), the bacterial communities in TM-1 and TM-2 shared 91.8%
379 similarity while those of HM-1 and HM-2 only shared 60.0%. Interestingly, although
380 altering the temperature of TM-1 into HM-1 did not cause any great shift in bacteria, the
381 second stage began to degenerate towards MAD. Archaeal clustering showed the closeness
382 between HM-2 and MAD with only 1.7% of difference. The archaeal dissimilarity
383 between TM-1 and TM-2 was 16.8%, while between HM-1 and HM-2, it was 49.7%. The
384 archaeal difference between HM-1 and HM-2 presented the largest among the digesters.
385 A principal component analysis (PCA) was conducted based on the clone numbers (since
386 their total clone numbers were almost the same) in the bacterial and archaeal 16S rRNA
387 gene clone library as classified in the head column in Table 3 and Table 4. (i.e. at a class
388 level for Proteobacteria, Firmicutes and Bacteroidetes, at a phylum level for the other
389 bacteria, and at a genus level for archaea). The results are presented in Figure 6.
390 Figure 6
391 In Figure 6 (a), the principal component 1 (PC1) and PC2 were extracted accounting for
392 90.7% of the cumulative contributions. Their negative relationships with Clostridia and
393 Betaproteobacteria were shown respectively. This was propably due to the unfavorable
394 factors represented by these two classes of bacteria. The locations of points on the score
395 map in Figure 6 (b) illustrate the observations of the bacterial communities. Notably,
396 MAD was closer to WAS55 and WAS70 in PC1 due to its low abundance of Clostridia.
397 The loading map in Figure 6 (c) for methanogens indicates two evenly matched principal
398 components. According to their correlations with the main methanogens, PC1 was likely to
399 represent the optimal temperatures for methanogens if WAS55, TM-2 and WAS70 are
401 to the positive axis of PC2 while coccoid genera are projected to the negative axis. It
402 should also be noted that an 11% variances were exhibited by PC3. With loadings majorly
403 from Methanoculleus, PC3 is considered to indicate and modify the distributions of the
405 In Figure 6 (d), WAS55 and WAS70 had neutral spots in PC1 and shifted a little in PC2,
406 which might be explained by the high evenness and fluctuations of the WAS. The
407 thermophilic TM-1 lay in the fourth quadrant, at the opposite of MAD. While the
408 downstream TM-2 seemed to be moving towards MAD, it remained closer to TM-1.
409 Comparatively, the hyperthermophilic HM-1 went far in the first quadrant from the origin
410 but HM-2 almost coincided with MAD on this map and also in PC3. This suggests that the
411 archaeal changes to the mesophilic digester were bigger from the thermophilic effluent
412 rather than the hyperthermophilic. The structure of methanogens in TM-2 was diversified
415 structures. Because the second stage was inoculated with the same seed sludge as MAD,
416 its archaeal communities were expected to be similar to those of MAD during the TM
417 period. In fact, however, TM-2 tenured a large quantity of thermophilic methanogens from
419 methanogenesis. Similarly, an earlier study discovered that putting the sludge from the
420 second stage of TM-TPAD in the thermophilic condition without adaption resulted in an
421 activity level as high as that in the sludge from the first stage (Vandenburgh & Ellis, 2002).
422 In the following HM period, it was assumed that there should have been two possible
424 continued to thrive in HM-2 thus HM-2 was similar to HM-1; and 2)
425 Methanothermobacter could not thrive, and therefore HM-2 was similar to TM-2. In this
426 study, the results shown in Figure 6 (d), however, indicated that the methanogenic cultures
427 in HM-2 “regressed” to be similar with MAD. These results might be explained that the
428 mesophilic methanogenic community was established in the form of MAD, unless a mass
429 of active thermophilic methanogens were continually introduced. Yet, the influenced
430 mesophilic structure would return to the spontaneous form of MAD as long as the
431 upstream digester was not introducing the “invading” thermophilic methanogens.
433 Protein is the major component of the WAS. It is primarily hydrolyzed into amino acids
434 and, via either direct deamination or the Stickland reaction, further converted into VFA
435 before being passed down to methane. It is considered that the inspection into the VFA
436 compositions within the hyperthermophilic reactor offers a fine section view, thus enabling
439 (To editor: the footnotes of Table 6 were place here since they have the hyperlinks to
440 reference)
441 ΔrH° is the standard enthalpy change of the reaction, ΔrG’310.15K and ΔrG’343.15K are the
442 Gibbs free energy change at the respective temperature, K310.15K and K343.15K are the
443 equilibrium constants at the respective temperature.
444 a
They were calculated with data from the Design Institute for Physical Properties (DIPPR)
445 Project 801 (Linstrom & Mallard, 2001), which was presented indirectly by Vatani et al.
446 (2007), regardless of the differences of isomers in enthalpy changes during the dilution and
447 the ionization of carboxylic protons.
448 b
The data were obtained from Yu et al. (2004) at 37 °C.
449
450 As shown in Table 5, the VFA composition in HM-1 was HAc > HPr > i-HVa > i-HBu >
451 HBu > HVa. This order agrees with the findings presented in previous studies, one of
452 which involved the 70 °C fermentation of WAS (Bolzonella et al., 2007) and another
453 reported the chemical hydrolysis of WAS at room temperature (Chen et al., 2007). The
454 proportions of HAc, HPr and i-HVa, which were the major components, were also similar
455 to those reported in the studies; at around 50%, 20% and 15% of the total VFA. With
456 respect to isomers in the VFA, according to Table 6, the thermodynamically stable ratio of
457 the isomer to the normal forms of butyrate or valerate was approximately 2:1 at 70 °C.
458 However, the isomer ratios from this study, as well as the results by Bolzonella et al.
459 (2007), were not consistent with the theoretical values. This disparity has been reported to
460 result from the transformation of iso-butyrate to n-butyrate did not occur when
461 methanogenesis was inhibited (Angelidaki & Ahring, 1995). At 70 °C, the
462 hydrogenotrophic Methanothermobacter may serve as the hydrogen sink for the
463 β-oxidation of n-butyrate allowing the fast conversion of both butyrate and iso-butyrate
464 into acetate. In fact, however, an accumulation of both butyrate and iso-butyrate, albeit at
465 lower percentages, suggesting β-oxidation was weak at 70 °C. These observations suggest
466 that the majority of the profile of the accumulated VFA in the hyperthermophilic digester
469 Two-phase anaerobic digestion (2PAD), which was first developed in 1971, is known for
470 separating the acidogenic and methanogenic phase to optimize their respective
471 microorganisms. The separation of the two phases is the key to make a 2PAD system.
472 (Pohland & Ghosh, 1971) In the first stage of HM-TPAD system, both the acidification
473 and the richness of the major protein hydrolyzer Coprothermobacter were observed. The
474 abundance of Archaea was low and the methanogenic structure mainly consisted of the
476 was the highest among all the digesters, with the methanogenic structure close to the
477 mesophilic single-stage digestion, while the population of Coprothermobacter fell along
478 with the decrease in hydrolysis efficiency. This is well-expressed in Figure 2. These
479 phenomena in HM-TPAD were consistent with the characteristics of 2PAD and were the
480 most essential differences from the conventional TM-TPAD. However, 5% of total COD
481 were produced as methane in the first (acidogenic) stage, indicating that the phase
482 separation was incomplete and can only be regarded as partial (Beccari et al., 1998) .
483 According to current knowledge, due to the high nitrogen content and the diversity of
484 methanogens in WAS, acidification requires extra manipulation, such as the employment
485 of physical and chemical pretreatment. In the previous 2PAD of WAS, the status of phase
486 separation was maintained by means of lowering the pH, dosing methanogenic inhibitors
487 or washing out the methanogens in the first stage to make an acidogenic reactor
488 (Kobayashi et al., 2012). The washing out of methanogens requires a HRT generally
489 shorter than 3 days (Metcalf et al., 2013), whereas a longer retention time is favorable for
490 the hydrolysis of WAS. In this study, by utilizing hyperthermophilic condition, the pH in
491 the digester was maintained at approximately 6.5 without shortening the retention time.
492 The elimination of the requirement for pH adjustment represents a significant cost saving
493 measure, by rendering the use of chemical reagents unnecessary. As such, it can be
496 4. Conclusions
497 It was observed that the temperature of the first stage varied the functioning pattern of the
498 TPAD system. In TM-TPAD, both stages were methanogenic with similar microbial
499 structures. Via hyperthermophilic condition, HM-TPAD achieved acidification in the first
500 stage. The abundance of Coprothermobacter in the first stage was higher than those in the
501 second stage. Archaeal population and the abundance of aceticlastic methanogen in the
502 methanogenesis were the highest. Thus, HM-TPAD basically separated the acidogenic and
505 Acknowledgement
506 This study was accomplished during the period when the first author was financially
509 E-supplementary data for this work can be found in e-version of this paper online.
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666
667 Table 1 Characteristics of waste activated sludge in this study.
668
Unit TM period HM period
pH - 5.82±0.05 5.80±0.04
Alkalinity g-CaCO3/L 0.95±0.06 2.07±0.20
Ammonia g-N/L 0.54±0.03 0.43±0.13
Total VFA mg-HAc/L 0.83±0.32 1.89±0.51
TS % 4.61±0.07 4.66±0.13
VS % 3.61±0.06 3.46±0.20
SS % 4.03±0.08 4.04±0.07
VSS % 3.37±0.06 3.25±0.06
Total COD g/L 57.1±1.7 52.3±0.8
Soluble COD g/L 7.48±2.86 5.37±0.95
Total proteins g/L 20.37±1.5 17.08±0.92
Soluble proteins g/L 1.74±0.50 1.20±0.45
Table 2 Performance of biogas production and organic removals on the steady states. In order to compare the removal abilities between the two stages of
TPAD, their contributions to the total removal through the process was converted to percentages and showed in brackets
TM period HM period
TM-TPAD HM-TPAD
Unit MAD MAD
TM-1 TM-2 Total HM-1 HM-2 Total
Biogas components CH4 % 63.0 60.6 66.6 62.3 61.0 39.4 67.6 63.3
CO2 % 35.9 38.8 32.6 37.1 38.0 58.3 31.9 36.0
Biogas productions rate L/L/d 0.39 2.15 0.21 0.60 0.42 0.39 0.40 0.40
Biogas recovery efficiency L/L-WASfed 11.8 12.9 5.02 17.9 12.7 2.35 9.60 12.0
L/g-VSfed 0.33 0.36 0.14 0.50 0.35 0.07 0.27 0.33
L/g-VSremoved 0.79 1.00 1.05 1.01 0.95 0.24 1.09 0.64
L/g-CODfed 0.21 0.23 0.09 0.31 0.22 0.04 0.17 0.21
Removals TS % 34.5 (68) (32) 39.5 31.5 (52) (48) 43.3
VS % 41.0 (73) (27) 49.0 37.3 (53) (47) 51.8
COD % 44.7 (70) (30) 52.7 39.5 (9) (91) 53.6
Total proteins % 45.9 (88) (12) 58.4 44.9 (64) (36) 65.4
SS % 39.0 (79) (21) 49.4 33.8 (70) (30) 49.6
VSS % 44.8 (83) (17) 57.6 41.7 (70) (30) 58.6
Particle COD % 46.0 (93) (7) 58.7 42.5 (61) (39) 59.1
Particle proteins % 47.9 (97) (3) 63.7 37.4 (88) (12) 62.1
1 Table 3 Bacterial communities and their diversity indices at the phylum (class) level
2
TM period HM period
WAS55 MAD TM-1 TM-2 WAS70 HM-1 HM-2
Number of copies 128 128 128 127 126 127 127
Number of OTUs 93 78 58 70 85 67 71
Shannon's diversity index 4.52 4.12 3.52 3.68 4.28 3.60 3.93
Pielou's evenness index 1.00 0.95 0.87 0.87 0.96 0.86 0.92
Proteobacteria
├ Betaproteobacteria 21.0 22.0 24.6 16.9 31.0 26.5 1.7
├ Alphaproteobacteria 8.1 8.1 7.6 10.2 5.6 5.1 1.7
├ Gammaproteobacteria 5.6 8.1 1.7 2.5 7.9 11.1 2.5
├ Deltaproteobacteria 0.8 5.7 0.8 5.1 1.6 0.9 10.1
└ Epsilonproteobacteria 0.8 - - - - - -
Firmicutes
├ Clostridia 8.1 8.9 49.2 40.7 11.1 33.3 35.3
├ Negativicutes 0.8 1.6 - 0.8 - 0.9 5.0
├ Bacilli - 0.8 - 2.5 - 0.9 0.8
└ Erysipelotrichia - - - - - - 1.7
Bacteroidetes
├ Sphingobacteriia 16.9 13.0 1.7 4.2 13.5 6.8 10.1
├ Flavobacteriia 8.1 3.3 1.7 - 2.4 - 4.2
├ Bacteroidetes inc sed - 0.8 - 4.2 - 1.7 10.1
├ Bacteroidia 3.2 2.4 0.8 0.8 2.4 3.4 3.4
└ Cytophagia 2.4 0.8 0.8 0.8 0.8 0.9 0.8
Actinobacteria 5.6 5.7 6.8 6.8 0.8 0.9 3.4
Chloroflexi 2.4 8.9 0.8 1.7 3.2 0.9 1.7
Verrucomicrobia 8.1 - - - 4.8 - 1.7
Ignavibacteriae 4.0 2.4 0.8 - 2.4 - -
Planctomycetes 1.6 0.8 - 0.8 3.2 2.6 -
Acidobacteria 1.6 0.8 - - 3.2 2.6 -
Nitrospirae - 0.8 - - 4.8 1.7 -
Armatimonadetes - - - - - - 4.2
Candidatus Saccharibacteria - - 1.7 - 1.6 - -
Synergistetes - 1.6 0.8 - - - -
Atribacteria - 0.8 - 0.8 - - -
WPS-2 - 0.8 - - - - 0.8
Hydrogenedentes 0.8 - - - - - 0.8
33
Cloacimonetes - 1.6 - - - - -
Spirochaetes - - - 0.8 - - -
3
34
4 Table 4 Archaeal communities and their diversity indices at the genus level
5
TM period HM period
WAS55 MAD TM-1 TM-2 WAS70 HM-1 HM-2
Number of copies 64 64 64 64 64 64 64
Number of OTUs 18 5 4 10 20 6 7
Shannon's diversity index 2.08 1.02 0.83 1.57 2.46 0.66 1.15
Pielou's evenness index 0.72 0.63 0.60 0.68 0.82 0.37 0.59
Methanosarcina 47.5 - 44.4 54.1 16.7 4.8 -
Methanosaeta 10.2 52.4 1.6 14.8 16.7 6.3 58.3
Methanolinea - 42.9 - 16.4 - - 36.7
Methanothermobacter - - - 3.3 - 87.3 3.3
Methanoculleus - - 54.0 9.8 - - -
Methanobrevibacter 6.8 - - 1.6 26.7 - -
Methanomethylovorans 5.1 - - - 21.7 1.6 -
Methanocorpusculum 16.9 - - - 1.7 - -
Methanospirillum 1.7 - - - 8.3 - -
Methanosphaera 3.4 4.8 - - 1.7 - -
Methanobacterium 1.7 - - - 3.3 - 1.7
Methanosphaerula 3.4 - - - - - -
Methanolobus 1.7 - - - - - -
Methanoregula - - - - 1.7 - -
Thermogymnomonas 1.7 - - - - - -
Nitrososphaera - - - - 1.7 - -
6
7
35
8 Table 5 Comparison of VFA profiles with previous research
9
Unit This study (Bolzonella et al., 2007) (Chen et al., 2007)
Temperature °C 70 70 70 70 70 21 21 21
a a
pH - 6.43 6.8 6.8 6.8 6.7 6 7 10 a
HRT days 6 5 3 2 1 8b 8b 8b
total VFA g/L 6.32 6.31 5.91 4.69 4.89 0.54 0.84 2.71
HAc % 50.3 63.9 52.3 52.0 51.8 35.2 45.9 45.8
HPr % 19.8 15.7 16.8 19.7 21.6 27.9 23.2 16.5
i-HBu % 8.1 4.4 4.7 8.2 7.9 7.5 7.6 9.2
HBu % 4.6 4.4 4.7 5.9 5.6 11.7 5.5 11.0
i-HVa % 15.7 10.9 20.7 13.6 13.0 14.1 15.0 15.7
HVa % 1.5 0.8 0.8 0.5 0.1 3.6 2.9 1.9
Acidificationc % 18.3 21.2 19.9 15.8 16.5 5.0 7.7 25.1
10
36
11 Table 6 Thermodynamic data for the isomerization of butyrate and valerate
12
ΔrH° a ΔrG’310.15K b K310.15K ΔrG’343.15K K343.15K
Reaction
(kJ/mol) (kJ/mol) (kJ/mol)
butyrate → iso-butyrate +2.8 -1.98 2.155 -1.89 1.940
valerate → iso-valerate +4.2 -2.02 2.189 -1.79 1.872
13
14
15
16
17
37
18 Highlights
19
20 The mechanism of the TPAD system changed with the temperature combinations.
25
38