3.1. Sludge and Inoculum Characteristics
shows the properties of the three batches of combined sludge and BMP inoculum samples that were used in this study. The properties of the sludge sampled from the Guelph WWTP at different times varied in TCOD contents, sCOD/TCOD ratios, and VFA contents. The combined sludge was a mixture of equal volumes of the primary and secondary sludge. The TS and TCOD contents of the second batch of sludge were significantly higher than those of the first and third batches of sludge. The VS/TS ratio of the first, second, and third batches of combined sludge were 68%, 70%, and 69%, respectively, and their sCOD/TCOD ratios were 4.2%, 2.7%, and 4%, respectively. On the other hand, the characteristics of the BMP inoculum sludge were relatively consistent. Of the BMP inoculum used in the first, second, and third batches of BMP experiments, the ratios of VS/TS were 54.2%, 58.8%, and 58.3%, respectively, and the ratios of sCOD/TCOD were 3.1%, 3.0%, and 2.7%, respectively.
The effects of TH and BH treatments on sludge solubilization and biogas production were similar in experiments on each of the three batches of sludge. However, for simplicity, the results obtained for the second batch of sludge are discussed in detail in Section 3.2
, Section 3.3
, Section 3.4
, Section 3.5
and Section 3.7
, while a comparison of the results obtained from the three batches of sludge is summarized separately.
3.2. Effect of TH and BH Treatments on Sludge Solubilization
shows the percent changes in the concentrations of TS, VS, TSS, VSS, and TCOD caused by the TH and BH treatments on the combined sludge. The percent changes were determined by normalizing the concentration changes with the corresponding initial concentrations. The change in TS caused by the TH treatment was negligible. In contrast, the BH treatments caused a 4% to 6% reduction in TS, which was likely due to the BH-induced biogas release and the oven drying-induced VFA loss during the TS measurement. The GC analysis showed that the gases generated during the BH treatments consisted mainly of carbon dioxide (CO2
), whereas the methane (CH4
) content was negligible. This result is because no inoculum was added to the BH bottles, and only a short anaerobic reaction time was applied in BH treatments. The ratios of TCOD to TS maintained a very close range of 1.24 ± 0.007 for the raw as well as the TH and BH-treated sludge.
The VS reduction caused by the treatment of TH, BH42, BH42+55, BH55+42, and BH55 were 1.8%, 17.7%, 16.6%, 16.2%, and 13.1%, respectively (Figure 1
). Compared to the TH treatment, the BH treatment resulted in a considerable reduction in the VS content of sludge. The TS mass balance for the BH treatment can be formulated as in Equation (2):
is the total TS reduction, ∆VS
is the VS reduction, and ∆iS
is the inorganic solids change.
When the ∆TSloss
observed in the BH treatments was in the range of 4% to 6%, the BH treatments resulted in a significant conversion of the VS content to the inorganic solids (iS
). The gravimetric analysis confirmed that the iS
content of the sludge increased by 17% to 22% after the BH treatments (Figure 2
). The main fermentation products of carbohydrates and proteins included short-chain VFAs, NH4
O, and CO2
. The CO2
generated from the fermentation reaction would mainly exist in the form of bicarbonate under the pH condition in this case. Therefore, both of the released NH4
from the sludge hydrolysis could contribute to the increase in the inorganic content of the BH-treated sludge. It is worth noting that the VS reduction caused by the TH treatment was close to the TH-induced TS reduction (Figure 1
and Figure 2
), implying that TH treatment at 165 °C might only degrade particles and macromolecules into smaller, soluble, organic molecules with only an insignificant production of CO2
Both TH and BH treatments resulted in a significant destruction of the suspended solids content (TSS) of the sludge. The TH, BH42, BH42+55, BH55+42, and BH55 treatments caused the TSS reductions of 16.9%, 12.0%, 17.4%, 16.3%, and 17.1%, respectively, and the VSS reductions of 22.6%, 17.5%, 24.6%, 23.1%, and 25.9%, respectively (Figure 1
). These results showed that the BH42+55, BH55+42, and BH55 treatments achieved comparable suspended-solid solubilization to the TH treatment. The relatively lower TSS and VSS reductions obtained with BH42 might suggest that the mesophilic BH treatments could be less effective for the solubilization of sludge suspended solids. The reduction of TSS is the sum of the reductions in VSS and inorganic suspended solids. The solids measurement showed that more than 98% of the TSS reduction is caused by the VSS reduction in both TH and BH treatment. This is understandable, because the TH and BH treatments could exert only a negligible impact on the solubilization of inorganic solids.
With the degree of sludge solubilization defined in this study as the ratio of ∆sCOD/(TCOD − sCODinitial
) to reflect the change in sCOD caused solely by hydrolysis, the TH treatment showed the highest degree of sludge solubilization (17%) followed by BH55 (14%), BH55+42 (13%), BH42+55 (13%), and BH42 (10%). In a study carried out to enhance anaerobic digestion by Yang et al. [37
] for excess sludge hydrolysis using enzyme addition, similar sludge solubilization results were achieved.
3.3. Effect of TH and BH Treatment on sCOD, VFA, and Alkalinity
shows the effects of the TH and BH treatments on the change in the sCOD, VFA, and alkalinity contents of the hydrolyzed sludge. The concentrations of sCOD, VFA, and alkalinity of the BH and TH-treated sludge were considerably higher than those of the untreated sludge. The highest sCOD increase of 377.5% was achieved with the TH treatment, followed by a 323.8% increase with BH55, 301.3% with BH42+55, 286.9% with BH55+42, and 221.7% with BH42 (Figure 3
). The ratios of sCOD increase (∆sCOD) to VSS reduction (∆VSS) were in the range of 1.00 ± 0.013 for all of the BH treatments, showing consistent conversion rates of sCOD to VSS for these different treatment conditions. For the TH treatment, the ∆sCOD/∆VSS ratio was 1.34 ± 0.024 (Figure 4
), which was higher than those observed with the BH treatment, suggesting that the compositions of sCOD of the BH and TH-treated sludge were different.
Despite the considerable increase in sCOD in the TH-treated sludge, the VFA increase for the TH-treated sludge was only 40%, which was significantly lower than the BH-treated sludge. As shown in Figure 4
, the ratios of ∆VFA (mg/L as acetic acid) to ∆sCOD (mg/L) were determined to be 39%, 40%, 37%, 40%, and 6% for the BH42, BH42+55, BH55+42, BH55, and TH treatments, respectively.
GC analysis showed that the main VFA species formed at the end of the three-day BH treatment were acetic, propionic, isobutyric, butyric and isovaleric acids. However, as shown in Table 2
, the main VFA compositions of the untreated, TH-treated, and BH-treated sludge were different. Both the TH-treated and untreated sludge contained mainly acetic and propionic acids, although the concentrations of these acids were much higher in the TH-treated sludge than in the untreated sludge. It seems that the TH treatment at 165 °C did not cause significant changes in the VFA species, but rather only increased the concentrations of acetic and propionic acids. However, BH treatment increased the total VFA concentrations in the sludge, and produced various VFAs that were not detected in the untreated and TH-treated sludge. Acetic, propionic, isobutyric, butyric, and isovaleric acids were detected in the BH-treated sludge, with acetic and propionic acids as the major VFA products. In descending order, the VFA production capacity of the BH treatment methods were BH42, BH55+42, BH42+55, and BH55, showing a positive impact of hydrolysis temperature on VFA production. Compared to the VFA concentrations in the sludge treated by BH42+55 and BH55+42, it seems that a later stage thermophilic hydrolysis would be beneficial to VFA production. Overall, the BH treatment resulted in much higher VFA productions than the TH treatment.
The BH treatment also resulted in a significant increase in sludge alkalinity (Figure 3
). Comparing the alkalinity (ALK) changes caused by the different BH methods, the sludge treated by BH55+42 showed the highest alkalinity increase, although BH42+55 and BH55 produced slightly higher VFAs. The main products of sludge hydrolysis could include VFAs, CO2
, soluble microbial products (SMP), and NH3
released from the hydrolysis of proteins. The production of CO2
and VFAs will decrease the pH, while the production of NH3
will increase the pH. In this study, the initial pH of the second batch of combined sludge was 6.79, while the pH values after the TH, BH42, BH55+42, BH42+55, and BH55 treatments were 6.58, 6.78, 6.75, 6.78, and 6.45, respectively. The insignificant change in pH suggests that the pH reduction caused by CO2
and VFA productions was well balanced by NH3
release from protein hydrolysis. The main alkalinity contributors in the hydrolyzed sludge could include carbonate, VFA, and SMP. Since the acid dissociation constant (pK
a) values of VFAs are between pH 4.7–4.8, VFAs in the hydrolyzed sludge will exist in deprotonated forms, and can thus contribute to alkalinity. SMP can also contribute to alkalinity, because SMP polysaccharides, proteins, and humic acids have negatively charged proton-binding sites [38
]. However, among these alkalinity contributors, carbonate was the main species to buffer the sludge pH change in the pH range relevant to the sludge hydrolysis by TH and BH treatments.
3.4. Effects of TH and BH Treatments on Methane Production
The effects of the TH and BH treatments on methane production were assessed by conducting 30-day BMP tests at 35 °C. Figure 5
a,b show the COD and VS-based methane yields over the operational period of 30 days. In the first two days, the methane productions by the TH-treated and untreated sludge (control) were higher than those from the BH-treated sludge, which was likely due to inhibition from the high VFA concentrations in the BH-treated sludge to the activities of methanogens. However, the accumulated methane production from the BH-treated sludge samples started to exceed that of the untreated sludge after the third day. The TH-treated sludge reached its highest methane yield at the fifth and sixth days of the BMP tests. Eventually, sludge treated by TH and BH55+42 attained similar methane yields. Overall, for all the sludge samples that were tested, 72–83% of the total methane gas produced in the 30-day BMP tests was generated in the first five days of the BMP tests.
The BMP tests showed that BH temperature could significantly affect methane production enhancement. As shown in Figure 5
a,b, sludge treated by BH55+42 produced highest methane of out all the BH conditions. Methane production with the BH55+42 treatment was also slightly higher than that with the TH treatment after 10 days of BMP tests. At the 15th day of the BMP test, the standardized (standard temperature and pressure, STP) methane yields of sludge treated by BH55+42 and TH were 23% and 20% higher than the control, respectively, while the other BH treatment conditions led to enhancement between 12–15%.
The 30-day methane yields at STP for the untreated, TH, BH42, BH42+55, BH55+42, and BH55 sludge samples were 223.80 NmLCH4/g COD, 259.45 NmLCH4/g COD, 239.54 NmLCH4/g COD, 240.6 NmLCH4/g COD, 266.4 NmLCH4/g COD, and 259.71 NmLCH4/g COD fed, respectively and 404.9 NmLCH4/g VS, 469.4 NmLCH4/g VS, 433.4 NmLCH4/g VS, 435.4 NmLCH4/g VS, 482.0 NmLCH4/g VS, and 469.9 NmLCH4/g VS fed, respectively.
This study also assessed sludge biodegradability, which is defined by the ratio of the actual CH4 produced to the theoretical CH4 per gram COD. Based on the methane gas volume obtained during the first 15 days of the BMP tests, the biodegradability of the sludge treated by BH55+42 and TH was determined to be 73% and 71%, respectively, compared to 59% for the untreated sludge. Similarly, sludge biodegradability measurements obtained at the end of the 30-day BMP tests for the sludge treated for the BH55+42 and TH were 76% and 74%, respectively, but only 64% for the untreated sludge. Accordingly, sludge digestion coupled with BH55+42 or TH pretreatments can achieve a higher biodegradability in 15 days than sludge digestion without pretreatment in 30 days.
3.5. VSS Reduction by Anaerobic Digestion
presents the reduction of VSS concentrations in sludge at the 10th, 20th, and 30th days of the BMP tests. At the end of the BMP tests, total VSS reductions in the sludge-treated BH42, BH42+55, BH55+42, BH55, and TH, as well as that of the untreated sludge, were 45.8%, 46.3%, 47.7%, 43.9%, 43.1%, and 42.7%, respectively, (Figure 6
). Thus, the BH and TH pretreatment did not demonstrate an evident enhancement of the overall VSS reduction in this study. The change in the VSS content of BMP mixed liquor reflected the growth of anaerobic microbes in the anaerobic digestion system and the destruction rate of VSS contents of the feedstock. VSS destruction during the BMP stage might be offset by the growth of anaerobic microbes. Thus, the reduction of VSS in the anaerobic digestion stage can be affected by the feed sludge solid contents, sludge composition, solid loading rates, SRT, and microbial community structures of the anaerobic digestion systems. Thus, further studies are needed to determine the optimal process conditions of anaerobic digestion for VSS reduction.
3.6. Comparison of the Results with the Three Batches of Sludge for Selected TH/BH-AD Conditions
The first batch test on BH and TH treatments showed that BH55+42 can result in a higher methane production than BH42+55, and comparable methane production enhancement can be achieved by TH and BH treatment. In order to versify these results, two other batches of tests were conducted using sludge taken from the same sources at different times. Among the total three batches of tests, TH was included in the first two batches while the third batch did not include TH but a BH treatment at 75 °C (BH75). As shown in Table 1
, the raw sludge used in the second batch test had higher TS, TCOD, sCOD, VFA, and TSS concentrations than the first and third batches of sludge. Similar hydrolysis performance trends during the BH and TH treatment tests were observed in terms of VSS reduction and the production of sCOD and VFAs. The sludge solubilization for TH, BH42+55, BH55+42, and BH42 for the batch 1 and 2 treatments were 17%, 13%, 13%, and 10% for batch 1, and 19%, 15.8%, 15.4% and 12.7% for batch 2, respectively. For the third batch, the sludge solubilization was 11% for BH42, 13.6% for BH42+55, 14.6% for BH55+42, 15.3% for BH55, and 20.6% for BH75. These results demonstrate the consistent hydrolysis performance trend caused by the BH and TH treatment for sludge solubilization.
The BMP tests that were conducted using all three batches of sludge consistently showed that the sludge treated by BH55+42 had the highest methane production enhancement (Table 3
). Methane yields from the first, second, and third batches of the sludge treated by BH55+42 were 282 ± 9.5 NmL/g, 255 ± 3.4 NmL/g, and 267 ± 3 (with an overall average of 268) NmL/g COD fed at the 15th day of BMP incubation, which was 21%, 23%, and 19% higher, respectively, than those with the controls. Accordingly, methane yields from the two batches of TH-treated sludge (the third batch did not have a TH condition) were 266 ± 2.4 NmL/g and 249 ± 3.7 NmL/g COD fed, corresponding to 14% and 20% enhancement, respectively, compared to the controls. Similar tendencies were also observed at the 30th day of the BMP test. Overall, although methane yields with the first batch sludge were slightly higher than the other two batches, all three batches of sludge showed that the BH55+42 treatment had the highest methane production enhancement compared to other BH treatment conditions.
3.7. Microbial Community Structures of the Anaerobic Mixed Liquor
Selected samples were taken from the BMP bottles for sequencing by the Illumina MiSeq system to characterize the microbial community structures under different BH-AD conditions. Figure 7
a shows the dominant bacteria phyla identified in the control (untreated sludge), and sludge treated with BH42+55, and BH55+42. The bacterial phyla of Bacteroidetes
, and Verrucomicrobia
were identified as the dominant phyla in all of the tested sludge samples, but the distribution of these phyla varied with the BH methods. The phyla of Bacteroidetes
, and Firmicutes
showed the highest OTU percentages in the untreated sludge, followed by the sludge treated with BH55+42, and then BH42+55.
At the genus level, Sphingobacterium
, and Bellilinea
presented in all the tested samples with the OTU percentages higher than 3% (Figure 7
b). The Sphingobacterium
genus contained bacteria that are non-formative and non-proteolytic, but some Sphingobacterium
can hydrolyze carbohydrates [39
]. The genus of Flavobacterium
contains aerobic bacteria, but some species, (e.g., F. hydatis
and F. succinicans
) can grow anaerobically under certain conditions. Most of the Flavobacterium
species can degrade polysaccharides and proteins [40
Compared to other tested sludge, sludge treated with BH42+55 showed higher proportions of Pedobacter
, and Clostridium
bacteria are aerobic chemoorganotrophic with an oxidative type of metabolism, and the mechanism that it presented in the BMP sludge is not clear. The genus Sedimentibacter
consists of amino acid and pyruvate-utilizing anaerobic bacterium [41
], while Clostridium
contains many species that are acetogens, which can produce acetate from H2
and glucose-fermenting pathways [42
was only detected as a major genus (OUT > 3%) in the sludge treated with BH42+55 and the control.
were the two most dominant methanogenic genera in all of the tested sludge samples. The members of Methanosaeta
are acetoclastic methanogens that split acetate methane and CO2
], while Methanolinea
are hydrogenotrophic methanogens that utilize H2
and formate for growth and methane production [44
]. Acetate-utilizing methanogens normally dominate in mesophilic digestion systems. However, the presence of hydrogenotrophic methanogens in mesophilic anaerobic digestion systems is also critical for achieving high methane production, because they can help maintain a low system H2
partial pressure by serving as H2
scavengers. Methanogens can only use acetate, CO2
, and some one-carbon organics. The conversions of some fermentation productions (e.g., propionic and butyric acids) to methane are only thermodynamically favorable at low H2
partial pressure. Thus, the presence of H2
-consuming organisms in anaerobic digestion systems is important to achieve an effective conversion from organics to methane gas.
This study focused on the effects of BH and TH pretreatment on the sludge solubilization and methane production of the combined primary and secondary sludge. The pretreatment results showed that the TH treatment at 165 °C for 30 min degraded around 17% of particulate COD to sCOD, and the BH treatment at temperatures between 42–55 °C caused around 10% to 14% of particulate COD dissolution. VFAs were identified as an important end product of both the BH and TH treatment of the combined sludge. For the TH treatment, VFAs are mainly produced from the degradation of unsaturated lipids [7
], while for the BH treatment, VFAs are produced in the acidogenesis and acetogenesis of the sludge. The GC analysis showed that there was a significant amount of propionic acid presented in both TH and BH-treated sludge. The accumulation of propionic acid in anaerobic digestion could cause the inhibition of the activity of fermenting bacteria and methanogens, causing process instability. In this study, the ratios of propionic to acetic acids were determined to be 0.83 for the TH treatment, and between 0.7–0.73 for the BH treatments (Table 2
). These values are higher than those reported in activated sludge treated by BH at conditions similar to this study. Ding et al. [28
] and Chen and Chang [25
] showed that the ratios of acetic acid to propionic acid were 0.29 and 0.41, respectively, in activated sludge treated by BH at 42 °C. The high propionic acid concentration in the combined sludge could be caused by the high propionic acid concentration in primary sludge. In this study, the combined sludge initially contained 492.0 mg/L of propionic acid, compared to only 18.7 mg/L in activated sludge reported in [28
]. The relative high propionic concentrations in the BH-treated sludge also indicates the limited degradation of propionic acid during the three-day BH period. The biological oxidation of propionic acid to acetic acid has a standard free energy change of +76 kJ/mol, and is only possible at a hydrogen partial pressure lower than 10−4
]. Since H2
is one of the main end products of fermentation, the degradation of propionic acid can only be achieved by an effective syntrophic association between propionic-oxidizing bacteria and hydrogen-consuming methanogens. In the BH treatment, the short retention time could have limited the population of slow growing H2
-consuming methanogens, which could cause a low degradation of propionic acid in the BH-treated sludge. However, there was no accumulation of propionic acid observed in the subsequent BMP test, indicating that there was a balanced population of syntrophic propionate-oxidizing bacteria and hydrogen-consuming methanogens. The Illumina sequencing also confirmed the co-existence of the syntrophic bacteria group (Figure 7
b) and hydrogenotrophic methanogens in the BMP mixed liquid.
With a direct comparison between TH and BH, this study showed that the BH treatment at temperatures between 42–55 °C for three days and the TH treatment at 165 °C for 30 min can achieve comparable biogas production enhancements that range from 10% to 23%, which are comparable to the results reported by other research studies. Ferrera et al. [46
] reported that BH treatment at 70 °C for nine hours could enhance biogas production by 30% in a 20-day thermophilic AD treatment of the thickened combined sludge. Bolzonella et al. [47
] reported that the BH pretreatment at 65 °C for two days in the thermophilic AD (55 °C) of activated sludge enhanced methane production by 8.9%, while Ge et al. [29
] showed a 25% enhancement of methane production by BH in the mesophilic digestion (35 °C) of primary sludge. For the TH pretreatment, Valo et al. [20
] reported that the TH treatment at temperatures between 130–170 °C for 30 min could enhance methane production by 21% to 45% in the 20-day mesophilic AD (35 °C) of activated sludge, while Wilson et al. [17
] reported a biogas enhancement up to 24% to 59% by the TH pretreatment at 150 °C and 170 °C, respectively, in the anaerobic digestion of mixed sludge at temperatures between 35–42 °C. While these studies showed that the TH treatment can achieve more than 50% enhancement in methane production, Ding et al. [28
] reported around 10% enhancement on methane production by both TH treatment at 165 °C for 30 min and BH treatment between 42–55 °C. Thus, the enhancement of biogas production by the BH and TH treatments could vary significantly with the pretreatment conditions, sludge properties, mixture ratio of primary and secondary sludge, and AD process conditions.
This study also showed that BH55+42 could achieve better methane production enhancement than BH42+55. The apparent difference in biogas production enhancement achieved by BH55+42 and BH42+55 might suggest that the carryover of the microbial population from the BH stage to the AD stage might affect the performance of the whole BH-AD process. Chen and Chang [25
] showed that the protein-fermenting and carbohydrate-fermenting bacteria obtained in BH35, BH42, and BH55 were significantly different, and that the bacterial community structure that was developed at BH 42 °C had a higher similarity to that formed at BH 35 °C than the one developed at BH 55 °C. The BH55+42-AD treatment includes BH treatment at 55 °C for 1.5 days, followed by BH at 42 °C for 1.5 days, and then, the 30-day BMP test at 35 °C AD. With such a treatment sequence, an initial higher BH temperature could accelerate the dissolution of extracellular polymeric substances (EPS), and the disintegration of sludge floc aggregates. Meanwhile, BH at 42 °C could establish a microbial community that can be easily adapted to the subsequent 35 °C AD condition. Additionally, Nakasaki et al. [48
] found that the methane production rate of AD can be correlated with archaea cell density in the digester, whereas no clear relationship was found between bacterial population and methane production. The BH55+42 and BH42+55 conditions could result in different archaea to bacterial ratios in the subsequent AD processes, which could further affect the methane production. Although the Illumina sequencing method in this study identified the dominant bacterial and methanogen genera, the population densities of different organism groups are yet to be determined in the future study.
In this study, mass balance analyses were also conducted to determine the ratios of conversion of COD to methane. The average COD conversions to CH4
were determined to be 0.20 NLCH4
/g COD, 0.30 NLCH4
/g COD, 0.31 NLCH4
/g COD, and 0.32 NLCH4
/g COD removal for the control, BH42+55, BH55+42, and TH pretreatments, respectively. These values were lower than the ideal COD to methane conversion rate of 0.35 LCH4
/g COD, implying that around 8.6% to 15.0% COD might be consumed through other bacterial metabolism pathways rather than by methanogenesis. For example, it is well known that acetate can be completely oxidized to CO2
by sulfate reducers through the acetyl–CoA pathway or a modified acetyl–CoA pathway [49
]. In addition, some organisms, such as anaerobic methanotrophic archaea (ANME), were recently identified to be able to oxidize methane in coupling with a sulfate reducer through a two-step reaction: the formation of methyl sulfide from CH4
by archaea and the subsequent consumption of methyl sulfide by sulfate reducers [50
]. Thus, the microbiological mechanisms of the methane production enhancement by TH and BH treatments still needs to be explored in order to understand the relationship between methane production and microbial consortia in BH-AD systems.