- Email: [email protected]
Enrichment of acetogenic bacteria in high rate anaerobic reactors under mesophilic and thermophilic conditions
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
Enrichment of acetogenic bacteria in high rate anaerobic reactors under mesophilic and thermophilic conditions P. Ryan*, C. Forbes, S. McHugh, C. O’Reilly, G.T.A. Fleming, E. Colleran Environmental Microbiology Research Unit, Department of Microbiology, National University of Ireland, Galway, Ireland
article info
abstract
Article history:
The objective of the current study was to expand the knowledge of the role of acetogenic
Received 4 January 2010
Bacteria in high rate anaerobic digesters. To this end, acetogens were enriched by supplying
Received in revised form
a variety of acetogenic growth supportive substrates to two laboratory scale high rate
14 May 2010
upflow anaerobic sludge bed (UASB) reactors operated at 37 C (R1) and 55 C (R2). The
Accepted 25 May 2010
reactors were initially fed a glucose/acetate influent. Having achieved high operational
Available online 1 June 2010
performance and granular sludge development and activity, both reactors were changed to homoacetogenic bacterial substrates on day 373 of the trial. The reactors were initially fed
Keywords:
with sodium vanillate as a sole substrate. Although % COD removal indicated that the 55 C
Acetogens
reactor out performed the 37 C reactor, effluent acetate levels from R2 were generally
UASB
higher than from R1, reaching values as high as 5023 mg l1. Homoacetogenic activity in
Thermophilic
both reactors was confirmed on day 419 by specific acetogenic activity (SAA) measurement,
Anaerobic
with higher values obtained for R2 than R1. Sodium formate was introduced as sole substrate to both reactors on day 464. It was found that formate supported acetogenic activity at both temperatures. By the end of the trial, no specific methanogenic activity (SMA) was observed against acetate and propionate indicating that the methane produced was solely by hydrogenotrophic Archaea. Higher SMA and SAA values against H2/CO2 suggested development of a formate utilising acetogenic population growing in syntrophy with hydrogenotrophic methanogens. Throughout the formate trial, the mesophilic reactor performed better overall than the thermophilic reactor. ª 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Anaerobic digestion is a natural process which has been exploited for over a century for the effective treatment of organic wastes and is currently recognised as a major technology for remediation of municipal and industrial wastewaters. Anaerobic digestion has advantages over the more conventional aerobic process including less excess sludge production, lower space requirements as well as the recovery of
energy from the waste material in the form of a renewable energy, biogas (Lettinga, 1995). In recent years, the application of this technology to the treatment of a variety of domestic and industrial waste streams as well as for the remediation of various recalcitrant compounds such as chlorinated aliphatics and other xenobiotics, has increased at full scale as a result of a greater understanding of the treatment process and the development of high rate, granular sludge based reactor designs such as the UASB (Guiot et al., 1992; Field et al., 1995; Frankin,
* Corresponding author. Present address: Innovation Centre, Bord na Mo´na, Main Street, Newbridge, Co. Kildare, Ireland. Tel.: þ353 45439382; fax: þ353 45434207. E-mail address: [email protected] (P. Ryan). 0043-1354/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2010.05.033
4262
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
2001). The success of the UASB design for the treatment of high strength liquid wastes hinges upon the formation and maintenance of anaerobic sludge granules in which Bacteria and Archaea are juxtaposed, facilitating interspecies transfer of substrates necessary for the complete methanogenesis of the organic constituents of the wastewater (Grotenhuis et al., 1991; O’Flaherty, 1997; Stams and Plugge, 2009). A relatively understudied distinguished group involved in the complex anaerobic digestion process is the acetogenic Bacteria. When acetogens were first isolated they were thought of as being metabolically limited and thermodynamically disadvantaged, but in fact, it is now acknowledged that quite the opposite is true (Drake and Daniel, 2004). Under standard redox potential conditions, whereby acetogens and methanogens compete for substrates, the reductive synthesis of acetate from CO2 by acetogenesis is thermodynamically less favourable than methanogenesis, which is the often cited reason used to explain out-competition of acetogens by methanogens in anaerobic sludge. However, it is now clear that acetogens are capable of a highly diverse range of metabolic transformations (Xu et al., 2009). The three underlying metabolic features of these organisms are (i) the use of chemolithoautotrophic substrates (H2/CO2 or CO/CO2) as sole sources of carbon and energy, (ii) heterotrophic conversion of certain sugars stoichiometrically to acetate, and (iii) the ability to oxidise methoxylated aromatic compounds via the acetyl-CoA pathway. Many, but not all acetogens, display all three of these metabolic capabilities. Under certain conditions, autotrophic growth on H2/CO2 can be substituted for heterotrophic metabolism of a wide range of sugars, alcohols, methoxylated aromatic compounds and single carbon compounds, such as methanol and formate (Li et al., 1994). This diverse range of electron donors is oxidised by acetogens to shunt a reductant to the acetyl-CoA pathway, conferring these organisms with a competitive edge and ensuring their proliferation in anaerobic sludge. It is now clear that acetogens are arguably the most metabolically diverse group of obligate anaerobes characterized and that they present some interesting challenges, in particular with reference to their role within the anaerobic digestion process. H2-utilising acetogens are known to be present in mesophilic digester sludge with numbers in the range of 105e107 CFU/ml (Zhang and Noike, 1994) which estimates to approximately 3e11% of total Bacteria in anaerobic sludge (Wang et al., 2007). It has yet to be demonstrated whether these organisms play an autotrophic or heterotrophic role in anaerobic digestion within different temperature ranges, although it has been shown that in certain complex microbial habitats, the metabolic interactions of acetogens are influenced by environmental factors such as pH and temperature and are strain specific (Kotsyurbenko et al., 2001). The objectives of the current study focused on enrichment of acetogens in two UASB reactors, R1 and R2, operated at mesophilic (37 C) and thermophilic (55 C) conditions, which were provided with influents composed of substrates chosen to promote acetate production by acetogens. Three individual liquid influent streams were supplied separately to the reactors, each designed to promote either the heterotrophic or autotrophic metabolism of acetogens. These objectives aimed to facilitate an understanding of acetogenic behaviour and reactions in the anaerobic digestion process which could
potentially be applied to large scale facilities treating wastewaters of similar composition.
2.
Materials and methods
2.1.
Reactor design and operation
Two glass laboratory scale anaerobic upflow sludge bed (UASB) reactors with an active volume of 4.25 l were employed for this study. The reactors were seeded with anaerobic granular sludge originating from an internal circulation (IC) reactor treating wastewater from a commercial milk processing plant at 37 C (Carbery Milk Products, Ballineen, Co. Cork, Ireland). The conveniently available sludge had been stored, unfed, at 4 C for 14 months prior to this study. An initial in-reactor seed volatile suspended solid (VSS) concentration of 11.5 g l1 as recommended by de Zeeuw (1984) was applied. The remainder of the reactor active volume was filled with a mixture of sucrose/acetate at a ratio of 60:40, on a COD basis, to a total of 10 g COD l1. Both reactors were started up at 37 C and the sucrose/acetate influent was initially supplied at a volumetric loading rate (VLR) of 5 g COD l1 d1 at a hydraulic retention time (HRT) of 2 days. This HRT was maintained throughout the trial. The trial operational parameters and results from the mesophilic reactor (R1) and thermophilic reactor (R2) are summarised in Tables 1 and 2. Throughout the trial periods, the COD:N:P ratio in the influent was maintained at 1000:5:0.5 by supplementation with NH4Cl and KH2PO4 to the required concentrations. The pH of each influent mixture was buffered by the addition of NaHCO3 at a concentration of 8 g l1 and supplemented with macro and micro nutrients (1 ml l1), as recommended by Shelton and Tiedje (1984).
2.2.
Specific methanogenic activity (SMA)
The specific methanogenic activities of the seed sludge and reactor sludges sampled on days 62, 256, 419 and 588 were determined using the pressure transducer technique to indirectly measure gas production in hypovials containing 2e5 g VSS l1 of sludge biomass (Colleran et al., 1992; Coates et al., 1996). Briefly, the test procedure involved the measurement of the biogas pressure increase developing in sealed vials fed with the non-gaseous substrates, ethanol (30 mM), propionate (30 mM), butyrate (15 mM) and acetate (30 mM), or of the pressure decrease in vials pressurized with H2/CO2 (80:20) to 1 atm. On completion of the test, millivolt readings were converted to ml biogas, the methane content of the vial headspace analysed and the VSS of the test vial determined. Analysis of these data and the rate data (ml biogas produced/ utilised per hour) allowed expression of specific methanogenic activity as ml CH4 (STP) g1 VSS d1. Assays on the seed sludge and sludge from the R1 were performed at 37 C; assays on sludge from the R2 were performed at 55 C.
2.3.
Specific acetogenic (SAA) activity tests
A modified version of the SMA test was used to determine the specific acetogenic activity (SAA) of sludge samples. A specific
4263
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
Table 1 e Operational parameters and average performance data of the mesophilic reactor (R1). Values are the mean of triplicates ± standard deviation. Figures in brackets represent the maximum and minimum values in that period. Period Days Influent
1 1e128 sucrose/ acetate
2a 128e240 sucrose/ acetate
2b 240e373 sucrose/ acetate
3a 373e391 sodium vanillate
3b 391e464 sodium vanillate
4a 464e479 sodium formate
4b 479e496 sodium formate
4c 496e588 sodium formate
Influent Parameters Temperature C COD (g l1) HRT (days) VLR g COD l1 d1
37 10 2 5
37 10 2 5
37 20 2 10
37 1 2 0.5
37 5 2 2.5
37 1 2 0.5
37 2.5 2 1.25
37 5 2 2.5
Effluent parameters % COD removal pH CH4 (%)
93.8 6.8 7.7 0.5 70.9 5.4
97.1 7.1 7.6 0.2 65.0 6.1
93.3 8.0 7.7 0.4 72.4 7.9
63.5 9.2 8.1 0.9 72.1 7.0
30.5 10.5 8.6 0.8 80.8 8.2
71.8 12.6 9.0 0.7 87.8 8.3
79.4 9.9 9.1 0.6 93.1 8.8
83.8 11.3 9.4 0.8 98.8 8.7
VFA (mg l1) Acetate mg l1 (minemax) Propionate mg l1 (minemax) Butyrate mg l1 (minemax)
90.1 (2.7e598.3) 75.2 (2.9e397.5) 11.8 (1.3e80.6)
29.2 (4.0e263.8) 12.2 (1.3e62.8) 6.8 (1.3e14.3)
85.2 (2.8e192.5) 26.3 (1.4e92.7) 8.1 (1.2e56.1)
745.6 (151e2130) 267.2 (67.8e511.1) 40.5 (18.3e59.4)
1567.5 (526e3015) 464.9 (34e1055) 106.2 (11.2e421)
614.5 (339.1e896) 389.6 (254e652) 72.8 (22e145)
99.8 (9.4e179) 89.8 (18.1e203.3) 22.1 (1.5e57.3)
75.5 (7.7e475.6) 36.8 (5.1e122.5) 6.6 (1.3e20.1)
methanogenic inhibitor, 2-bromoethane sulfonate (BES), (Bouwer and McCarty, 1983; Chidthaisong and Conrad, 2000), was included at a concentration of 100 mM in the test vials. The vials were pressurised with H2/CO2 (80:20) to 1 atm. Pressure decrease in the vials was measured over time with the pressure transducer device. Parallel vials were set up under the same parameters and sacrificed for acetate analysis by gas chromatography. On completion of the test the methane content of the vial headspace was analysed and the VSS of the test vials was determined. Analysis of the data and the rate of acetate production allowed expression of acetogenic activity as ml acetate g1 VSS d1.
2.4.
Analytical methods
Reactor effluent/influent samples were regularly analysed for COD, pH and VFA content according to Standard Methods (APHA, 1998). Methane concentration in the biogas was analysed by gas chromatography (GC) using a glass column packed with Poropak Q 100e120 mesh in a Philips PYE-Unicam Series 304 chromatograph fitted with a gas sampling port and a flame ionisation detector. The column temperature was maintained at 35 C. The injection port and detector temperatures were 105 C and 100 C, respectively (McHugh et al., 2005). Volatile fatty acids (VFAs) and ethanol were quantified using a gas chromatograph (6890 Plus, Agilent, Palo Alto, CA) equipped with an Innowax capillary column (Agilent) and a flame ionization detector.
3.
Results
3.1.
Bioreactor performance
The start-up phase for both reactors in period 1, showed COD removal efficiencies reaching averages of 90% after 10 days of operation (Fig. 1) and the % methane in biogas averaging 70%
from both reactors after 14 days (Tables 1 and 2). Operation of R2 at 55 C commenced on day 128 (period 2) and caused an immediate reduction in COD removal efficiency and methane content of the biogas from this reactor (Table 2). The % methane recorded deteriorated to 12% by day 135 and a minimum COD removal of 26.3% was recorded on day 139. This period of R2 instability also caused a rise in VFA discharge with acetate levels reaching up to 1500 mg l1 (Fig. 2). The reactor recovered, achieving >90% COD removal by day 167 after just 39 days of operation at 55 C (Fig. 1) and VFA levels remained negligible for the remainder of period 2 (Fig. 2). On day 240, the VLR was increased to 10 g COD l1 d1 (Fig. 1). An initial negative effects on the performance of both reactors was, however, shortlived, with COD removal efficiencies returning to approximately 90% for R1 and R2 after 40 and 60 days respectively (Figs. 1 and 2). The sucrose/acetate influent was changed to a sodium vanillate feed on day 373 (period 3a, Tables 1 and 2) at an initial concentration of 1 g COD l1. Since the HRT remained unchanged at 2 days, this resulted in an initial VLR of 0.5 g COD l1 d1. Despite this low loading rate, COD removal efficiencies decreased sharply for both reactors (Fig. 1), with associated increases in effluent acetate levels (Fig. 2). When COD removal efficiency was shown to improve for both reactors, the sodium vanillate influent concentration was increased to 5 g COD l1 on day 391, resulting in an increased VLR of 2.5 g COD l1 d1. This had a very negative impact on the performance of both reactors. The % COD removal for the mesophilic R1 fluctuated considerably between days 391 and 463 (Period 3b, Table 1 and Fig. 1), with a low of 3.8 observes on day 399 and an average value of 30% throughout this period. Effluent acetate also fluctuated during this period, reaching a peak of 3015 mg l1 on day 401 and an average value of >1560 mg l1 throughout the 72 day test period (Fig. 2 and Table 1). Similarly, for thermophilic R2, this feed period was marked by considerable instability. The % COD removal decreased to 16.5% on day 401, averaging 53.2% throughout period 3b (Fig. 1).
4264
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
Table 2 e Operational parameters and average performance data of the thermophilic reactor (R2). Values are the mean of triplicates ± standard deviation. Figures in brackets represent the maximum and minimum values in that period. Period Days Influent
1 1e128 sucrose/ acetate
2a 128e240 sucrose/ acetate
2b 240e373 sucrose/ acetate
3a 373e391 sodium vanillate
3b 391e464 sodium vanillate
4a 464e479 sodium formate
4b 479e496 sodium formate
4c 496e588 sodium formate
Influent Parameters Temperature C COD (g l1) HRT (days) VLR g COD l1 d1
37 10 2 5
55 10 2 5
55 20 2 10
55 1 2 0.5
55 5 2 2.5
55 1 2 0.5
55 2.5 2 1.25
55 5 2 2.5
Effluent parameters % COD removal pH CH4 (%)
93.9 8.2 7.7 1.2 68.0 6.2
87.2 9.3 7.7 1.1 59.0 5.4
89.8 12.9 7.7 1.8 71.5 6.8
64.8 11.6 8.1 0.5 61.3 5.8
53.2 8.0 8.5 0.8 65.0 7.7
81.4 9.9 9.0 1.0 75.3 8.1
73.5 8.5 9.1 0.6 85.4 8.6
65.2 9.2 9.4 1.1 80.1 7.9
134.8 (4.1e986.3) 235.8 (6.0e1138.2) 26.3 (1.9e244.6)
238.7 (2.0e1593.6) 124.3 (1.0e885.2) 71.7 (0.8e478.0)
85.4 (3.1e56.6) 36.9 (1.3e31.1) 11.5 (1.2e19.7)
1157.7 (209.7e2663) 175.5 (43.9e722) 28.7 (20.3e88.9)
1241.6 (143e5023) 271.6 (104.3e1776) 51.3 (22.1e455.3)
85.3 (43.1e214.9) 73.9 (31.7e222.5) 7.5 (1.3e25.6)
177.09 (56.8e90.8) 66.8 (13e104.1) 44.6 (1.3e72)
934.6 (11.1e1886.3) 82.4 (6.2e225.6) 26.7 (1.2e119.2)
VFA (mg l1) Acetate mg l1 (minemax) Propionate mg l1 (minemax) Butyrate mg l1 (minemax)
By the end of the sodium vanillate trial, however, R2 was achieving >85% COD removal efficiency. Effluent acetate levels from R2 showed a peak of >5000 mg l1 on day 427 and averaged 1242 mg l1 during the 72 day trial period (Fig. 2 and Table 2). The % methane of the biogas from both R1 and R2 increased, however, during period 3b (Tables 1 and 2). On day 464, sodium formate was introduced as sole substrate to the reactors, at a COD concentration of 1 g COD l1 and a VLR of 0.5 g COD l1 d1 (Tables 1 and 2). Biomass adaptation to the new stream was apparent after one HRT (2 days) and the average % COD removal during period 4a increased to 71.8% in R1 and 81.4% in R2 (Tables 1 and 2). The % methane content of the biogas produced by both reactors continued to rise during this period while effluent VFAs decreased sharply from the previous period (Fig. 2). The VLR of both reactors was increased to 1.25 g COD l1 d1 on day 479
and then to 2.5 g COD l1 d1 on day 496. R1 adapted readily to the increased VLR with a COD removal efficiency of 92% and biogas methane content of 98.9% recorded on day 533. R2 performance, however, deteriorated in response to the increased VLR, with a reduction in average COD removal efficiency and a two fold increase in average acetate discharge values between periods 4a and 4b which sharply increased during period 4c (Fig. 2, Tables 1 and 2). However by the end of the formate trial, there was little difference between the operational performances of both reactors.
3.2.
Physiological assessment of biomass
The specific methanogenic activity (SMA) and specific acetogenic activity (SAA) values generated from testing of the seed sludge and sludges removed intermittently from the reactors
Fig. 1 e % soluble COD removal by R1 and R2 throughout the trial period ( mesophilic R1, ). Operational periods defined in Tables 1 and 2 are indicated by arrows. (
thermophilic R2); VLR
4265
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
Fig. 2 e Effluent acetate levels (mg lL1) throughout the trial period ( mesophilic R1, Operational periods defined in Tables 1 and 2 are indicated on the graph.
are presented in Tables 3 and 4. The SMA values generated from the seed sludge against acetate, ethanol, propionate, butyrate and H2/CO2 were 10.6, 25.3, 41.8, 21.3 and 100.6 ml CH4 (STP) g1 VSS d1 respectively (Table 3). These results reflect the fact that prior to seeding the reactors, the sludge had been stored long term at 4 C and so was relatively inactive. The SMA values recorded for sludge removed on day 62 during start-up of R1 and R2 at 37 C are illustrated in Table 3. All activities were all significantly higher compared to seed sludge profiles and indicate the activity of all trophic groups necessary for successful syntrophic and methanogenic populations. SAA results remained negligible at this point, indicating minimal activity of H2/CO2 utilising acetogens (Table 4). By day 256 (Period 2b), the operational temperature of R2 had been changed and R1 and R2 were performing well under a high VLR of 10 g COD l1 d1 (Tables 1 and 2; Fig. 1). This was reflected in the SMA profile of the sludges with notable increases in activity on all substrates in comparison to the seed sludge activity assay (Table 3). The decreased SMA in the R2 sludge on day 256 in comparison with sludge removed from this reactor on day 62 against acetate, propionate and butyrate is mostly likely due to the emergence of a thermophilic population in response to the increase in operational temperature. A marked increase in the capacity of the R2 sludge to degrade H2/CO2 was noted on day 256, however. Evidence that acetogenic populations were active in the thermophilic sludge was supported by the high SAA activity of 159.3 ml acetate g1 VSS d1 recorded on day 256, a nine fold increase from the previous sample on day 62 (Table 3). On day 419, the ability of the reactor sludges to degrade many of the substrates tested had deteriorated since day 256 which was expected given the decrease in reactor performances associated with the change in influent substrate to vanillate (Fig. 1; Table 3). Interestingly, the SMA values for H2/ CO2 conversion by R1 and R2 remained consistent, which may indicate that methane was produced from H2/CO2 during this period. It has been previously reported that hydrogenophilic methanogens are less susceptible to VFA induced inhibition
thermophilic R2).
than acetoclastic methanogens or acetogens (O’Flaherty et al., 1999). This theory is supported by the increased acetogenic activity recorded in this period from R1 and R2 biomass (Table 4) which was associated with elevated acetate discharge from the reactors (Fig. 2) and may have resulted in inhibition of the acetoclastic methanogens. The SAA values of the mesophilic and thermophilic takedown sludge on day 588 after 124 days of operation on sodium formate remained high at 349.3 and 426.8 ml acetate g1 VSS d1, respectively (Table 4). The SMA values on this day, however, indicated that there was reduced methanogenic activity upon all substrates tested, with the exception of H2/ CO2. This was similar to the results generated from previous tests where activity of the reactor sludges against acetate, propionate and butyrate had been gradually decreasing as the trial progressed until the test on day 588 when a total lack of activity against acetate and propionate by both R1 and R2 biomass was evident (Table 3).
4.
Discussion
The objective to enrichment acetogenic Bacteria and the effect of this on the anaerobic process at mesophilic and thermophilic temperatures was demonstrated in this paper. The development of stable well-functioning anaerobic communities was crucial during the start-up of this trial. Therefore, a sugar based influent which supports the growth and maintenance of the fermentative, acidogenic and acetogenic bacterial groups to produce acetate and H2/CO2, direct methanogenic precursors, was applied. This influent was composed of sucrose which supports the growth of a wide range of characterised acetogenic isolates converting it to acetate via the acetyl-CoA pathway (Drake et al., 2006). The addition of acetate in the start-up period was to encourage both sludge granulation (Hulshoff Pol et al., 2004) and maintenance of the activity of the acetoclastic methanogenic Archaea.
4266
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
Table 3 e Specific methanogenic activity (SMA) values of R1 and R2 biomass sampled throughout the trial. Values are the mean of triplicates ± standard deviation. Reactor
Day
Substrates (ml CH4 (STP) g1 VSS d1)
Temp C Acetate
Seed R1 R1 R1 R1 R2 R2 R2 R2
0 62 256 419 588 62 256 419 588
37 37 37 37 37 37 55 55 55
Ethanol
10.6 1.1 98.6 2.5 53.0 3.6 8.4 0.6 0 100.5 10.9 43.6 1.8 4.5 0.5 0
C C C C C C C C C
The sucrose influent was readily degraded by both R1 and R2 during period 1 (Tables 1 and 2). SMA values obtained from sludge sampled on day 62 during period 1 indicated the development of methanogenic consortia in R1 and R2. Operation of R2 at 55 C caused an immediate initial reduction in COD removal efficiency and % methane content of the biogas. However, stable reactor performance was resumed after 20 HRTs. This start-up transition of thermophilic operation from mesophilic sludge in a single step increment has previously been observed (Forbes et al., 2009). The VLR increase on day 240 after a lengthy period of stable performance in both R1 and R2 proved that the thermophilic reactor was slower to recover in comparison to the mesophilic reactor in terms of COD removal efficiency. R2 biomass exhibited a higher capacity for methanogenic activity on H2/CO2 by day 256 and an elevated acetogenic activity value (Table 3). This confirmed the cultivation of an active acetogenic consortium at 55 C achieving a major objective set at the beginning of the trial. The introduction of vanillate on day 391 had an immediate negative effect on the COD removal efficiency in both R1 and R2 (Fig. 1). Vanillate is a methoxylated aromatic compound and a natural product of plants, microorganisms and animals (Mechichi et al., 2005). The effect of direct application of a methoxylated aromatic compound on the anaerobic process in a bioreactor has not been previously researched. The ability to utilize methoxyl groups of aromatic compounds is a specific metabolic capability of some acetogens, such as Clostridium
Table 4 e Specific acetogenic activity (SAA) values of R1 and R2 biomass sampled throughout the trial. Values are the mean of triplicates ± standard deviation. Reactor
Day
Temp C
Seed R1 R1 R1 R1 R2 R2 R2 R2
0 62 256 419 588 62 256 419 588
37 37 37 37 37 37 55 55 55
C C C C C C C C C
H2/CO2 ml acetate g1 VSS d1 11.4 24.1 74.3 143.6 349.3 18.0 159.3 354.8 426.8
0.5 1.2 3.3 12.8 31.4 0.8 11.7 22.9 39.6
25.3 115.9 78.2 82.9 78.7 124.3 91.2 56.8 54.7
2.0 15.8 7.3 4.7 7.1 11.9 6.6 6.5 2.8
Propionate
Butyrate
41.8 3.3 76.7 7.1 51.6 4.8 4.4 0.5 0 74.1 6.7 24.3 3.5 8.4 0.9 0
21.3 55.9 78.1 9.1 3.6 59.2 26.8 7.3 12.7
1.2 3.7 4.8 1.1 0.8 2.8 3.0 0.5 0.09
H2/CO2 100.6 198.3 232.5 122.9 179.1 175.4 307.6 187.7 273.1
9.9 14.7 20.8 10.5 18.4 15.8 30.8 16.6 22.8
coccoides, Sporomusa silvacetica and Clostridium thermoaceticum (Bache and Pfennig, 1981; Daniel et al., 1991; Frazer et al., 1995). All these species possess the ability to O-demethylate methoxylated aromatic compounds like vanillate and metabolise the O-methyl group via the acetyl-CoA pathway. SAA results from day 419 indicated that the level of acetogenic activity in R2 which had been developed during the application of sucrose and acetate in period 2 continued to increase during the application of sodium vanillate in period 3 (Table 4). During this period, R2 out performed its mesophilic counterpart in terms of COD removal efficiency and levels of VFAs discharged in the reactor effluent (Figs. 1 and 2). The elevated levels of acetate discharged by both reactors during period 3 demonstrated the degradation of sodium vanillate to form acetate and thus confirmed the enrichment of acetogenic consortia in the reactors. Because of this, it is also assumed that excess acetate was not a result of propionate and butyrate oxidation to acetate as it is thermodynamically unfavorable for propionate and butyrate to be degraded until acetate and H2 are eliminated by the methanogens (van Lier et al., 1996). The SMA profile of both reactor sludges from day 419 on H2/CO2 was maintained during period 3 despite the reduction in reactor performances (Fig. 1; Table 3). van Lier et al. (1993) stipulated that the number of viable hydrogenotrophic methanogens is important for efficient hydrogen removal to balance the syntrophic acetate degradation and in turn maintain low VFA levels. SMA against the soluble substrates, acetate, propionate and butyrate on the same day were significantly reduced (Table 3), most likely due to accumulation of acetate from the acetogenic Bacteria which is known to inhibit propionate and butyrate degradation (Gorris et al., 1989). The high SMA on H2/CO2 and SAA values recorded on day 419 (Tables 3 and 4) indicated the maintenance of low hydrogen levels in both reactors which, in turn, resulted in low propionate discharge, despite accumulation of acetate. This highlights the importance of autotrophic acetogenesis during periods of stress in reactors. The autotrophic metabolic capabilities of the acetogenic Bacteria in a mixed culture were assessed in the final period (period 4) of the reactor trial when sodium formate was supplied as sole substrate (Tables 1 and 2). Although formate is a substrate suitable for heterotrophic metabolism, its initial conversion to H2/CO2 promotes the proliferation of specific acetogenic species that are capable of autotrophic growth on H2/CO2. Formate also plays an important role in interspecies
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
H2/formate transfer (Thiele and Zeikus, 1988). One of the critical steps in the anaerobic digestion process is the oxidation of organic acids, by electron transfer via hydrogen or formate, from acetogenic Bacteria to methanogens. This syntrophic microbiological process is strongly restricted by a thermodynamic limitation on the concentration of hydrogen or formate present (Batstone et al., 2006). The presence of the sodium formate influent stream pushed this syntrophic relationship to operate at a high rate due to the amounts of available formate/H2. Results from period 4 indicate that the mesophilic reactor performed better overall in comparison to the thermophilic reactor when formate was the substrate in terms of COD removal (Fig. 1). At the initial low VLR the thermophilic biomass operated well with low loading rates, but, once the VLR was increased on day 479, the % COD removal efficiencies deteriorated until the end of the trial (Fig. 1). By the final day on formate, day 588, SMA tests indicated that neither reactor sludge displayed methanogenic activity on acetate or propionate (Table 3). This suggests inhibition of the acetoclastic methanogens and propionate syntrophs during period 4 which may be explained by the effect of formate in interspecies H2/formate transfer. Similarly, Voolapalli and Stuckey (2001) reported that hydrogen or formate accumulation did not overload the process dynamics, but the resulting slow kinetics of VFA degradation was problematic. These authors suggested that monitoring acetate rather than hydrogen accumulation would give a better understanding of reactor stability. From a reaction point of view there is no difference between hydrogen and formate. However there are two important physiochemical differences between them. Firstly, hydrogen has a higher diffusivity than formate and secondly formate is more soluble than hydrogen (Batstone et al., 2006). Below a critical H2/formate level, the hydrogen consumers do not obtain enough energy for growth and above a certain level the H2 producers run into similar problems (Dolfing, 2001). The high percentage of methane recorded in the biogas at this stage of the trial, coupled with the elevated methanogenic activity on H2/CO2, suggests that the hydrogenotrophic methanogenic consortia present in the thermophilic and mesophilic sludges were utilising the excess accessible formate and out-competing the acetoclastic methanogens. It has also been reported that the rates of acetogenesis are stimulated by the addition of formate, which can result in outcompetition of methanogens by acetogens (Tholen and Brune, 1999). This is also a likely scenario based on SAA profiles from the reactors biomass at the conclusion of the trial (Table 4). The elevated reactor pH (Tables 1 and 2) and high internal acetate concentrations from the final period of anaerobic treatment of sodium formate, particularly in the thermophilic reactor, indicated the activation of syntrophic acetate oxidation which is the mechanism by which acetate is oxidised to carbon dioxide by a proton reducing bacterium (homoacetogen) followed by a reduction of the carbon dioxide by hydrogen to methane by a methanogenic bacterium (Schnu¨rer et al., 1999). Factors relating to the occurrence of syntrophic acetate oxidation include a high salt concentration and a high concentration of VFAs resulting in inhibition of acetate methanogenesis (Schnurer et al., 1996). The formate supplied as the influent stream to both reactors was a sodium based
4267
compound which increased the levels of salt within both reactors. Although the levels of acetate during this period were reduced in comparison to the previous period when vanillate was supplied, they still remained elevated, particularly in R2 towards the end of the trial when the VLR was increased (Fig. 2). The final factor supporting this theory is the lack of methanogenic activity recorded on acetate in both R1 and R2 on day 588 (Table 3). The high levels of hydrogenotrophic methanogenic activity at this time would help to maintain the reduced hydrogen partial pressures that are favourable for syntrophic acetate oxidation. Schnurer et al. (1996) isolated a novel acetogenic bacterium capable of oxidising acetate in syntrophic association with a H2-utilising bacterium, and this acetogen was subsequently named Clostridium ultunese. In co-culture with a hydrogen utilising methanogen, C. ultunese degraded acetate stoichiometrically to methane and the oxidation of acetate appears to occur via a reversal of the acetyl-CoA pathway (Schnu¨rer et al., 1997). The growth supportive substrates of this strain include formate as well as glucose, pyruvate, ethylene glycol and cysteine. The resulting end products are acetate, formate and H2. Practical application of these findings demonstrate that the development and enrichment of heterotrophic and autotrophic acetogens in granular sludges would facilitate the high rate methanogenic conversion of industrial waste streams containing methylated aromatics and single carbon compounds, such as methanol and formate.
5.
Conclusions
These results are significant with regards to the enrichment of homoacetogenic and acetogenic species in mesophilic and thermophilic anaerobic sludge granules, a finding which has not been previously reported. The substrates used during this trial successfully demonstrated the three metabolic capabilities of acetogens in a mixed culture; - heterotrophic conversion of sugars to acetate, the ability to demethylate a methoxylated aromatic compound, vanillate and the use of the autotrophic substrate formate as a carbon source, as confirmed by SMA and SAA results. On average, the thermophilic reactor performed better, displaying higher COD removal efficiencies in comparison to its mesophilic counterpart during the treatment of sodium vanillate. The thermophilic reactor was much slower to achieve stable performance following the change of influent from sodium vanillate to sodium formate. The continued decline of the acetoclastic methanogenic populations from vanillate was evident in period 4. The high sodium concentration, introduced through the sodium formate influent, further inhibited recovery of the acetate and propionate degraders in both thermophilic and mesophilic reactors. The elevated SMA in period 4 on H2/CO2, particularly in the thermophilic reactor, indicates that methane production was as a result of hydrogenotrophic methanogenesis which supports evidence of syntrophic acetate oxidation activity.
4268
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
Monitoring acetate accumulation is suggested as a responsible method to assess reactor stability.
references
Bache, R., Pfennig, N., 1981. Selective isolation of Acetobacterium woodii on methoxylated aromatic acids and determination of growth yields. Archives of Microbiology 130 (3), 255e261. Batstone, D.J., Picioreanu, C., van Loosdrecht, M.C.M., 2006. Multidimensional modelling to investigate interspecies hydrogen transfer in anaerobic biofilms. Water Research 40 (16), 3099e3108. Bouwer, E.J., McCarty, P.L., 1983. Effects of 2-bromoethanesulfonic acid and 2- chloroethanesulfonic acid on acetate utilization in a continuous-flow methanogenic fixed-film column. Applied and Environmental Microbiology 45 (4), 1408e1410. Chidthaisong, A., Conrad, R., 2000. Specificity of chloroform, 2bromoethanesulfonate and fluoroacetate to inhibit methanogenesis and other anaerobic processes in anoxic rice field soil. Soil Biology and Biochemistry 32 (7), 977e988. Coates, J.D., Coughlan, M.F., Colleran, E., 1996. Simple method for the measurement of the hydrogenotrophic methanogenic activity of anaerobic sludges. Journal of Microbiological Methods 26 (3), 237e246. Colleran, E., Concannon, F., Golden, T., Geoghegan, F., Cru, lish, B., Killilea, E., Henry, M., Coates, J.D., 1992. Use of methanogenic activity tests to characterize anaerobic sludges, screen for anaerobic biodegradability and determine toxicity thresholds against individual anaerobic trophic groups and species. Water Science and Technology 25 (7), 31e40. Daniel, S.L., Keith, E.S., Yang, H., Lin, Y.-S., Drake, H.L., 1991. Utilization of methoxylated aromatic compounds by the acetogen Clostridium thermoaceticum: expression and specificity of the co-dependent O-demethylating activity. Biochemical and Biophysical Research Communications 180 (1), 416e422. Dolfing, J., 2001. The microbial logic behind the prevalence of incomplete oxidation of organic compounds by acetogenic bacteria in methanogenic environments. Microbial Ecology 41 (2), 83e89. Drake, H.L., Daniel, S.L., 2004. Physiology of the thermophilic acetogen Moorella thermoacetica. Research in Microbiology 155 (10), 869e883. Drake, H., Ku¨sel, K., Matthies, C., 2006. Acetogenic prokaryotes. In: The Prokaryotes. Springer, New York, pp. 354e420. Field, J.A., Stams, A.J.M., Kato, M., Schraa, G., 1995. Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia. Antonie Van Leeuwenhoek 67 (1), 47e77. Forbes, C., O’Reilly, C., McLaughlin, L., Gilleran, G., Tuohy, M., Colleran, E., 2009. Application of high rate, high temperature anaerobic digestion to fungal thermozyme hydrolysates from carbohydrate wastes. Water Research 43 (9), 2531e2539. Frankin, R.J., 2001. Full-scale experiences with anaerobic treatment of industrial wastewater. Water Science and Technology 44 (8), 1e6. Frazer, A.C., Coschigano, P.W., Young, L.Y., 1995. Toluene metabolism under anaerobic conditions: a review. Anaerobe 1 (6), 293e303. Gorris, L.G.M., van Deursen, J.M.A., van der Drift, C., Vogels, G.D., 1989. Inhibition of propionate degradation by acetate in methanogenic fluidized bed reactors. Biotechnology Letters 11 (1), 61e66. Grotenhuis, J.T.C., Kissel, J.C., Plugge, C.M., Stams, A.J.M., Zehnder, A.J.B., 1991. Role of substrate concentration in particle size distribution of methanogenic granular sludge in UASB reactors. Water Research 25 (1), 21e27.
Guiot, S.R., Arcand, Y., Chavarie, C., 1992. Advantages of fluidization on granule size and activity development in upflow anaerobic sludge bed reactors. Water Science and Technology 26 (33), 897e906. Hulshoff Pol, L.W., de Castro Lopes, S.I., Lettinga, G., Lens, P.N.L., 2004. Anaerobic sludge granulation. Water Research 38 (6), 1376e1389. Kotsyurbenko, O.R., Glagolev, M.V., Nozhevnikova, A.N., Conrad, R., 2001. Competition between homoacetogenic bacteria and methanogenic archaea for hydrogen at low temperature. FEMS Microbiology Ecology 38 (2e3), 153e159. Lettinga, G., 1995. Anaerobic digestion and wastewater treatment systems. Antonie Van Leeuwenhoek 67 (1), 3e28. Li, Y., Fang, H.P., Noike, T. 1994. Recent developments in microbiology and biochemistry of homoacetogens and syntrophic acetogens. In: Proceedings of the seventh International Symposium on Anaerobic Digestion: Oral Paper Preprints, Cape Town, South Africa, pp. 54e57. van Lier, J.B., Brummeler, E., Lettinga, G., 1993. Thermo-tolerant anaerobic degradation of volatile fatty acids by digested organic fraction of municipal solid waste. Journal of Fermentation and Bioengineering 76 (2), 140e144. van Lier, J.B., Martin, J.L.S., Lettinga, G., 1996. Effect of temperature on the anaerobic thermophilic conversion of volatile fatty acids by dispersed and granular sludge. Water Research 30 (1), 199e207. McHugh, S., Collins, C., O’Flaherty, V., 2005. Long-term, high-rate anaerobic biological treatment of whey wastewaters at psychrophilic temperatures. Bioresource Technology 97 (14), 1669e1678. Mechichi, T., Patel, B.K.C., Sayadi, S., 2005. Anaerobic degradation of methoxylated aromatic compounds by Clostridium methoxybenzovorans and a nitrate-reducing bacterium Thauera sp. strain Cin3,4. International Biodeterioration and Biodegradation 56 (4), 224e230. O’Flaherty, V. 1997. Microbial Interactions during Anaerobic Treatment of Sulphate-containing Wastewaters. PhD thesis. National University of Ireland, Galway. O’Flaherty, V., Colohan, S., Mulkerrins, D., Colleran, E., 1999. Effect of sulphate addition on volatile fatty acid and ethanol degradation in an anaerobic hybrid reactor. II: microbial interactions and toxic effects. Bioresource Technology 68 (2), 109e120. Schnurer, A., Schink, B., Svensson, B.H., 1996. Clostridium ultunense sp. nov., a mesophilic bacterium oxidizing acetate in syntrophic association with a hydrogenotrophic methanogenic bacterium. International Journal of Systematic and Evolutionary Microbiology 46, 1145e1152. Schnu¨rer, A., Svensson, B.H., Schink, B., 1997. Enzyme activities in and energetics of acetate metabolism by the mesophilic syntrophically acetate oxidizing anaerobe Clostridium ultunense. FEMS Microbiology Letters 154 (2), 331e336. Schnu¨rer, A., Zellner, G., Svensson, B.H., 1999. Mesophilic syntrophic acetate oxidation during methane formation in biogas reactors. FEMS Microbiology Ecology 29 (3), 249e261. Shelton, D.R., Tiedje, J.M., 1984. General method for determining anaerobic biodegradation potential. Applied and Environmental Microbiology 47 (4), 850e857. Stams, A.J.M., Plugge, C.M., 2009. Electron transfer in syntrophic communities of anaerobic bacteria and archaea. Nature Reviews, Microbiology 7 (8), 568e577. Thiele, J.H., Zeikus, J.G., 1988. Control of interspecies electron flow during anaerobic digestion: significance of formate transfer versus hydrogen transfer during syntrophic methanogenesis in flocs. Applied and Environmental Microbiology 54 (1), 20e29. Tholen, A., Brune, A., 1999. Localization and in-situ activities of homoacetogenic bacteria in the highly compartmentalized hindgut of soil-feeding higher termites
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
(Cubitermes spp.). Applied and Environmental Microbiology 65 (10), 4497e4505. Voolapalli, R.K., Stuckey, D.C., 2001. Hydrogen production in anaerobic reactors during shock loads e Influence of formate production and H2 kinetics. Water Research 35 (7), 1831e1841. Wang, J., Zhang, Z., Zhang, Z., Zheng, P., Li, C., 2007. The influence of anaerobic sludge retention time on anaerobic co-digestion of dyeing and printing wastewater and sewage sludge. African Journal of Biotechnology 6 (7), 902e907.
4269
Xu, K., Liu, H., Du, G., Chen, J., 2009. Real-time PCR assay targeting formyltetrahydrofolate synthetase gene to enumerate acetogens in natural and engineered environments. Anaerobe 15 (5), 204e213. de Zeeuw, W. 1984. Acclimatisation of anaerobic sludge for UASB reactor start-up. Ph.D. thesis. Agricultural University, Wageningen, The Netherlands. Zhang, T.C., Noike, T., 1994. Influence of retention time on reactor performance and bacterial trophic populations in anaerobic digestion processes. Water Research 28 (1), 27e36.
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
Enrichment of acetogenic bacteria in high rate anaerobic reactors under mesophilic and thermophilic conditions P. Ryan*, C. Forbes, S. McHugh, C. O’Reilly, G.T.A. Fleming, E. Colleran Environmental Microbiology Research Unit, Department of Microbiology, National University of Ireland, Galway, Ireland
article info
abstract
Article history:
The objective of the current study was to expand the knowledge of the role of acetogenic
Received 4 January 2010
Bacteria in high rate anaerobic digesters. To this end, acetogens were enriched by supplying
Received in revised form
a variety of acetogenic growth supportive substrates to two laboratory scale high rate
14 May 2010
upflow anaerobic sludge bed (UASB) reactors operated at 37 C (R1) and 55 C (R2). The
Accepted 25 May 2010
reactors were initially fed a glucose/acetate influent. Having achieved high operational
Available online 1 June 2010
performance and granular sludge development and activity, both reactors were changed to homoacetogenic bacterial substrates on day 373 of the trial. The reactors were initially fed
Keywords:
with sodium vanillate as a sole substrate. Although % COD removal indicated that the 55 C
Acetogens
reactor out performed the 37 C reactor, effluent acetate levels from R2 were generally
UASB
higher than from R1, reaching values as high as 5023 mg l1. Homoacetogenic activity in
Thermophilic
both reactors was confirmed on day 419 by specific acetogenic activity (SAA) measurement,
Anaerobic
with higher values obtained for R2 than R1. Sodium formate was introduced as sole substrate to both reactors on day 464. It was found that formate supported acetogenic activity at both temperatures. By the end of the trial, no specific methanogenic activity (SMA) was observed against acetate and propionate indicating that the methane produced was solely by hydrogenotrophic Archaea. Higher SMA and SAA values against H2/CO2 suggested development of a formate utilising acetogenic population growing in syntrophy with hydrogenotrophic methanogens. Throughout the formate trial, the mesophilic reactor performed better overall than the thermophilic reactor. ª 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Anaerobic digestion is a natural process which has been exploited for over a century for the effective treatment of organic wastes and is currently recognised as a major technology for remediation of municipal and industrial wastewaters. Anaerobic digestion has advantages over the more conventional aerobic process including less excess sludge production, lower space requirements as well as the recovery of
energy from the waste material in the form of a renewable energy, biogas (Lettinga, 1995). In recent years, the application of this technology to the treatment of a variety of domestic and industrial waste streams as well as for the remediation of various recalcitrant compounds such as chlorinated aliphatics and other xenobiotics, has increased at full scale as a result of a greater understanding of the treatment process and the development of high rate, granular sludge based reactor designs such as the UASB (Guiot et al., 1992; Field et al., 1995; Frankin,
* Corresponding author. Present address: Innovation Centre, Bord na Mo´na, Main Street, Newbridge, Co. Kildare, Ireland. Tel.: þ353 45439382; fax: þ353 45434207. E-mail address: [email protected] (P. Ryan). 0043-1354/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2010.05.033
4262
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
2001). The success of the UASB design for the treatment of high strength liquid wastes hinges upon the formation and maintenance of anaerobic sludge granules in which Bacteria and Archaea are juxtaposed, facilitating interspecies transfer of substrates necessary for the complete methanogenesis of the organic constituents of the wastewater (Grotenhuis et al., 1991; O’Flaherty, 1997; Stams and Plugge, 2009). A relatively understudied distinguished group involved in the complex anaerobic digestion process is the acetogenic Bacteria. When acetogens were first isolated they were thought of as being metabolically limited and thermodynamically disadvantaged, but in fact, it is now acknowledged that quite the opposite is true (Drake and Daniel, 2004). Under standard redox potential conditions, whereby acetogens and methanogens compete for substrates, the reductive synthesis of acetate from CO2 by acetogenesis is thermodynamically less favourable than methanogenesis, which is the often cited reason used to explain out-competition of acetogens by methanogens in anaerobic sludge. However, it is now clear that acetogens are capable of a highly diverse range of metabolic transformations (Xu et al., 2009). The three underlying metabolic features of these organisms are (i) the use of chemolithoautotrophic substrates (H2/CO2 or CO/CO2) as sole sources of carbon and energy, (ii) heterotrophic conversion of certain sugars stoichiometrically to acetate, and (iii) the ability to oxidise methoxylated aromatic compounds via the acetyl-CoA pathway. Many, but not all acetogens, display all three of these metabolic capabilities. Under certain conditions, autotrophic growth on H2/CO2 can be substituted for heterotrophic metabolism of a wide range of sugars, alcohols, methoxylated aromatic compounds and single carbon compounds, such as methanol and formate (Li et al., 1994). This diverse range of electron donors is oxidised by acetogens to shunt a reductant to the acetyl-CoA pathway, conferring these organisms with a competitive edge and ensuring their proliferation in anaerobic sludge. It is now clear that acetogens are arguably the most metabolically diverse group of obligate anaerobes characterized and that they present some interesting challenges, in particular with reference to their role within the anaerobic digestion process. H2-utilising acetogens are known to be present in mesophilic digester sludge with numbers in the range of 105e107 CFU/ml (Zhang and Noike, 1994) which estimates to approximately 3e11% of total Bacteria in anaerobic sludge (Wang et al., 2007). It has yet to be demonstrated whether these organisms play an autotrophic or heterotrophic role in anaerobic digestion within different temperature ranges, although it has been shown that in certain complex microbial habitats, the metabolic interactions of acetogens are influenced by environmental factors such as pH and temperature and are strain specific (Kotsyurbenko et al., 2001). The objectives of the current study focused on enrichment of acetogens in two UASB reactors, R1 and R2, operated at mesophilic (37 C) and thermophilic (55 C) conditions, which were provided with influents composed of substrates chosen to promote acetate production by acetogens. Three individual liquid influent streams were supplied separately to the reactors, each designed to promote either the heterotrophic or autotrophic metabolism of acetogens. These objectives aimed to facilitate an understanding of acetogenic behaviour and reactions in the anaerobic digestion process which could
potentially be applied to large scale facilities treating wastewaters of similar composition.
2.
Materials and methods
2.1.
Reactor design and operation
Two glass laboratory scale anaerobic upflow sludge bed (UASB) reactors with an active volume of 4.25 l were employed for this study. The reactors were seeded with anaerobic granular sludge originating from an internal circulation (IC) reactor treating wastewater from a commercial milk processing plant at 37 C (Carbery Milk Products, Ballineen, Co. Cork, Ireland). The conveniently available sludge had been stored, unfed, at 4 C for 14 months prior to this study. An initial in-reactor seed volatile suspended solid (VSS) concentration of 11.5 g l1 as recommended by de Zeeuw (1984) was applied. The remainder of the reactor active volume was filled with a mixture of sucrose/acetate at a ratio of 60:40, on a COD basis, to a total of 10 g COD l1. Both reactors were started up at 37 C and the sucrose/acetate influent was initially supplied at a volumetric loading rate (VLR) of 5 g COD l1 d1 at a hydraulic retention time (HRT) of 2 days. This HRT was maintained throughout the trial. The trial operational parameters and results from the mesophilic reactor (R1) and thermophilic reactor (R2) are summarised in Tables 1 and 2. Throughout the trial periods, the COD:N:P ratio in the influent was maintained at 1000:5:0.5 by supplementation with NH4Cl and KH2PO4 to the required concentrations. The pH of each influent mixture was buffered by the addition of NaHCO3 at a concentration of 8 g l1 and supplemented with macro and micro nutrients (1 ml l1), as recommended by Shelton and Tiedje (1984).
2.2.
Specific methanogenic activity (SMA)
The specific methanogenic activities of the seed sludge and reactor sludges sampled on days 62, 256, 419 and 588 were determined using the pressure transducer technique to indirectly measure gas production in hypovials containing 2e5 g VSS l1 of sludge biomass (Colleran et al., 1992; Coates et al., 1996). Briefly, the test procedure involved the measurement of the biogas pressure increase developing in sealed vials fed with the non-gaseous substrates, ethanol (30 mM), propionate (30 mM), butyrate (15 mM) and acetate (30 mM), or of the pressure decrease in vials pressurized with H2/CO2 (80:20) to 1 atm. On completion of the test, millivolt readings were converted to ml biogas, the methane content of the vial headspace analysed and the VSS of the test vial determined. Analysis of these data and the rate data (ml biogas produced/ utilised per hour) allowed expression of specific methanogenic activity as ml CH4 (STP) g1 VSS d1. Assays on the seed sludge and sludge from the R1 were performed at 37 C; assays on sludge from the R2 were performed at 55 C.
2.3.
Specific acetogenic (SAA) activity tests
A modified version of the SMA test was used to determine the specific acetogenic activity (SAA) of sludge samples. A specific
4263
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
Table 1 e Operational parameters and average performance data of the mesophilic reactor (R1). Values are the mean of triplicates ± standard deviation. Figures in brackets represent the maximum and minimum values in that period. Period Days Influent
1 1e128 sucrose/ acetate
2a 128e240 sucrose/ acetate
2b 240e373 sucrose/ acetate
3a 373e391 sodium vanillate
3b 391e464 sodium vanillate
4a 464e479 sodium formate
4b 479e496 sodium formate
4c 496e588 sodium formate
Influent Parameters Temperature C COD (g l1) HRT (days) VLR g COD l1 d1
37 10 2 5
37 10 2 5
37 20 2 10
37 1 2 0.5
37 5 2 2.5
37 1 2 0.5
37 2.5 2 1.25
37 5 2 2.5
Effluent parameters % COD removal pH CH4 (%)
93.8 6.8 7.7 0.5 70.9 5.4
97.1 7.1 7.6 0.2 65.0 6.1
93.3 8.0 7.7 0.4 72.4 7.9
63.5 9.2 8.1 0.9 72.1 7.0
30.5 10.5 8.6 0.8 80.8 8.2
71.8 12.6 9.0 0.7 87.8 8.3
79.4 9.9 9.1 0.6 93.1 8.8
83.8 11.3 9.4 0.8 98.8 8.7
VFA (mg l1) Acetate mg l1 (minemax) Propionate mg l1 (minemax) Butyrate mg l1 (minemax)
90.1 (2.7e598.3) 75.2 (2.9e397.5) 11.8 (1.3e80.6)
29.2 (4.0e263.8) 12.2 (1.3e62.8) 6.8 (1.3e14.3)
85.2 (2.8e192.5) 26.3 (1.4e92.7) 8.1 (1.2e56.1)
745.6 (151e2130) 267.2 (67.8e511.1) 40.5 (18.3e59.4)
1567.5 (526e3015) 464.9 (34e1055) 106.2 (11.2e421)
614.5 (339.1e896) 389.6 (254e652) 72.8 (22e145)
99.8 (9.4e179) 89.8 (18.1e203.3) 22.1 (1.5e57.3)
75.5 (7.7e475.6) 36.8 (5.1e122.5) 6.6 (1.3e20.1)
methanogenic inhibitor, 2-bromoethane sulfonate (BES), (Bouwer and McCarty, 1983; Chidthaisong and Conrad, 2000), was included at a concentration of 100 mM in the test vials. The vials were pressurised with H2/CO2 (80:20) to 1 atm. Pressure decrease in the vials was measured over time with the pressure transducer device. Parallel vials were set up under the same parameters and sacrificed for acetate analysis by gas chromatography. On completion of the test the methane content of the vial headspace was analysed and the VSS of the test vials was determined. Analysis of the data and the rate of acetate production allowed expression of acetogenic activity as ml acetate g1 VSS d1.
2.4.
Analytical methods
Reactor effluent/influent samples were regularly analysed for COD, pH and VFA content according to Standard Methods (APHA, 1998). Methane concentration in the biogas was analysed by gas chromatography (GC) using a glass column packed with Poropak Q 100e120 mesh in a Philips PYE-Unicam Series 304 chromatograph fitted with a gas sampling port and a flame ionisation detector. The column temperature was maintained at 35 C. The injection port and detector temperatures were 105 C and 100 C, respectively (McHugh et al., 2005). Volatile fatty acids (VFAs) and ethanol were quantified using a gas chromatograph (6890 Plus, Agilent, Palo Alto, CA) equipped with an Innowax capillary column (Agilent) and a flame ionization detector.
3.
Results
3.1.
Bioreactor performance
The start-up phase for both reactors in period 1, showed COD removal efficiencies reaching averages of 90% after 10 days of operation (Fig. 1) and the % methane in biogas averaging 70%
from both reactors after 14 days (Tables 1 and 2). Operation of R2 at 55 C commenced on day 128 (period 2) and caused an immediate reduction in COD removal efficiency and methane content of the biogas from this reactor (Table 2). The % methane recorded deteriorated to 12% by day 135 and a minimum COD removal of 26.3% was recorded on day 139. This period of R2 instability also caused a rise in VFA discharge with acetate levels reaching up to 1500 mg l1 (Fig. 2). The reactor recovered, achieving >90% COD removal by day 167 after just 39 days of operation at 55 C (Fig. 1) and VFA levels remained negligible for the remainder of period 2 (Fig. 2). On day 240, the VLR was increased to 10 g COD l1 d1 (Fig. 1). An initial negative effects on the performance of both reactors was, however, shortlived, with COD removal efficiencies returning to approximately 90% for R1 and R2 after 40 and 60 days respectively (Figs. 1 and 2). The sucrose/acetate influent was changed to a sodium vanillate feed on day 373 (period 3a, Tables 1 and 2) at an initial concentration of 1 g COD l1. Since the HRT remained unchanged at 2 days, this resulted in an initial VLR of 0.5 g COD l1 d1. Despite this low loading rate, COD removal efficiencies decreased sharply for both reactors (Fig. 1), with associated increases in effluent acetate levels (Fig. 2). When COD removal efficiency was shown to improve for both reactors, the sodium vanillate influent concentration was increased to 5 g COD l1 on day 391, resulting in an increased VLR of 2.5 g COD l1 d1. This had a very negative impact on the performance of both reactors. The % COD removal for the mesophilic R1 fluctuated considerably between days 391 and 463 (Period 3b, Table 1 and Fig. 1), with a low of 3.8 observes on day 399 and an average value of 30% throughout this period. Effluent acetate also fluctuated during this period, reaching a peak of 3015 mg l1 on day 401 and an average value of >1560 mg l1 throughout the 72 day test period (Fig. 2 and Table 1). Similarly, for thermophilic R2, this feed period was marked by considerable instability. The % COD removal decreased to 16.5% on day 401, averaging 53.2% throughout period 3b (Fig. 1).
4264
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
Table 2 e Operational parameters and average performance data of the thermophilic reactor (R2). Values are the mean of triplicates ± standard deviation. Figures in brackets represent the maximum and minimum values in that period. Period Days Influent
1 1e128 sucrose/ acetate
2a 128e240 sucrose/ acetate
2b 240e373 sucrose/ acetate
3a 373e391 sodium vanillate
3b 391e464 sodium vanillate
4a 464e479 sodium formate
4b 479e496 sodium formate
4c 496e588 sodium formate
Influent Parameters Temperature C COD (g l1) HRT (days) VLR g COD l1 d1
37 10 2 5
55 10 2 5
55 20 2 10
55 1 2 0.5
55 5 2 2.5
55 1 2 0.5
55 2.5 2 1.25
55 5 2 2.5
Effluent parameters % COD removal pH CH4 (%)
93.9 8.2 7.7 1.2 68.0 6.2
87.2 9.3 7.7 1.1 59.0 5.4
89.8 12.9 7.7 1.8 71.5 6.8
64.8 11.6 8.1 0.5 61.3 5.8
53.2 8.0 8.5 0.8 65.0 7.7
81.4 9.9 9.0 1.0 75.3 8.1
73.5 8.5 9.1 0.6 85.4 8.6
65.2 9.2 9.4 1.1 80.1 7.9
134.8 (4.1e986.3) 235.8 (6.0e1138.2) 26.3 (1.9e244.6)
238.7 (2.0e1593.6) 124.3 (1.0e885.2) 71.7 (0.8e478.0)
85.4 (3.1e56.6) 36.9 (1.3e31.1) 11.5 (1.2e19.7)
1157.7 (209.7e2663) 175.5 (43.9e722) 28.7 (20.3e88.9)
1241.6 (143e5023) 271.6 (104.3e1776) 51.3 (22.1e455.3)
85.3 (43.1e214.9) 73.9 (31.7e222.5) 7.5 (1.3e25.6)
177.09 (56.8e90.8) 66.8 (13e104.1) 44.6 (1.3e72)
934.6 (11.1e1886.3) 82.4 (6.2e225.6) 26.7 (1.2e119.2)
VFA (mg l1) Acetate mg l1 (minemax) Propionate mg l1 (minemax) Butyrate mg l1 (minemax)
By the end of the sodium vanillate trial, however, R2 was achieving >85% COD removal efficiency. Effluent acetate levels from R2 showed a peak of >5000 mg l1 on day 427 and averaged 1242 mg l1 during the 72 day trial period (Fig. 2 and Table 2). The % methane of the biogas from both R1 and R2 increased, however, during period 3b (Tables 1 and 2). On day 464, sodium formate was introduced as sole substrate to the reactors, at a COD concentration of 1 g COD l1 and a VLR of 0.5 g COD l1 d1 (Tables 1 and 2). Biomass adaptation to the new stream was apparent after one HRT (2 days) and the average % COD removal during period 4a increased to 71.8% in R1 and 81.4% in R2 (Tables 1 and 2). The % methane content of the biogas produced by both reactors continued to rise during this period while effluent VFAs decreased sharply from the previous period (Fig. 2). The VLR of both reactors was increased to 1.25 g COD l1 d1 on day 479
and then to 2.5 g COD l1 d1 on day 496. R1 adapted readily to the increased VLR with a COD removal efficiency of 92% and biogas methane content of 98.9% recorded on day 533. R2 performance, however, deteriorated in response to the increased VLR, with a reduction in average COD removal efficiency and a two fold increase in average acetate discharge values between periods 4a and 4b which sharply increased during period 4c (Fig. 2, Tables 1 and 2). However by the end of the formate trial, there was little difference between the operational performances of both reactors.
3.2.
Physiological assessment of biomass
The specific methanogenic activity (SMA) and specific acetogenic activity (SAA) values generated from testing of the seed sludge and sludges removed intermittently from the reactors
Fig. 1 e % soluble COD removal by R1 and R2 throughout the trial period ( mesophilic R1, ). Operational periods defined in Tables 1 and 2 are indicated by arrows. (
thermophilic R2); VLR
4265
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
Fig. 2 e Effluent acetate levels (mg lL1) throughout the trial period ( mesophilic R1, Operational periods defined in Tables 1 and 2 are indicated on the graph.
are presented in Tables 3 and 4. The SMA values generated from the seed sludge against acetate, ethanol, propionate, butyrate and H2/CO2 were 10.6, 25.3, 41.8, 21.3 and 100.6 ml CH4 (STP) g1 VSS d1 respectively (Table 3). These results reflect the fact that prior to seeding the reactors, the sludge had been stored long term at 4 C and so was relatively inactive. The SMA values recorded for sludge removed on day 62 during start-up of R1 and R2 at 37 C are illustrated in Table 3. All activities were all significantly higher compared to seed sludge profiles and indicate the activity of all trophic groups necessary for successful syntrophic and methanogenic populations. SAA results remained negligible at this point, indicating minimal activity of H2/CO2 utilising acetogens (Table 4). By day 256 (Period 2b), the operational temperature of R2 had been changed and R1 and R2 were performing well under a high VLR of 10 g COD l1 d1 (Tables 1 and 2; Fig. 1). This was reflected in the SMA profile of the sludges with notable increases in activity on all substrates in comparison to the seed sludge activity assay (Table 3). The decreased SMA in the R2 sludge on day 256 in comparison with sludge removed from this reactor on day 62 against acetate, propionate and butyrate is mostly likely due to the emergence of a thermophilic population in response to the increase in operational temperature. A marked increase in the capacity of the R2 sludge to degrade H2/CO2 was noted on day 256, however. Evidence that acetogenic populations were active in the thermophilic sludge was supported by the high SAA activity of 159.3 ml acetate g1 VSS d1 recorded on day 256, a nine fold increase from the previous sample on day 62 (Table 3). On day 419, the ability of the reactor sludges to degrade many of the substrates tested had deteriorated since day 256 which was expected given the decrease in reactor performances associated with the change in influent substrate to vanillate (Fig. 1; Table 3). Interestingly, the SMA values for H2/ CO2 conversion by R1 and R2 remained consistent, which may indicate that methane was produced from H2/CO2 during this period. It has been previously reported that hydrogenophilic methanogens are less susceptible to VFA induced inhibition
thermophilic R2).
than acetoclastic methanogens or acetogens (O’Flaherty et al., 1999). This theory is supported by the increased acetogenic activity recorded in this period from R1 and R2 biomass (Table 4) which was associated with elevated acetate discharge from the reactors (Fig. 2) and may have resulted in inhibition of the acetoclastic methanogens. The SAA values of the mesophilic and thermophilic takedown sludge on day 588 after 124 days of operation on sodium formate remained high at 349.3 and 426.8 ml acetate g1 VSS d1, respectively (Table 4). The SMA values on this day, however, indicated that there was reduced methanogenic activity upon all substrates tested, with the exception of H2/ CO2. This was similar to the results generated from previous tests where activity of the reactor sludges against acetate, propionate and butyrate had been gradually decreasing as the trial progressed until the test on day 588 when a total lack of activity against acetate and propionate by both R1 and R2 biomass was evident (Table 3).
4.
Discussion
The objective to enrichment acetogenic Bacteria and the effect of this on the anaerobic process at mesophilic and thermophilic temperatures was demonstrated in this paper. The development of stable well-functioning anaerobic communities was crucial during the start-up of this trial. Therefore, a sugar based influent which supports the growth and maintenance of the fermentative, acidogenic and acetogenic bacterial groups to produce acetate and H2/CO2, direct methanogenic precursors, was applied. This influent was composed of sucrose which supports the growth of a wide range of characterised acetogenic isolates converting it to acetate via the acetyl-CoA pathway (Drake et al., 2006). The addition of acetate in the start-up period was to encourage both sludge granulation (Hulshoff Pol et al., 2004) and maintenance of the activity of the acetoclastic methanogenic Archaea.
4266
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
Table 3 e Specific methanogenic activity (SMA) values of R1 and R2 biomass sampled throughout the trial. Values are the mean of triplicates ± standard deviation. Reactor
Day
Substrates (ml CH4 (STP) g1 VSS d1)
Temp C Acetate
Seed R1 R1 R1 R1 R2 R2 R2 R2
0 62 256 419 588 62 256 419 588
37 37 37 37 37 37 55 55 55
Ethanol
10.6 1.1 98.6 2.5 53.0 3.6 8.4 0.6 0 100.5 10.9 43.6 1.8 4.5 0.5 0
C C C C C C C C C
The sucrose influent was readily degraded by both R1 and R2 during period 1 (Tables 1 and 2). SMA values obtained from sludge sampled on day 62 during period 1 indicated the development of methanogenic consortia in R1 and R2. Operation of R2 at 55 C caused an immediate initial reduction in COD removal efficiency and % methane content of the biogas. However, stable reactor performance was resumed after 20 HRTs. This start-up transition of thermophilic operation from mesophilic sludge in a single step increment has previously been observed (Forbes et al., 2009). The VLR increase on day 240 after a lengthy period of stable performance in both R1 and R2 proved that the thermophilic reactor was slower to recover in comparison to the mesophilic reactor in terms of COD removal efficiency. R2 biomass exhibited a higher capacity for methanogenic activity on H2/CO2 by day 256 and an elevated acetogenic activity value (Table 3). This confirmed the cultivation of an active acetogenic consortium at 55 C achieving a major objective set at the beginning of the trial. The introduction of vanillate on day 391 had an immediate negative effect on the COD removal efficiency in both R1 and R2 (Fig. 1). Vanillate is a methoxylated aromatic compound and a natural product of plants, microorganisms and animals (Mechichi et al., 2005). The effect of direct application of a methoxylated aromatic compound on the anaerobic process in a bioreactor has not been previously researched. The ability to utilize methoxyl groups of aromatic compounds is a specific metabolic capability of some acetogens, such as Clostridium
Table 4 e Specific acetogenic activity (SAA) values of R1 and R2 biomass sampled throughout the trial. Values are the mean of triplicates ± standard deviation. Reactor
Day
Temp C
Seed R1 R1 R1 R1 R2 R2 R2 R2
0 62 256 419 588 62 256 419 588
37 37 37 37 37 37 55 55 55
C C C C C C C C C
H2/CO2 ml acetate g1 VSS d1 11.4 24.1 74.3 143.6 349.3 18.0 159.3 354.8 426.8
0.5 1.2 3.3 12.8 31.4 0.8 11.7 22.9 39.6
25.3 115.9 78.2 82.9 78.7 124.3 91.2 56.8 54.7
2.0 15.8 7.3 4.7 7.1 11.9 6.6 6.5 2.8
Propionate
Butyrate
41.8 3.3 76.7 7.1 51.6 4.8 4.4 0.5 0 74.1 6.7 24.3 3.5 8.4 0.9 0
21.3 55.9 78.1 9.1 3.6 59.2 26.8 7.3 12.7
1.2 3.7 4.8 1.1 0.8 2.8 3.0 0.5 0.09
H2/CO2 100.6 198.3 232.5 122.9 179.1 175.4 307.6 187.7 273.1
9.9 14.7 20.8 10.5 18.4 15.8 30.8 16.6 22.8
coccoides, Sporomusa silvacetica and Clostridium thermoaceticum (Bache and Pfennig, 1981; Daniel et al., 1991; Frazer et al., 1995). All these species possess the ability to O-demethylate methoxylated aromatic compounds like vanillate and metabolise the O-methyl group via the acetyl-CoA pathway. SAA results from day 419 indicated that the level of acetogenic activity in R2 which had been developed during the application of sucrose and acetate in period 2 continued to increase during the application of sodium vanillate in period 3 (Table 4). During this period, R2 out performed its mesophilic counterpart in terms of COD removal efficiency and levels of VFAs discharged in the reactor effluent (Figs. 1 and 2). The elevated levels of acetate discharged by both reactors during period 3 demonstrated the degradation of sodium vanillate to form acetate and thus confirmed the enrichment of acetogenic consortia in the reactors. Because of this, it is also assumed that excess acetate was not a result of propionate and butyrate oxidation to acetate as it is thermodynamically unfavorable for propionate and butyrate to be degraded until acetate and H2 are eliminated by the methanogens (van Lier et al., 1996). The SMA profile of both reactor sludges from day 419 on H2/CO2 was maintained during period 3 despite the reduction in reactor performances (Fig. 1; Table 3). van Lier et al. (1993) stipulated that the number of viable hydrogenotrophic methanogens is important for efficient hydrogen removal to balance the syntrophic acetate degradation and in turn maintain low VFA levels. SMA against the soluble substrates, acetate, propionate and butyrate on the same day were significantly reduced (Table 3), most likely due to accumulation of acetate from the acetogenic Bacteria which is known to inhibit propionate and butyrate degradation (Gorris et al., 1989). The high SMA on H2/CO2 and SAA values recorded on day 419 (Tables 3 and 4) indicated the maintenance of low hydrogen levels in both reactors which, in turn, resulted in low propionate discharge, despite accumulation of acetate. This highlights the importance of autotrophic acetogenesis during periods of stress in reactors. The autotrophic metabolic capabilities of the acetogenic Bacteria in a mixed culture were assessed in the final period (period 4) of the reactor trial when sodium formate was supplied as sole substrate (Tables 1 and 2). Although formate is a substrate suitable for heterotrophic metabolism, its initial conversion to H2/CO2 promotes the proliferation of specific acetogenic species that are capable of autotrophic growth on H2/CO2. Formate also plays an important role in interspecies
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
H2/formate transfer (Thiele and Zeikus, 1988). One of the critical steps in the anaerobic digestion process is the oxidation of organic acids, by electron transfer via hydrogen or formate, from acetogenic Bacteria to methanogens. This syntrophic microbiological process is strongly restricted by a thermodynamic limitation on the concentration of hydrogen or formate present (Batstone et al., 2006). The presence of the sodium formate influent stream pushed this syntrophic relationship to operate at a high rate due to the amounts of available formate/H2. Results from period 4 indicate that the mesophilic reactor performed better overall in comparison to the thermophilic reactor when formate was the substrate in terms of COD removal (Fig. 1). At the initial low VLR the thermophilic biomass operated well with low loading rates, but, once the VLR was increased on day 479, the % COD removal efficiencies deteriorated until the end of the trial (Fig. 1). By the final day on formate, day 588, SMA tests indicated that neither reactor sludge displayed methanogenic activity on acetate or propionate (Table 3). This suggests inhibition of the acetoclastic methanogens and propionate syntrophs during period 4 which may be explained by the effect of formate in interspecies H2/formate transfer. Similarly, Voolapalli and Stuckey (2001) reported that hydrogen or formate accumulation did not overload the process dynamics, but the resulting slow kinetics of VFA degradation was problematic. These authors suggested that monitoring acetate rather than hydrogen accumulation would give a better understanding of reactor stability. From a reaction point of view there is no difference between hydrogen and formate. However there are two important physiochemical differences between them. Firstly, hydrogen has a higher diffusivity than formate and secondly formate is more soluble than hydrogen (Batstone et al., 2006). Below a critical H2/formate level, the hydrogen consumers do not obtain enough energy for growth and above a certain level the H2 producers run into similar problems (Dolfing, 2001). The high percentage of methane recorded in the biogas at this stage of the trial, coupled with the elevated methanogenic activity on H2/CO2, suggests that the hydrogenotrophic methanogenic consortia present in the thermophilic and mesophilic sludges were utilising the excess accessible formate and out-competing the acetoclastic methanogens. It has also been reported that the rates of acetogenesis are stimulated by the addition of formate, which can result in outcompetition of methanogens by acetogens (Tholen and Brune, 1999). This is also a likely scenario based on SAA profiles from the reactors biomass at the conclusion of the trial (Table 4). The elevated reactor pH (Tables 1 and 2) and high internal acetate concentrations from the final period of anaerobic treatment of sodium formate, particularly in the thermophilic reactor, indicated the activation of syntrophic acetate oxidation which is the mechanism by which acetate is oxidised to carbon dioxide by a proton reducing bacterium (homoacetogen) followed by a reduction of the carbon dioxide by hydrogen to methane by a methanogenic bacterium (Schnu¨rer et al., 1999). Factors relating to the occurrence of syntrophic acetate oxidation include a high salt concentration and a high concentration of VFAs resulting in inhibition of acetate methanogenesis (Schnurer et al., 1996). The formate supplied as the influent stream to both reactors was a sodium based
4267
compound which increased the levels of salt within both reactors. Although the levels of acetate during this period were reduced in comparison to the previous period when vanillate was supplied, they still remained elevated, particularly in R2 towards the end of the trial when the VLR was increased (Fig. 2). The final factor supporting this theory is the lack of methanogenic activity recorded on acetate in both R1 and R2 on day 588 (Table 3). The high levels of hydrogenotrophic methanogenic activity at this time would help to maintain the reduced hydrogen partial pressures that are favourable for syntrophic acetate oxidation. Schnurer et al. (1996) isolated a novel acetogenic bacterium capable of oxidising acetate in syntrophic association with a H2-utilising bacterium, and this acetogen was subsequently named Clostridium ultunese. In co-culture with a hydrogen utilising methanogen, C. ultunese degraded acetate stoichiometrically to methane and the oxidation of acetate appears to occur via a reversal of the acetyl-CoA pathway (Schnu¨rer et al., 1997). The growth supportive substrates of this strain include formate as well as glucose, pyruvate, ethylene glycol and cysteine. The resulting end products are acetate, formate and H2. Practical application of these findings demonstrate that the development and enrichment of heterotrophic and autotrophic acetogens in granular sludges would facilitate the high rate methanogenic conversion of industrial waste streams containing methylated aromatics and single carbon compounds, such as methanol and formate.
5.
Conclusions
These results are significant with regards to the enrichment of homoacetogenic and acetogenic species in mesophilic and thermophilic anaerobic sludge granules, a finding which has not been previously reported. The substrates used during this trial successfully demonstrated the three metabolic capabilities of acetogens in a mixed culture; - heterotrophic conversion of sugars to acetate, the ability to demethylate a methoxylated aromatic compound, vanillate and the use of the autotrophic substrate formate as a carbon source, as confirmed by SMA and SAA results. On average, the thermophilic reactor performed better, displaying higher COD removal efficiencies in comparison to its mesophilic counterpart during the treatment of sodium vanillate. The thermophilic reactor was much slower to achieve stable performance following the change of influent from sodium vanillate to sodium formate. The continued decline of the acetoclastic methanogenic populations from vanillate was evident in period 4. The high sodium concentration, introduced through the sodium formate influent, further inhibited recovery of the acetate and propionate degraders in both thermophilic and mesophilic reactors. The elevated SMA in period 4 on H2/CO2, particularly in the thermophilic reactor, indicates that methane production was as a result of hydrogenotrophic methanogenesis which supports evidence of syntrophic acetate oxidation activity.
4268
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
Monitoring acetate accumulation is suggested as a responsible method to assess reactor stability.
references
Bache, R., Pfennig, N., 1981. Selective isolation of Acetobacterium woodii on methoxylated aromatic acids and determination of growth yields. Archives of Microbiology 130 (3), 255e261. Batstone, D.J., Picioreanu, C., van Loosdrecht, M.C.M., 2006. Multidimensional modelling to investigate interspecies hydrogen transfer in anaerobic biofilms. Water Research 40 (16), 3099e3108. Bouwer, E.J., McCarty, P.L., 1983. Effects of 2-bromoethanesulfonic acid and 2- chloroethanesulfonic acid on acetate utilization in a continuous-flow methanogenic fixed-film column. Applied and Environmental Microbiology 45 (4), 1408e1410. Chidthaisong, A., Conrad, R., 2000. Specificity of chloroform, 2bromoethanesulfonate and fluoroacetate to inhibit methanogenesis and other anaerobic processes in anoxic rice field soil. Soil Biology and Biochemistry 32 (7), 977e988. Coates, J.D., Coughlan, M.F., Colleran, E., 1996. Simple method for the measurement of the hydrogenotrophic methanogenic activity of anaerobic sludges. Journal of Microbiological Methods 26 (3), 237e246. Colleran, E., Concannon, F., Golden, T., Geoghegan, F., Cru, lish, B., Killilea, E., Henry, M., Coates, J.D., 1992. Use of methanogenic activity tests to characterize anaerobic sludges, screen for anaerobic biodegradability and determine toxicity thresholds against individual anaerobic trophic groups and species. Water Science and Technology 25 (7), 31e40. Daniel, S.L., Keith, E.S., Yang, H., Lin, Y.-S., Drake, H.L., 1991. Utilization of methoxylated aromatic compounds by the acetogen Clostridium thermoaceticum: expression and specificity of the co-dependent O-demethylating activity. Biochemical and Biophysical Research Communications 180 (1), 416e422. Dolfing, J., 2001. The microbial logic behind the prevalence of incomplete oxidation of organic compounds by acetogenic bacteria in methanogenic environments. Microbial Ecology 41 (2), 83e89. Drake, H.L., Daniel, S.L., 2004. Physiology of the thermophilic acetogen Moorella thermoacetica. Research in Microbiology 155 (10), 869e883. Drake, H., Ku¨sel, K., Matthies, C., 2006. Acetogenic prokaryotes. In: The Prokaryotes. Springer, New York, pp. 354e420. Field, J.A., Stams, A.J.M., Kato, M., Schraa, G., 1995. Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia. Antonie Van Leeuwenhoek 67 (1), 47e77. Forbes, C., O’Reilly, C., McLaughlin, L., Gilleran, G., Tuohy, M., Colleran, E., 2009. Application of high rate, high temperature anaerobic digestion to fungal thermozyme hydrolysates from carbohydrate wastes. Water Research 43 (9), 2531e2539. Frankin, R.J., 2001. Full-scale experiences with anaerobic treatment of industrial wastewater. Water Science and Technology 44 (8), 1e6. Frazer, A.C., Coschigano, P.W., Young, L.Y., 1995. Toluene metabolism under anaerobic conditions: a review. Anaerobe 1 (6), 293e303. Gorris, L.G.M., van Deursen, J.M.A., van der Drift, C., Vogels, G.D., 1989. Inhibition of propionate degradation by acetate in methanogenic fluidized bed reactors. Biotechnology Letters 11 (1), 61e66. Grotenhuis, J.T.C., Kissel, J.C., Plugge, C.M., Stams, A.J.M., Zehnder, A.J.B., 1991. Role of substrate concentration in particle size distribution of methanogenic granular sludge in UASB reactors. Water Research 25 (1), 21e27.
Guiot, S.R., Arcand, Y., Chavarie, C., 1992. Advantages of fluidization on granule size and activity development in upflow anaerobic sludge bed reactors. Water Science and Technology 26 (33), 897e906. Hulshoff Pol, L.W., de Castro Lopes, S.I., Lettinga, G., Lens, P.N.L., 2004. Anaerobic sludge granulation. Water Research 38 (6), 1376e1389. Kotsyurbenko, O.R., Glagolev, M.V., Nozhevnikova, A.N., Conrad, R., 2001. Competition between homoacetogenic bacteria and methanogenic archaea for hydrogen at low temperature. FEMS Microbiology Ecology 38 (2e3), 153e159. Lettinga, G., 1995. Anaerobic digestion and wastewater treatment systems. Antonie Van Leeuwenhoek 67 (1), 3e28. Li, Y., Fang, H.P., Noike, T. 1994. Recent developments in microbiology and biochemistry of homoacetogens and syntrophic acetogens. In: Proceedings of the seventh International Symposium on Anaerobic Digestion: Oral Paper Preprints, Cape Town, South Africa, pp. 54e57. van Lier, J.B., Brummeler, E., Lettinga, G., 1993. Thermo-tolerant anaerobic degradation of volatile fatty acids by digested organic fraction of municipal solid waste. Journal of Fermentation and Bioengineering 76 (2), 140e144. van Lier, J.B., Martin, J.L.S., Lettinga, G., 1996. Effect of temperature on the anaerobic thermophilic conversion of volatile fatty acids by dispersed and granular sludge. Water Research 30 (1), 199e207. McHugh, S., Collins, C., O’Flaherty, V., 2005. Long-term, high-rate anaerobic biological treatment of whey wastewaters at psychrophilic temperatures. Bioresource Technology 97 (14), 1669e1678. Mechichi, T., Patel, B.K.C., Sayadi, S., 2005. Anaerobic degradation of methoxylated aromatic compounds by Clostridium methoxybenzovorans and a nitrate-reducing bacterium Thauera sp. strain Cin3,4. International Biodeterioration and Biodegradation 56 (4), 224e230. O’Flaherty, V. 1997. Microbial Interactions during Anaerobic Treatment of Sulphate-containing Wastewaters. PhD thesis. National University of Ireland, Galway. O’Flaherty, V., Colohan, S., Mulkerrins, D., Colleran, E., 1999. Effect of sulphate addition on volatile fatty acid and ethanol degradation in an anaerobic hybrid reactor. II: microbial interactions and toxic effects. Bioresource Technology 68 (2), 109e120. Schnurer, A., Schink, B., Svensson, B.H., 1996. Clostridium ultunense sp. nov., a mesophilic bacterium oxidizing acetate in syntrophic association with a hydrogenotrophic methanogenic bacterium. International Journal of Systematic and Evolutionary Microbiology 46, 1145e1152. Schnu¨rer, A., Svensson, B.H., Schink, B., 1997. Enzyme activities in and energetics of acetate metabolism by the mesophilic syntrophically acetate oxidizing anaerobe Clostridium ultunense. FEMS Microbiology Letters 154 (2), 331e336. Schnu¨rer, A., Zellner, G., Svensson, B.H., 1999. Mesophilic syntrophic acetate oxidation during methane formation in biogas reactors. FEMS Microbiology Ecology 29 (3), 249e261. Shelton, D.R., Tiedje, J.M., 1984. General method for determining anaerobic biodegradation potential. Applied and Environmental Microbiology 47 (4), 850e857. Stams, A.J.M., Plugge, C.M., 2009. Electron transfer in syntrophic communities of anaerobic bacteria and archaea. Nature Reviews, Microbiology 7 (8), 568e577. Thiele, J.H., Zeikus, J.G., 1988. Control of interspecies electron flow during anaerobic digestion: significance of formate transfer versus hydrogen transfer during syntrophic methanogenesis in flocs. Applied and Environmental Microbiology 54 (1), 20e29. Tholen, A., Brune, A., 1999. Localization and in-situ activities of homoacetogenic bacteria in the highly compartmentalized hindgut of soil-feeding higher termites
w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 4 2 6 1 e4 2 6 9
(Cubitermes spp.). Applied and Environmental Microbiology 65 (10), 4497e4505. Voolapalli, R.K., Stuckey, D.C., 2001. Hydrogen production in anaerobic reactors during shock loads e Influence of formate production and H2 kinetics. Water Research 35 (7), 1831e1841. Wang, J., Zhang, Z., Zhang, Z., Zheng, P., Li, C., 2007. The influence of anaerobic sludge retention time on anaerobic co-digestion of dyeing and printing wastewater and sewage sludge. African Journal of Biotechnology 6 (7), 902e907.
4269
Xu, K., Liu, H., Du, G., Chen, J., 2009. Real-time PCR assay targeting formyltetrahydrofolate synthetase gene to enumerate acetogens in natural and engineered environments. Anaerobe 15 (5), 204e213. de Zeeuw, W. 1984. Acclimatisation of anaerobic sludge for UASB reactor start-up. Ph.D. thesis. Agricultural University, Wageningen, The Netherlands. Zhang, T.C., Noike, T., 1994. Influence of retention time on reactor performance and bacterial trophic populations in anaerobic digestion processes. Water Research 28 (1), 27e36.