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Effect of molybdate on methanogenic and sulfidogenic activity of biomass
Bioresource Technology 96 (2005) 1215–1222
Effect of molybdate on methanogenic and sulfidogenic activity of biomass S.K. Patidar a, Vinod Tare
b,*
a
b
Department of Civil Engineering, National Institute of Technology, Kurukshetra 136 119, India Environmental Engineering and Management Program, Department of Civil Engineering, Indian Institute of Technology, Kanpur 208 016, India Received in revised form 4 June 2004; accepted 5 November 2004 Available online 19 December 2004
Abstract The effect of molybdate, a sulfate analog, on the total methanogenic activity (TMA) and total sulfidogenic activity (TSA) of biomass metabolizing synthetic sucrose based substrate containing sulfate was investigated in batch assays. In Phase I of the study, TMA and TSA were assessed twice for four feed changes at a chemical oxygen demand to sulfate ðCOD=SO2 4 Þ ratio of 3.5. In Phase II, long-term experiments were conducted for 10–13 feed changes with varying chemical oxygen demand (COD) concentration, sulfate concentration, COD=SO2 4 ratio, molybdate dose and biomass with different growth histories. Assays with 3 mM molybdate showed TSA inhibition over 85%. Dose dependency was observed for sulfate concentration, COD=SO2 4 ratio, and biomass history. The minimum concentration that gave over 93% TSA inhibition was 0.25 mM. However, intermediate concentrations of molybdate inhibited methane producing bacteria (MPB) activity. TMA stimulation was observed at 0.75–2.0 mM molybdate. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Total methanogenic activity; Total sulfidogenic activity; Molybdate; Biomass; Methane producing bacteria; Sulfate reducing bacteria; Inhibition
1. Introduction Anaerobic degradation of sulfate laden organics involves competitive interaction between different microorganisms, including fermenters, obligate hydrogen producing acetogens, homoacetogens, methanogens and sulfate reducers. The terminal degradation reactions involve methanogens and sulfate reducers, which compete for the common substrates hydrogen and acetate. Success of high rate anaerobic treatment systems relies on means to achieve development and maintenance of well settleable granular and/or attached biomass with optimum microbial activity of each group of bacteria. Attempts have been made to understand and develop technological *
Corresponding author. Tel.: +91 512 2597792; fax: +91 512 2590260/2597395/2590007. E-mail address: [email protected] (V. Tare). 0960-8524/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2004.11.001
tools to steer competition between MPB and SRB in the desired direction to achieve the intended objectives of the anaerobic treatment process. A number of parameters such as substrate concentration and composition, sulfate concentration, COD=SO2 4 ratio, temperature, pH, sulfide toxicity, availability of nutrients, reactor system configuration, organic loading, hydraulic loading, velocity, immobilizing properties of bacteria, nature of biomass, type of seed, acclimation period and reactor operation, and presence of inhibitors affect the delicate system balance involving various groups of bacteria including MPB and SRB (Lens et al., 1998; Srinivasan and Viraraghavan, 1998). The generation of hydrogen sulfide affects biomass activity due to toxicity and precipitation of key essential metals. SRB are favored by thermodynamic and kinetic factors compared to methanogens (Colleran et al., 1995). However, sulfide toxicity, to both methanogens and sulfate reducers, inhibits microbial
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activity and affects substrate utilization. Studies involving use of inhibitors to suppress activity of SRB and promote growth of MPB with higher substrate utilization rates have been reported in literature. Molybdate, chromate, tungstate and selenate are analogs of the sulfate ion which inhibit sulfate reduction with the approximate order of 2 2 2 effectiveness CrO2 4 > MoO4 ¼ WO4 > SeO4 (Taylor and Oremland, 1979). Molybdate enters cells via a sulfate transport system and interferes with the formation of adenosine phosphosulfate (APS), leading to deprivation of reduced sulfur compounds for growth. It forms adenosine phospho-molybdate in the cell. Chromate toxicity is attributed to competition with the sulfate transport system. Chromate is toxic to most bacteria including MPB. Selenate enters cells via the sulfate transport system and forms adenosine phospho-selenate (Barton, 1992). A number of studies have demonstrated use of molybdate for sulfate reduction inhibition at different doses: 0.2–200 mM (Smith and Klug, 1981), 10 mM (Hilton and Archer, 1988), 2 mM (Lo and Liao, 1990), and 3.2 mM (Yadav and Archer, 1988). Some studies have indicated inhibition of MPB along with SRB at levels of 2–20 mM (Hilton and Archer, 1988; Lo and Liao, 1990; Yadav and Archer, 1988, 1989; Karhadkar et al., 1987). Continuous addition of molybdate at 3 mM or more from start-up was found more effective for inhibiting sulfate reduction and increasing methane production (Tanaka and Lee, 1997). Studies have reported SRB adaptation to molybdate and consequently higher dose requirement with time (Yadav and Archer, 1988, 1989). Clancy et al. (1992) have reported a non-specific effect of molybdate at low phosphate concentration. These studies demonstrated successful inhibition of sulfate reduction, but the dose applied varied over a very wide range of concentrations ranging from 0.2 to 200 mM. Various issues related to molybdate supplementation such as optimum dose, role of phosphate and non-specific effects of molybdate, etc., need to be interpreted better and are yet to be fully elucidated. In view of this, the present study was undertaken to investigate the effect of molybdate supplementation on the methanogenic and sulfidogenic activities of biomass metabolizing substrates containing sulfate.
2. Methods 2.1. Biomass The effects of molybdate were assessed in terms of the total methanogenic activity (TMA) and total sulfidogenic activity (TSA) of SRB deficient biomass collected from an upflow anaerobic sludge blanket (UASB) plant treating domestic wastewater at Kanpur, India and SRB enriched biomass from a laboratory scale UASB reactor treating sulfate-containing substrate.
2.2. Substrate Synthetic substrate was prepared by dissolving jaggery as a carbon source (a locally available form of sucrose, also known as gur or Indian sugar) and sodium sulfate in tape water. 2.3. Operating conditions The study was performed in Phase I and Phase II for 4 and 10–13 feed changes, respectively. In Phase I of the study, a 3 mM molybdate dose was tested twice to observe the effects of molybdate supplementation on the TMA and TSA of biomass. This was done to assess the adequacy of the applied dose to inhibit the TSA of biomass. Finally, in Phase II, a long duration assay study involving supplementation with varying molybdate doses for varying COD, COD=SO2 4 ratio, sulfate concentration and biomass with different histories of development was carried out. An overview of the batch assay study is presented in Table 1. 2.4. Batch assay procedure For performing the batch assay study, the methanogenic activity test suggested by Isa et al. (1993) was modified suitably in terms of change in substrate composition and measurement of sulfate as well as sulfide for each feed change to calculate both TMA and TSA of biomass. The biomass used for the study was well settleable and all liquor was decanted off before replenishing with fresh feed at each feeding. About 50–60 ml biomass was added to 500 ml serum bottles to get a volatile suspended solids (VSS) concentration in the range 1–2 g l1. Sufficient quantities of jaggery (1.0 g jaggery 1.015 g COD; 1–2 g jaggery) and Na2SO4 (0.43–1.905 g) were added to get initial COD concentration in the range 2000–4000 mg l1 and COD=SO2 ratio 0.66–3.5. A sufficient quantity of 4 NaHCO3 (1–2 g) was added to buffer the systems. The required quantity of sodium molybdate (0.12 g Na2MoO4 Æ 2H2O for 1.0 mM and equivalent for other doses) was added to obtain the desired molybdate dose. Finally, tap water (purged of oxygen with nitrogen gas) was added up to the 500 ml mark. The serum bottles were properly capped and connected to a liquid displacement system for recording methane production with time. A schematic view of the experimental setup is presented in Fig. 1. After 48 h, part of the supernatant from each serum bottle was transferred to a 125 ml glass bottle containing sufficient volumes of 6 N NaOH and zinc acetate solution for precipitating sulfide. The bottle was immediately sealed with no air under the stopper and sulfide concentration was estimated using the iodometric method as per Standard
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Table 1 Overview of batch assays study Phase
Set no.
No. of feeds
Assay details
I
I
4
1 2 Substrate: COD 2000 mg l1, SO2 4 571.43 mg l , COD=SO4 ratio 3.5 Molybdate dose: 3 mM Biomass: SRB deficient biomass No. of assays: control + assay with molybdate
II
I
11
1 2 Substrate: COD 2000 mg l1, SO2 4 571.43 mg l , COD=SO4 ratio 3.5 Biomass: SRB deficient biomass No. of assays: Control + 4 assays of 1.0 mM, 2.0 mM, 3.0 mM and 4.0 mM molybdate
II
11
1 2 Substrate: COD 4000 mg l1, SO2 4 1142.86 mg l , COD=SO4 ratio 3.5 Biomass: SRB enriched biomass No. of assays: assays with molybdate dose of 0.1, 0.25, 0.5, 0.75, 1.0 and 2.0 mM
III
11
1 2 Substrate: COD 4000 mg l1, SO2 4 1142.86 mg l , COD=SO4 ratio 3.5 Biomass: SRB deficient biomass No. of assays: control + assays with molybdate dose of 0.05, 0.075, 0.1, 0.25, 0.5 mM
IV
13
1 2 Substrate: COD 1700 mg l1, SO2 4 2575.75 mg l , COD=SO4 ratio 0.66 Biomass: SRB deficient biomass No. of assays: assays with molybdate dose of 0.25, 0.5, 0.75, 1.0, 2.0 mM
Rubber Tubing
Hypodermic Needle
Pinch Cock
Liquid Displacement System
Conical Funnel Reaction Mixture
Serum Bottle
Fig. 1. Schematic of batch assays set-up.
Methods (1998). The remaining portion of supernatant was decanted carefully to prevent loss of biomass and used for sulfate estimation after filtering through a micro glass fiber filter (grade GF/C). This constituted the first feeding. Likewise, the procedure was repeated for the required number of feedings (4 feed changes in Phase I and 11–13 feed changes in Phase II). The entire study was conducted at 35 ± 1 °C in a controlled temperature cabin. After completion of the test, the amount of VSS in each serum bottle was estimated by transferring the biomass to a silica crucible. 2.5. Analysis Volatile suspended solids, sulfate and sulfide concentrations were estimated according to Standard Methods (1998).
2.6. Data analysis and interpretation of results The maximum slope of the cumulative gas production curve yields total methanogenic activity of the biomass as gCH4–COD gVSS1 d1. Total sulfidogenic activity was calculated on the basis of sulfate reduced and dissolved sulfide produced and expressed as 1 1 gSO2 d . 4 -reduced gVSS TMA and TSA of the biomass in last feed were considered to assess the effect of molybdate supplementation in Phase I of the study. In Phase II, the first 4 feeds were considered as acclimation phase and data were omitted. TMA and TSA values were plotted with number of feeds to observe changes with time. Various least square parameters were estimated for statistical analysis of data. Null hypothesis was tested for significance of slope of each combination using t-test.
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In this phase of the study, an assay with pre-decided molybdate concentration of 3 mM was tested twice. Average TMA and TSA were calculated in terms of 1 gCH4–COD gVSS1 d1 and gSO2 4 -reduced gVSS 1 d , respectively. TMA in assays with molybdate supplementation was 0.77 gCH4–COD gVSS1 d1 relative to control TMA of 0.43 gCH4–COD gVSS1 d1. Assays with molybdate supplementation showed TSA 1 1 0.02 gSO2 d relative to control 4 -reduced gVSS 2 TSA of 0.14 gSO4 -reduced gVSS1 d1. Molybdate supplementation resulted in increased TMA relative to control. It showed no inhibition of MPB at 3 mM dose, rather it indicated stimulation of TMA. TSA in assays with molybdate was lowered relative to control. It showed over 85% inhibition in SRB activity due to molybdate. The results of this phase show that 3 mM molybdate inhibited SRB activity and enhanced MPB activity. 3.2. Phase II: assays with increased number of feeds under varying conditions 3.2.1. Set-I: effect of varying molybdate dose on methanogenic and sulfidogenic activity in SRB deficient biomass TMA and TSA variation with number of feed changes at varying doses of molybdate are presented in Fig. 2a and b and on the basis of these the following observations could be made. Effect on TMA: All assays showed no variation in TMA with number of feeds (Fig. 2a). Significantly higher TMA was observed at dosages of 1.0 and 2.0 mM relative to control but it decreased insignificantly with number of feeds. Doses of 3.0 mM and 4.0 mM did not show a significant effect on TMA. Effect on TSA: Variation in TSA of control and assay with 1.0 mM dose was negligible (Fig. 2b). TSA of all assays with molybdate supplementation was significantly lower than the control showing significant inhibition to SRB at all doses applied. No significant variation in TSA with number of feeds was observed except in the assay with 1.0 mM molybdate. Overall, molybdate supplementation at all doses applied caused almost complete SRB inhibition (>96%). There was also significant stimulation of MPB activity at 1.0 and 2.0 mM molybdate. Molybdate doses up to 4.0 mM did not produce any inhibition in MPB activity,
(a)
0.5 gCH4-COD gVSS-1 d-1
3.1. Phase I: assays with 4 feed changes
Total Methanogenic Activity
3. Results and discussion
0.6
0.4
0.3
0.2
0.1
(b)
0.14 -1 -1 2gSO4 -reduced gVSS d
Significance of slope and intercept was tested for each combination relative to control to which no molybdate was added.
Total Sulfidogenic Activity
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0.12 0.10 0.08 0.06 0.04 0.02 0.00 1
2
3
4
5
6
7
8
Feed Control 1.0 mM 2.0 mM
3.0 mM 4.0 mM
Fig. 2. Total methanogenic and sulfidogenic activity in Phase II, Set I.
but doses of 1.0 and 2.0 mM were adequate for substantial SRB activity without any effect on MPB activity. 3.2.2. Set-II: effect of varying molybdate dose on methanogenic and sulfidogenic activity in SRB enriched biomass TMA and TSA variations with number of feed changes at varying doses of molybdate are presented in Fig. 3a and b. The assay with the lowest dose of 0.1 mM was considered as control for interpretation of results as many studies have reported inhibition of sulfate reduction at doses 0.5 mM and higher. Effect on TMA: TMA in all assays did not show any variation with number of feeds (Fig. 3a). TMA in assays with doses of 0.75 mM and more were significantly higher relative to control without significant variation with number of feeds. Effect on TSA: TSA of all assays except the assay with 0.5 mM molybdate showed variation with number of feeds (Fig. 3b). TSA in all assays were significantly lower relative to control and decreased significantly with number of feeds relative to control. In the present set of assay experiments, SRB inhibition greater than 90% was observed at molybdate doses of 0.5 mM and higher with significant stimulation of MPB at doses of 0.75 mM and higher. However, assays
S.K. Patidar, V. Tare / Bioresource Technology 96 (2005) 1215–1222 0.7
(a) Total Methanogenic Activity gCH -COD gVSS-1 d-1
0.9 0.8 0.7 0.6 0.5 0.4
0.5
0.4
0.3
0.12
(b) Total Sulfidogenic Activity -1 -1 gSO24 -reduced gVSS d
(b)
0.15
0.10
4
Total Sulfidogenic Activity -1 gSO2--reduced gVSS-1 d
0.6
0.2
0.20
0.05
0.00 1
(a)
4
Total Methanogenic Activity -1 gCH4-COD gVSS-1 d
1.0
1219
2
3
4
5
6
7
8
Feed 0.10 mM 0.25 mM 0.50 mM
0.10 0.08 0.06 0.04 0.02 0.00 1
2
3
4
5
6
7
8
Feed
0.75 mM 1.00 mM 2.00 mM
Fig. 3. Total methanogenic and sulfidogenic activity in Phase-II, Set II.
with lower doses of 0.25 and 0.5 mM showed inhibition of MPB activity. It appears that sulfide toxicity and/or molybdate may have had some role in inhibiting MPB activity when the dose applied was insufficient to inhibit SRB activity substantially. 3.2.3. Set-III: effect of low molybdate dose on methanogenic and sulfidogenic activity in SRB deficient biomass In the previous set, significant SRB inhibition was observed at molybdate doses as low as 0.5 mM for SRB enriched biomass. In the present set, the effect of molybdate at dosages 0.5 mM and lower was assessed for SRB deficient biomass to ascertain dose dependency on nature of biomass. TMA and TSA variations with number of feeds were as presented in Fig. 4a and b. Effect on TMA: TMA in all assays showed no variation with number of feeds except for the assay with 0.5 mM molybdate (Fig. 4a). TMA in assays with 0.05 mM and 0.075 mM molybdate declined significantly with number of feeds and indicated MPB inhibition after the second and first feeds, respectively. TMA in other assays with higher molybdate doses of 0.1 and
Control 0.050 mM 0.075 mM
0.100 mM 0.250 mM 0.500 mM
Fig. 4. Total methanogenic and sulfidogenic activity in Phase II, Set III.
0.5 mM was significantly lower than the control and decreased significantly with number of feeds. Effect on TSA: TSA variation in all assays was observed with number of feeds except for the assay with 0.25 mM molybdate (Fig. 4b). TSA in all assays were significantly lower than the control, indicating significant inhibition of SRB. TSA decreased significantly with number of feeds in all assays except in the assay with 0.05 mM molybdate. TSA activity of SRB deficient biomass in the present set was inhibited by over 90% at a lower dose of 0.25 mM as compared to the 0.5 mM dose requirement in the previous set. It showed dose dependency on the nature of biomass. Results of the present set of assays indicated increase in inhibition of MPB with increase in molybdate dose. Possibly sulfide toxicity might have been responsible for MPB inhibition at lower concentrations of molybdate due to substantial sulfate reduction. However, higher MPB inhibition was observed at dosages of 0.25 and 0.5 mM, which inhibited SRB activity more than 90%. At these dosages only molybdate could have had a role in inhibiting MPB activity, as sulfide concentration was too insignificant to cause any inhibition. The
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results of previous set also showed a similar phenomenon, when some intermediate range of molybdate dose inhibited more than 90% SRB activity with some inhibition of MPB activity. However, at sufficiently higher dosages stimulation of TMA was observed. Further investigations are warranted to elucidate the effect of molybdate on MPB activity. 3.2.4. Set-IV: effect of molybdate dose on methanogenic and sulfidogenic activity in SRB deficient biomass at lower COD=SO2 4 ratio TMA and TSA variations with number of feeds for substrate with low COD=SO2 4 of 0.66 are presented in Fig. 5a and b. In this set the significance of slope and intercept was tested relative to the assay with the lowest dose of 0.25 mM. Effect on TMA: TMA of all assays showed variation with number of feeds (Fig. 5a). TMA in assays with 0.5 and 0.75 mM molybdate were significantly lower than in the assay with 0.25 mM indicating significant inhibition of MPB with increase in molybdate dose. Inhibition due to 0.75 mM molybdate increased significantly with number of feeds. TMA in the assay with 1.0 mM molybdate
4
Total Methanogenic Activity gCH -COD gVSS-1 d-1
0.35
(a)
0.30
4. Overview of results and discussion
0.25
(a) Effect of molybdate on different biomass: Supplementation of varying molybdate doses with SRB enriched biomass (Set II) and SRB deficient biomass (Set III) for the same sulfate concentration and COD=SO2 4 ratio indicated that doses of 0.75 mM and higher significantly stimulated TMA, whereas increased inhibition with increase in dose from 0.05 to 0.5 mM was observed for SRB deficient biomass (Table 2). The formation of MoO2 S2 and MoS2 complexes is toxic to methano2 4 gens. In SRB enriched biomass, due to higher molybdate uptake, complex formation might have been lower with less toxicity compared to SRB deficient biomass. More than 90% inhibition of SRB was observed at molybdate doses of 0.5 mM or higher in SRB enriched biomass, whereas similar inhibition was observed at the relatively lower dose of 0.25 mM in SRB deficient biomass. It clearly shows molybdate dose dependency on the nature of biomass and its history of evolution. (b) Effect of molybdate at varying sulfate concentrations: SRB inhibition over 90% was observed at the relatively lower dose of 0.25 mM at sulfate concentration of 1142.86 mg l1 (Set III) compared to the dose requirement of 2 mM at sulfate concentration of 2575.75 mg l1 (Set IV) for SRB deficient biomass (Table 2). The higher molybdate requirement at higher sulfate concentration in substrate may have been due to competitive inhibition by molybdate.
0.20 0.15 0.10 0.05 0.04
(b)
0.03
0.02
4
Total Sulfidogenic Activity gSO 2- -reduced gVSS-1 d-1
was significantly higher in the beginning but declined significantly, so that the value was lower than that for the assay with 0.25 mM after the fifth feed. TMA in the assay with 2.0 mM molybdate showed insignificant stimulation in the beginning with significant increase with number of feeds. Effect on TSA: TSA in all assays showed variation with number of feeds except for the assay with 1.0 mM molybdate (Fig. 5b). Increase in dose resulted in a decrease in TSA. TSA in the assays with 0.25 and 0.5 mM molybdate followed a similar trend. TSA in assays with dosages of 0.75, 1.0 and 2.0 mM showed significantly higher values relative to the 0.25 mM dose in the beginning but declined significantly with increase in number of feeds. Molybdate (0.75 and 1.0 mM) produced about 42% inhibition in the last feed, whereas 2.0 mM resulted in more than 90% inhibition relative to the lowest dose of 0.25 mM in the last feed. The higher dose requirements of 2.0 mM in the present set for substantial SRB inhibition indicated dose dependency on sulfate concentration and COD=SO2 4 ratio in the substrate. Increased MPB inhibition was observed as molybdate dose increased from 0.25 to 1.0 mM as observed in the previous sets. However, stimulation was observed at 2.0 mM molybdate.
0.01
0.00 2
4
6
8
10
Feed 0.25 mM 0.50 mM 0.75 mM
1.00 mM 2.00 mM
Fig. 5. Total methanogenic and sulfidogenic activity in Phase II, Set IV.
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Table 2 Comparative assessment of effect of molybdate for varying conditions Set II SRB enriched biomass, SO2 4 concentration 1142.86 mg l1, COD=SO2 4 ratio 3.5 SRB enriched vs SRB deficient biomass Molybdate dose (mM) 0.25 TMA M TSA #
0.5 M #
0.75 " #
1.0 " #
2.0 " #
Set III SRB deficient biomass, SO2 4 concentration 1142.86 mg l1, COD=SO2 4 ratio 3.5 Effect of molybdate for varying Molybdate dose (mM) TMA TSA
feed sulfate 0.05 M #
concentration 0.075 0.1 M # # #
0.25 # #
0.5 # #
Set II SRB enriched biomass, COD concentration 2000 mg l1, COD=SO2 4 ratio 3.5 Effect of molybdate for varying Molybdate dose (mM) TMA TSA
COD=SO2 4 0.25 M #
ratio 0.5 M #
0.75 " #
1.0 " #
2.0 " #
Set III SRB deficient biomass, SO2 4 concentration 1142.86 mg l1, COD=SO2 4 ratio 3.5 0.05 M #
0.075 M #
0.1 # #
0.25 # #
0.5 # #
Set IV SRB deficient biomass, SO2 4 concentration 2575.75 mg l1, COD=SO2 4 ratio 0.66 0.5 # M
0.75 # #
1.0 M #
2.0 M #
Set IV SRB deficient biomass, COD concentration 1700 mg l1, COD=SO2 4 ratio 0.66 0.5 # M
0.75 # #
1.0 M #
2.0 M #
": Significant increase relative to control/minimum dose; #: significant decrease relative to control/minimum dose; M: variable trend/insignificant increase/insignificant decrease relative to control/minimum dose.
For the same doses of 0.25 mM and 0.5 mM, higher TMA inhibition was observed at higher sulfate concentration. This may have been due to higher direct sulfide toxicity or higher concentrations of molybdate sulfide complexes. (c) Effect of molybdate at varying COD=SO2 4 ratio: A higher molybdate dose of 2 mM was required to inhibit TSA by over 90% at the lower COD=SO2 4 ratio of 0.66 (Set IV) compared to the dose requirement of 0.5 mM at COD=SO2 4 ratio of 3.5 (Set II) for substrate COD 1700–2000 mg l1 (Table 2). The above discussion clearly indicates dose dependency of molybdate on history of biomass evolution, sulfate concentration and COD=SO2 4 ratio in substrate.
5. Conclusions (1) Molybdate supplementation inhibited SRB activity considerably (>85%) at 3 mM. However, the lowest dose that inhibited more than 93% SRB activity was 0.25 mM for SRB deficient biomass. (2) Molybdate dose required for inhibition of SRB appears to depend upon SO2 concentration, 4 COD=SO2 4 ratio and history of biomass. (3) The effect of molybdate supplementation on MPB activity needs to be further investigated as increase in dose up to some intermediate concentration indicated inhibition and thereafter further increase caused stimulation of MPB activity.
References Barton, L.L., 1992. Sulfur metabolism. In: Lederberg, J. (Ed.), Encyclopedia of Microbiology, Vol. 4. Academic, New York, pp. 135–150. Clancy, P.B., Venkataraman, N., Lynd, L.R., 1992. Biochemical inhibition of sulfate reduction in batch and continuous anaerobic digesters. Water Sci. Technol. 25, 51–60. Colleran, E., Finnergan, S., Lens, P., 1995. Anaerobic treatment of sulfate containing waste streams. Antonie Van Leeuwenhoek 67, 29–46. Hilton, M.G., Archer, D.B., 1988. Anaerobic digestion of a sulfaterich molasses wastewater: inhibition of hydrogen sulfide production. Biotechnol. Bioeng. 31, 885–888. Isa, M.H., Farooqi, I.H., Siddiqi, R.H., 1993. Methanogenic activity test for study of anaerobic processes. Indian J. Environ. Health 35, 1–8. Karhadkar, P.P., Audice, J.-M., Faup, G.M., Khanna, P., 1987. Sulfide and sulfate inhibition of methanogenesis. Water Res. 21, 1061–1066. Lens, P.N.L., Visser, A., Janssen, A.J.H., Hulshoff Pol, L.W., Lettinga, G., 1998. Biotechnological treatment of sulfate rich wastewaters. Crit. Rev. Environ. Sci. Technol. 28, 41–88. Lo, K.V., Liao, P.H., 1990. Anaerobic treatment of bakers yeast wastewater. I. Start-up and sodium molybdate addition. Biomass 21, 207–218. Smith, R.L., Klug, M.J., 1981. Electron donors utilized by sulfatereducing bacteria in eutrophic lake sediments. Appl. Environ. Microbiol. 42, 116–121. Srinivasan, P.T., Viraraghavan, T., 1998. Anaerobic treatment of high sulfate content industrial wastewaters: a review. J. Indian Assoc. Environ. Manage. 25, 1–9. Standard Methods, 1998. In: Greenberg, A.E., Clesceri, L.S., Eaton, A.D. (Eds.), Standard Methods for Examination of Water and Wastewater, 20th ed. APHA, Washington, DC. Tanaka, S., Lee, Y.H., 1997. Control of sulfate reduction by molybdate in anaerobic digestion. Water Sci. Technol. 36 (12), 143–150.
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Taylor, B.F., Oremland, R.S., 1979. Depletion of adenosine triphosphate in Desulfovibrio by oxyanions of group VI elements. Curr. Microbiol. 3, 101–103. Yadav, V.K., Archer, D.B., 1988. Specific inhibition of sulfatereducing bacteria in methanogenic co-culture. Lett. Appl. Microbiol. 7 (6), 165–168.
Yadav, V.K., Archer, D.B., 1989. Sodium molybdate inhibits sulfate reduction in the anaerobic treatment of high sulfate molasses wastewater. Appl. Microbiol. Biotechnol. 31, 103–106.
Effect of molybdate on methanogenic and sulfidogenic activity of biomass S.K. Patidar a, Vinod Tare
b,*
a
b
Department of Civil Engineering, National Institute of Technology, Kurukshetra 136 119, India Environmental Engineering and Management Program, Department of Civil Engineering, Indian Institute of Technology, Kanpur 208 016, India Received in revised form 4 June 2004; accepted 5 November 2004 Available online 19 December 2004
Abstract The effect of molybdate, a sulfate analog, on the total methanogenic activity (TMA) and total sulfidogenic activity (TSA) of biomass metabolizing synthetic sucrose based substrate containing sulfate was investigated in batch assays. In Phase I of the study, TMA and TSA were assessed twice for four feed changes at a chemical oxygen demand to sulfate ðCOD=SO2 4 Þ ratio of 3.5. In Phase II, long-term experiments were conducted for 10–13 feed changes with varying chemical oxygen demand (COD) concentration, sulfate concentration, COD=SO2 4 ratio, molybdate dose and biomass with different growth histories. Assays with 3 mM molybdate showed TSA inhibition over 85%. Dose dependency was observed for sulfate concentration, COD=SO2 4 ratio, and biomass history. The minimum concentration that gave over 93% TSA inhibition was 0.25 mM. However, intermediate concentrations of molybdate inhibited methane producing bacteria (MPB) activity. TMA stimulation was observed at 0.75–2.0 mM molybdate. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Total methanogenic activity; Total sulfidogenic activity; Molybdate; Biomass; Methane producing bacteria; Sulfate reducing bacteria; Inhibition
1. Introduction Anaerobic degradation of sulfate laden organics involves competitive interaction between different microorganisms, including fermenters, obligate hydrogen producing acetogens, homoacetogens, methanogens and sulfate reducers. The terminal degradation reactions involve methanogens and sulfate reducers, which compete for the common substrates hydrogen and acetate. Success of high rate anaerobic treatment systems relies on means to achieve development and maintenance of well settleable granular and/or attached biomass with optimum microbial activity of each group of bacteria. Attempts have been made to understand and develop technological *
Corresponding author. Tel.: +91 512 2597792; fax: +91 512 2590260/2597395/2590007. E-mail address: [email protected] (V. Tare). 0960-8524/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2004.11.001
tools to steer competition between MPB and SRB in the desired direction to achieve the intended objectives of the anaerobic treatment process. A number of parameters such as substrate concentration and composition, sulfate concentration, COD=SO2 4 ratio, temperature, pH, sulfide toxicity, availability of nutrients, reactor system configuration, organic loading, hydraulic loading, velocity, immobilizing properties of bacteria, nature of biomass, type of seed, acclimation period and reactor operation, and presence of inhibitors affect the delicate system balance involving various groups of bacteria including MPB and SRB (Lens et al., 1998; Srinivasan and Viraraghavan, 1998). The generation of hydrogen sulfide affects biomass activity due to toxicity and precipitation of key essential metals. SRB are favored by thermodynamic and kinetic factors compared to methanogens (Colleran et al., 1995). However, sulfide toxicity, to both methanogens and sulfate reducers, inhibits microbial
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activity and affects substrate utilization. Studies involving use of inhibitors to suppress activity of SRB and promote growth of MPB with higher substrate utilization rates have been reported in literature. Molybdate, chromate, tungstate and selenate are analogs of the sulfate ion which inhibit sulfate reduction with the approximate order of 2 2 2 effectiveness CrO2 4 > MoO4 ¼ WO4 > SeO4 (Taylor and Oremland, 1979). Molybdate enters cells via a sulfate transport system and interferes with the formation of adenosine phosphosulfate (APS), leading to deprivation of reduced sulfur compounds for growth. It forms adenosine phospho-molybdate in the cell. Chromate toxicity is attributed to competition with the sulfate transport system. Chromate is toxic to most bacteria including MPB. Selenate enters cells via the sulfate transport system and forms adenosine phospho-selenate (Barton, 1992). A number of studies have demonstrated use of molybdate for sulfate reduction inhibition at different doses: 0.2–200 mM (Smith and Klug, 1981), 10 mM (Hilton and Archer, 1988), 2 mM (Lo and Liao, 1990), and 3.2 mM (Yadav and Archer, 1988). Some studies have indicated inhibition of MPB along with SRB at levels of 2–20 mM (Hilton and Archer, 1988; Lo and Liao, 1990; Yadav and Archer, 1988, 1989; Karhadkar et al., 1987). Continuous addition of molybdate at 3 mM or more from start-up was found more effective for inhibiting sulfate reduction and increasing methane production (Tanaka and Lee, 1997). Studies have reported SRB adaptation to molybdate and consequently higher dose requirement with time (Yadav and Archer, 1988, 1989). Clancy et al. (1992) have reported a non-specific effect of molybdate at low phosphate concentration. These studies demonstrated successful inhibition of sulfate reduction, but the dose applied varied over a very wide range of concentrations ranging from 0.2 to 200 mM. Various issues related to molybdate supplementation such as optimum dose, role of phosphate and non-specific effects of molybdate, etc., need to be interpreted better and are yet to be fully elucidated. In view of this, the present study was undertaken to investigate the effect of molybdate supplementation on the methanogenic and sulfidogenic activities of biomass metabolizing substrates containing sulfate.
2. Methods 2.1. Biomass The effects of molybdate were assessed in terms of the total methanogenic activity (TMA) and total sulfidogenic activity (TSA) of SRB deficient biomass collected from an upflow anaerobic sludge blanket (UASB) plant treating domestic wastewater at Kanpur, India and SRB enriched biomass from a laboratory scale UASB reactor treating sulfate-containing substrate.
2.2. Substrate Synthetic substrate was prepared by dissolving jaggery as a carbon source (a locally available form of sucrose, also known as gur or Indian sugar) and sodium sulfate in tape water. 2.3. Operating conditions The study was performed in Phase I and Phase II for 4 and 10–13 feed changes, respectively. In Phase I of the study, a 3 mM molybdate dose was tested twice to observe the effects of molybdate supplementation on the TMA and TSA of biomass. This was done to assess the adequacy of the applied dose to inhibit the TSA of biomass. Finally, in Phase II, a long duration assay study involving supplementation with varying molybdate doses for varying COD, COD=SO2 4 ratio, sulfate concentration and biomass with different histories of development was carried out. An overview of the batch assay study is presented in Table 1. 2.4. Batch assay procedure For performing the batch assay study, the methanogenic activity test suggested by Isa et al. (1993) was modified suitably in terms of change in substrate composition and measurement of sulfate as well as sulfide for each feed change to calculate both TMA and TSA of biomass. The biomass used for the study was well settleable and all liquor was decanted off before replenishing with fresh feed at each feeding. About 50–60 ml biomass was added to 500 ml serum bottles to get a volatile suspended solids (VSS) concentration in the range 1–2 g l1. Sufficient quantities of jaggery (1.0 g jaggery 1.015 g COD; 1–2 g jaggery) and Na2SO4 (0.43–1.905 g) were added to get initial COD concentration in the range 2000–4000 mg l1 and COD=SO2 ratio 0.66–3.5. A sufficient quantity of 4 NaHCO3 (1–2 g) was added to buffer the systems. The required quantity of sodium molybdate (0.12 g Na2MoO4 Æ 2H2O for 1.0 mM and equivalent for other doses) was added to obtain the desired molybdate dose. Finally, tap water (purged of oxygen with nitrogen gas) was added up to the 500 ml mark. The serum bottles were properly capped and connected to a liquid displacement system for recording methane production with time. A schematic view of the experimental setup is presented in Fig. 1. After 48 h, part of the supernatant from each serum bottle was transferred to a 125 ml glass bottle containing sufficient volumes of 6 N NaOH and zinc acetate solution for precipitating sulfide. The bottle was immediately sealed with no air under the stopper and sulfide concentration was estimated using the iodometric method as per Standard
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Table 1 Overview of batch assays study Phase
Set no.
No. of feeds
Assay details
I
I
4
1 2 Substrate: COD 2000 mg l1, SO2 4 571.43 mg l , COD=SO4 ratio 3.5 Molybdate dose: 3 mM Biomass: SRB deficient biomass No. of assays: control + assay with molybdate
II
I
11
1 2 Substrate: COD 2000 mg l1, SO2 4 571.43 mg l , COD=SO4 ratio 3.5 Biomass: SRB deficient biomass No. of assays: Control + 4 assays of 1.0 mM, 2.0 mM, 3.0 mM and 4.0 mM molybdate
II
11
1 2 Substrate: COD 4000 mg l1, SO2 4 1142.86 mg l , COD=SO4 ratio 3.5 Biomass: SRB enriched biomass No. of assays: assays with molybdate dose of 0.1, 0.25, 0.5, 0.75, 1.0 and 2.0 mM
III
11
1 2 Substrate: COD 4000 mg l1, SO2 4 1142.86 mg l , COD=SO4 ratio 3.5 Biomass: SRB deficient biomass No. of assays: control + assays with molybdate dose of 0.05, 0.075, 0.1, 0.25, 0.5 mM
IV
13
1 2 Substrate: COD 1700 mg l1, SO2 4 2575.75 mg l , COD=SO4 ratio 0.66 Biomass: SRB deficient biomass No. of assays: assays with molybdate dose of 0.25, 0.5, 0.75, 1.0, 2.0 mM
Rubber Tubing
Hypodermic Needle
Pinch Cock
Liquid Displacement System
Conical Funnel Reaction Mixture
Serum Bottle
Fig. 1. Schematic of batch assays set-up.
Methods (1998). The remaining portion of supernatant was decanted carefully to prevent loss of biomass and used for sulfate estimation after filtering through a micro glass fiber filter (grade GF/C). This constituted the first feeding. Likewise, the procedure was repeated for the required number of feedings (4 feed changes in Phase I and 11–13 feed changes in Phase II). The entire study was conducted at 35 ± 1 °C in a controlled temperature cabin. After completion of the test, the amount of VSS in each serum bottle was estimated by transferring the biomass to a silica crucible. 2.5. Analysis Volatile suspended solids, sulfate and sulfide concentrations were estimated according to Standard Methods (1998).
2.6. Data analysis and interpretation of results The maximum slope of the cumulative gas production curve yields total methanogenic activity of the biomass as gCH4–COD gVSS1 d1. Total sulfidogenic activity was calculated on the basis of sulfate reduced and dissolved sulfide produced and expressed as 1 1 gSO2 d . 4 -reduced gVSS TMA and TSA of the biomass in last feed were considered to assess the effect of molybdate supplementation in Phase I of the study. In Phase II, the first 4 feeds were considered as acclimation phase and data were omitted. TMA and TSA values were plotted with number of feeds to observe changes with time. Various least square parameters were estimated for statistical analysis of data. Null hypothesis was tested for significance of slope of each combination using t-test.
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In this phase of the study, an assay with pre-decided molybdate concentration of 3 mM was tested twice. Average TMA and TSA were calculated in terms of 1 gCH4–COD gVSS1 d1 and gSO2 4 -reduced gVSS 1 d , respectively. TMA in assays with molybdate supplementation was 0.77 gCH4–COD gVSS1 d1 relative to control TMA of 0.43 gCH4–COD gVSS1 d1. Assays with molybdate supplementation showed TSA 1 1 0.02 gSO2 d relative to control 4 -reduced gVSS 2 TSA of 0.14 gSO4 -reduced gVSS1 d1. Molybdate supplementation resulted in increased TMA relative to control. It showed no inhibition of MPB at 3 mM dose, rather it indicated stimulation of TMA. TSA in assays with molybdate was lowered relative to control. It showed over 85% inhibition in SRB activity due to molybdate. The results of this phase show that 3 mM molybdate inhibited SRB activity and enhanced MPB activity. 3.2. Phase II: assays with increased number of feeds under varying conditions 3.2.1. Set-I: effect of varying molybdate dose on methanogenic and sulfidogenic activity in SRB deficient biomass TMA and TSA variation with number of feed changes at varying doses of molybdate are presented in Fig. 2a and b and on the basis of these the following observations could be made. Effect on TMA: All assays showed no variation in TMA with number of feeds (Fig. 2a). Significantly higher TMA was observed at dosages of 1.0 and 2.0 mM relative to control but it decreased insignificantly with number of feeds. Doses of 3.0 mM and 4.0 mM did not show a significant effect on TMA. Effect on TSA: Variation in TSA of control and assay with 1.0 mM dose was negligible (Fig. 2b). TSA of all assays with molybdate supplementation was significantly lower than the control showing significant inhibition to SRB at all doses applied. No significant variation in TSA with number of feeds was observed except in the assay with 1.0 mM molybdate. Overall, molybdate supplementation at all doses applied caused almost complete SRB inhibition (>96%). There was also significant stimulation of MPB activity at 1.0 and 2.0 mM molybdate. Molybdate doses up to 4.0 mM did not produce any inhibition in MPB activity,
(a)
0.5 gCH4-COD gVSS-1 d-1
3.1. Phase I: assays with 4 feed changes
Total Methanogenic Activity
3. Results and discussion
0.6
0.4
0.3
0.2
0.1
(b)
0.14 -1 -1 2gSO4 -reduced gVSS d
Significance of slope and intercept was tested for each combination relative to control to which no molybdate was added.
Total Sulfidogenic Activity
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0.12 0.10 0.08 0.06 0.04 0.02 0.00 1
2
3
4
5
6
7
8
Feed Control 1.0 mM 2.0 mM
3.0 mM 4.0 mM
Fig. 2. Total methanogenic and sulfidogenic activity in Phase II, Set I.
but doses of 1.0 and 2.0 mM were adequate for substantial SRB activity without any effect on MPB activity. 3.2.2. Set-II: effect of varying molybdate dose on methanogenic and sulfidogenic activity in SRB enriched biomass TMA and TSA variations with number of feed changes at varying doses of molybdate are presented in Fig. 3a and b. The assay with the lowest dose of 0.1 mM was considered as control for interpretation of results as many studies have reported inhibition of sulfate reduction at doses 0.5 mM and higher. Effect on TMA: TMA in all assays did not show any variation with number of feeds (Fig. 3a). TMA in assays with doses of 0.75 mM and more were significantly higher relative to control without significant variation with number of feeds. Effect on TSA: TSA of all assays except the assay with 0.5 mM molybdate showed variation with number of feeds (Fig. 3b). TSA in all assays were significantly lower relative to control and decreased significantly with number of feeds relative to control. In the present set of assay experiments, SRB inhibition greater than 90% was observed at molybdate doses of 0.5 mM and higher with significant stimulation of MPB at doses of 0.75 mM and higher. However, assays
S.K. Patidar, V. Tare / Bioresource Technology 96 (2005) 1215–1222 0.7
(a) Total Methanogenic Activity gCH -COD gVSS-1 d-1
0.9 0.8 0.7 0.6 0.5 0.4
0.5
0.4
0.3
0.12
(b) Total Sulfidogenic Activity -1 -1 gSO24 -reduced gVSS d
(b)
0.15
0.10
4
Total Sulfidogenic Activity -1 gSO2--reduced gVSS-1 d
0.6
0.2
0.20
0.05
0.00 1
(a)
4
Total Methanogenic Activity -1 gCH4-COD gVSS-1 d
1.0
1219
2
3
4
5
6
7
8
Feed 0.10 mM 0.25 mM 0.50 mM
0.10 0.08 0.06 0.04 0.02 0.00 1
2
3
4
5
6
7
8
Feed
0.75 mM 1.00 mM 2.00 mM
Fig. 3. Total methanogenic and sulfidogenic activity in Phase-II, Set II.
with lower doses of 0.25 and 0.5 mM showed inhibition of MPB activity. It appears that sulfide toxicity and/or molybdate may have had some role in inhibiting MPB activity when the dose applied was insufficient to inhibit SRB activity substantially. 3.2.3. Set-III: effect of low molybdate dose on methanogenic and sulfidogenic activity in SRB deficient biomass In the previous set, significant SRB inhibition was observed at molybdate doses as low as 0.5 mM for SRB enriched biomass. In the present set, the effect of molybdate at dosages 0.5 mM and lower was assessed for SRB deficient biomass to ascertain dose dependency on nature of biomass. TMA and TSA variations with number of feeds were as presented in Fig. 4a and b. Effect on TMA: TMA in all assays showed no variation with number of feeds except for the assay with 0.5 mM molybdate (Fig. 4a). TMA in assays with 0.05 mM and 0.075 mM molybdate declined significantly with number of feeds and indicated MPB inhibition after the second and first feeds, respectively. TMA in other assays with higher molybdate doses of 0.1 and
Control 0.050 mM 0.075 mM
0.100 mM 0.250 mM 0.500 mM
Fig. 4. Total methanogenic and sulfidogenic activity in Phase II, Set III.
0.5 mM was significantly lower than the control and decreased significantly with number of feeds. Effect on TSA: TSA variation in all assays was observed with number of feeds except for the assay with 0.25 mM molybdate (Fig. 4b). TSA in all assays were significantly lower than the control, indicating significant inhibition of SRB. TSA decreased significantly with number of feeds in all assays except in the assay with 0.05 mM molybdate. TSA activity of SRB deficient biomass in the present set was inhibited by over 90% at a lower dose of 0.25 mM as compared to the 0.5 mM dose requirement in the previous set. It showed dose dependency on the nature of biomass. Results of the present set of assays indicated increase in inhibition of MPB with increase in molybdate dose. Possibly sulfide toxicity might have been responsible for MPB inhibition at lower concentrations of molybdate due to substantial sulfate reduction. However, higher MPB inhibition was observed at dosages of 0.25 and 0.5 mM, which inhibited SRB activity more than 90%. At these dosages only molybdate could have had a role in inhibiting MPB activity, as sulfide concentration was too insignificant to cause any inhibition. The
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results of previous set also showed a similar phenomenon, when some intermediate range of molybdate dose inhibited more than 90% SRB activity with some inhibition of MPB activity. However, at sufficiently higher dosages stimulation of TMA was observed. Further investigations are warranted to elucidate the effect of molybdate on MPB activity. 3.2.4. Set-IV: effect of molybdate dose on methanogenic and sulfidogenic activity in SRB deficient biomass at lower COD=SO2 4 ratio TMA and TSA variations with number of feeds for substrate with low COD=SO2 4 of 0.66 are presented in Fig. 5a and b. In this set the significance of slope and intercept was tested relative to the assay with the lowest dose of 0.25 mM. Effect on TMA: TMA of all assays showed variation with number of feeds (Fig. 5a). TMA in assays with 0.5 and 0.75 mM molybdate were significantly lower than in the assay with 0.25 mM indicating significant inhibition of MPB with increase in molybdate dose. Inhibition due to 0.75 mM molybdate increased significantly with number of feeds. TMA in the assay with 1.0 mM molybdate
4
Total Methanogenic Activity gCH -COD gVSS-1 d-1
0.35
(a)
0.30
4. Overview of results and discussion
0.25
(a) Effect of molybdate on different biomass: Supplementation of varying molybdate doses with SRB enriched biomass (Set II) and SRB deficient biomass (Set III) for the same sulfate concentration and COD=SO2 4 ratio indicated that doses of 0.75 mM and higher significantly stimulated TMA, whereas increased inhibition with increase in dose from 0.05 to 0.5 mM was observed for SRB deficient biomass (Table 2). The formation of MoO2 S2 and MoS2 complexes is toxic to methano2 4 gens. In SRB enriched biomass, due to higher molybdate uptake, complex formation might have been lower with less toxicity compared to SRB deficient biomass. More than 90% inhibition of SRB was observed at molybdate doses of 0.5 mM or higher in SRB enriched biomass, whereas similar inhibition was observed at the relatively lower dose of 0.25 mM in SRB deficient biomass. It clearly shows molybdate dose dependency on the nature of biomass and its history of evolution. (b) Effect of molybdate at varying sulfate concentrations: SRB inhibition over 90% was observed at the relatively lower dose of 0.25 mM at sulfate concentration of 1142.86 mg l1 (Set III) compared to the dose requirement of 2 mM at sulfate concentration of 2575.75 mg l1 (Set IV) for SRB deficient biomass (Table 2). The higher molybdate requirement at higher sulfate concentration in substrate may have been due to competitive inhibition by molybdate.
0.20 0.15 0.10 0.05 0.04
(b)
0.03
0.02
4
Total Sulfidogenic Activity gSO 2- -reduced gVSS-1 d-1
was significantly higher in the beginning but declined significantly, so that the value was lower than that for the assay with 0.25 mM after the fifth feed. TMA in the assay with 2.0 mM molybdate showed insignificant stimulation in the beginning with significant increase with number of feeds. Effect on TSA: TSA in all assays showed variation with number of feeds except for the assay with 1.0 mM molybdate (Fig. 5b). Increase in dose resulted in a decrease in TSA. TSA in the assays with 0.25 and 0.5 mM molybdate followed a similar trend. TSA in assays with dosages of 0.75, 1.0 and 2.0 mM showed significantly higher values relative to the 0.25 mM dose in the beginning but declined significantly with increase in number of feeds. Molybdate (0.75 and 1.0 mM) produced about 42% inhibition in the last feed, whereas 2.0 mM resulted in more than 90% inhibition relative to the lowest dose of 0.25 mM in the last feed. The higher dose requirements of 2.0 mM in the present set for substantial SRB inhibition indicated dose dependency on sulfate concentration and COD=SO2 4 ratio in the substrate. Increased MPB inhibition was observed as molybdate dose increased from 0.25 to 1.0 mM as observed in the previous sets. However, stimulation was observed at 2.0 mM molybdate.
0.01
0.00 2
4
6
8
10
Feed 0.25 mM 0.50 mM 0.75 mM
1.00 mM 2.00 mM
Fig. 5. Total methanogenic and sulfidogenic activity in Phase II, Set IV.
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Table 2 Comparative assessment of effect of molybdate for varying conditions Set II SRB enriched biomass, SO2 4 concentration 1142.86 mg l1, COD=SO2 4 ratio 3.5 SRB enriched vs SRB deficient biomass Molybdate dose (mM) 0.25 TMA M TSA #
0.5 M #
0.75 " #
1.0 " #
2.0 " #
Set III SRB deficient biomass, SO2 4 concentration 1142.86 mg l1, COD=SO2 4 ratio 3.5 Effect of molybdate for varying Molybdate dose (mM) TMA TSA
feed sulfate 0.05 M #
concentration 0.075 0.1 M # # #
0.25 # #
0.5 # #
Set II SRB enriched biomass, COD concentration 2000 mg l1, COD=SO2 4 ratio 3.5 Effect of molybdate for varying Molybdate dose (mM) TMA TSA
COD=SO2 4 0.25 M #
ratio 0.5 M #
0.75 " #
1.0 " #
2.0 " #
Set III SRB deficient biomass, SO2 4 concentration 1142.86 mg l1, COD=SO2 4 ratio 3.5 0.05 M #
0.075 M #
0.1 # #
0.25 # #
0.5 # #
Set IV SRB deficient biomass, SO2 4 concentration 2575.75 mg l1, COD=SO2 4 ratio 0.66 0.5 # M
0.75 # #
1.0 M #
2.0 M #
Set IV SRB deficient biomass, COD concentration 1700 mg l1, COD=SO2 4 ratio 0.66 0.5 # M
0.75 # #
1.0 M #
2.0 M #
": Significant increase relative to control/minimum dose; #: significant decrease relative to control/minimum dose; M: variable trend/insignificant increase/insignificant decrease relative to control/minimum dose.
For the same doses of 0.25 mM and 0.5 mM, higher TMA inhibition was observed at higher sulfate concentration. This may have been due to higher direct sulfide toxicity or higher concentrations of molybdate sulfide complexes. (c) Effect of molybdate at varying COD=SO2 4 ratio: A higher molybdate dose of 2 mM was required to inhibit TSA by over 90% at the lower COD=SO2 4 ratio of 0.66 (Set IV) compared to the dose requirement of 0.5 mM at COD=SO2 4 ratio of 3.5 (Set II) for substrate COD 1700–2000 mg l1 (Table 2). The above discussion clearly indicates dose dependency of molybdate on history of biomass evolution, sulfate concentration and COD=SO2 4 ratio in substrate.
5. Conclusions (1) Molybdate supplementation inhibited SRB activity considerably (>85%) at 3 mM. However, the lowest dose that inhibited more than 93% SRB activity was 0.25 mM for SRB deficient biomass. (2) Molybdate dose required for inhibition of SRB appears to depend upon SO2 concentration, 4 COD=SO2 4 ratio and history of biomass. (3) The effect of molybdate supplementation on MPB activity needs to be further investigated as increase in dose up to some intermediate concentration indicated inhibition and thereafter further increase caused stimulation of MPB activity.
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