effect of molybdate on microbial consortium - Wiley Online Library

2 downloads 0 Views 1MB Size Report
Sep 4, 2013 - Soraya Zahedi,a∗ Diego Sales,a Luis-Isidoro Romerob and Rosario Soleraa. Abstract. BACKGROUND: The effect of molybdate on a microbial ...
Research Article Received: 15 May 2013

Revised: 27 August 2013

Accepted article published: 4 September 2013

Published online in Wiley Online Library: 4 October 2013

(wileyonlinelibrary.com) DOI 10.1002/jctb.4215

Biomethanization from sulfate-containing municipal solid waste: effect of molybdate on microbial consortium Soraya Zahedi,a∗ Diego Sales,a Luis-Isidoro Romerob and Rosario Soleraa Abstract BACKGROUND: The effect of molybdate on a microbial consortium of dry thermophilic anaerobic digestion (AD) from sulfate-containing municipal solid waste was studied. RESULTS: The study showed molybdate inhibition affecting hydrolysis, acidogenesis, and methanogenesis, but the main microbial populations in AD can sustain the molybdate load over a short period of time. Molybdate was not a bactericide for all members of the principal groups, only a few of them are affected. Although all the microbial populations decreased, the weakest groups were sulfate reducing bacteria (SRBs) and butyrate-utilising acetogens (BUAs) and the most resistant group was propionate-utilising acetogens (PUAs). Prior to using molybdate, the relative percentages were: Eubacteria:Archaea 80:20; acetogens 25% (BUAs 11% and PUAs 14%) and SRBs 17%. After molybdate addition the relative percentages were: Eubacteria:Archaea were 83:17, acetogens remained over 28% (BUAs 8% and PUAs 20%) and SRBs around 11%. The relation between acetate-utilising methanogens (AUMs) and hydrogen-utilising methanogens (HUMs) changed slightly from 55:45 to 53:47. CONCLUSION: All biochemical reactions in AD were inhibited and an increase in food:microorganism(F:M) ratio was observed. The microorganism’s removal rates and growth inhibition rates are indicators of inhibitory effect in all analysed populations by inhibitor addition. c 2013 Society of Chemical Industry  Keywords: biomethanization; inhibition; sulfate-containing solid waste; microbial community structure

INTRODUCTION The data published by the National Integrated Plan of Wastes (NIPW) (2008–2015) in Spain indicates that in Andaluc´ıa more than 4 000 000 tons of urban waste is generated each year. Anaerobic digestion (AD) is an attractive treatment strategy for the organic fraction of municipal solid waste (OFMSW) and is of great benefit from an environmental point of view. Therefore much research work has been carried out on biogas technologies.1 – 3

J Chem Technol Biotechnol 2014; 89: 1379–1387



Correspondence to: Soraya Zahedi D´ıaz, Department of Environmental Technologies. Faculty of Marine and Environmental Sciences (CASEM). University of C´adiz. Pol, R´ıo San Pedro s/n, 11510 Puerto Real (C´adiz), Espa˜na. E-mail: [email protected]

a Department of Environmental Technologies. Faculty of Marine and Environmental Sciences (CASEM), University of C´adiz, Pol, R´ıo San Pedro s/n, 11510, Puerto Real (C´adiz), Spain b Department of Chemical Engineering and Food Technology. Faculty of Sciences, University of C´adiz. Pol, R´ıo San Pedro s/n, 11510, Puerto Real (C´adiz), Spain

www.soci.org

c 2013 Society of Chemical Industry 

1379

The AD of OFMSW, which is ultimately converted into methane (CH4 ) and carbon dioxide (CO2 ), is carried out by the coordinated action of various groups of microorganisms and passes through several intermediate stages. In the first and second steps, hydrolysis and acidification take place by hydrolytic-acidogenic bacteria (HABs), and intermediate products (volatile fatty acids (VFAs), hydrogen (H2 ) and CO2 ) are generated. In the third step, VFAs are transformed into acetate, H2 and CO2 by acetogens bacteria. Propionate-utilising acetogens (PUAs) and butyrateutilising acetogens (BUAs) are the majority of the acetogens in the anaerobic reactors. In the last step, acetate-utilising methanogens (AUMs) and hydrogen-utilising methanogens (HUMs) are capable of converting acetate or H2 and CO2 to CH4 . Inhibitory substances, such as sulfate, are often found to be the leading cause of anaerobic reactor failure, since they are often present in substantial concentrations in waste. Due to the inhibition process, there is usually a decrease of CH4 production and an accumulation of VFAs. Moreover, the biogas produced

during anaerobic treatment of sulfate-containing OFMSW holds around 1% (v/v) sulfide (H2 S). H2 S is a corrosive gas which makes it problematic to use biogas as a fuel for boilers or electricity generation in a biogas engine.4 Sulfate reduction is performed by sulfate reducing bacteria (SRBs) which reduces compounds (long and branch-chained fatty acids, ethanol, and other alcohols, organic acids, and aromatic compounds) to acetate and CO2 (incomplete oxidizers) or convert acetate to CO2 and HCO3 - . The affinity of SRBs for reduced substrates has been ranked in the order of H2 > propionate > other organic electron donors.5 As SRBs can use a wide range of different substrates, they can compete with several different types of microorganisms involved in AD: methanogens, acetogens or HABs.6,7 Nevertheless, literature data related to the outcome of competition between SRBs and the microorganisms mentioned above are contradictory.8 The

www.soci.org considerable variation in the inhibition/toxicity levels reported for sulfate is due to the complexity of the AD process, where mechanisms such as antagonisms, synergism, acclimation and complexing could significantly affect inhibition. Studies based on the use of inhibitors to suppress the activity of SRBs and, consequently, promote growth of methanogens have been reported in the literature.4,8 – 10 Several researchers have used molybdate for the selective inhibition of SRBs because apart from being an SRB inhibitor, it is considered a nutrient for methanogens.4,10 The feasibility of using molybdate to control sulfate reduction in a biological reactor, however, is not well established as some published results indicate an adverse effect on Archaea and Eubacteria. Therefore, some authors conclude that this substance does not inhibit HABs, but only methanogens and SRBs.4 Other authors argue that inhibition is greater in SRBs than in methanogens.9 There are no results about the effect on acetogenic bacteria. In addition, there are disagreements on the bactericidal or bacteriostatic nature of the inhibition. Issues related with molybdate supplementation need to be better explained and fully elucidated. Taking into consideration the background research, the present study was undertaken to investigate the effect of molybdate supplementation on the AD of sulfate-containing OFMSW. The effect of a continuous dose of inhibitor on HABs, SRBs, acetogens and both methanogen populations (AUMs and HUMs) was examined. The evolution of the anaerobic consortia was analysed by fluorescent in situ hybridization (FISH), employing different oligonucleotide probes. With this technique we can determine the evolution of the different populations growing in a single-phase AD, so the effect of molybdate on them can be evaluated independently. The food:microorganism (F:M) ratio is a key factor controlling AD.11,12 Under given conditions, the bacterial consortia can consume only a limited amount of food each day. This ratio is the controlling factor in all biological treatment processes. A lower F:M ratio will result in a greater percentage of waste being converted into gas. Therefore, an increase in F:M ratio is indicative of destabilization of the AD process. In this article, the effect of operational parameter modifications on chemical soluble demand (CODD ), VFAs, total volatile solid (TVS), microbiological population, net growth, CH4 and H2 S production and F:M ratio have been considered to study the effect of molybdate in the anaerobic process from sulfate-containing real municipal solid waste. Moreover, different relations between physico-chemical and microbiological parameters have also been considered.

MATERIALS AND METHODS Digester, start-up and operational conditions Dry AD of OFMSW was carried out as a single phase system at thermophilic temperature (55◦ C). The reactor (5 L) was equipped with a thermostatic water bath (7 L), a stirring paddle (23 rpm), a biogas outlet and a feed inlet. As a biomass retention system has not been used, the hydraulic retention time (HRT) is equal to the solids retention time in the reactor. The HRT assayed was 6.5 days. The organic loading rate (OLR) of this digester was 11 kg TVS m-3 day− (3.4 kg CODD m-3 day− ). Regarding the feeding regime, the reactor was fed once a day (semi-continuous).

1380

Inoculum, food waste and feeding Anaerobic digester effluent from a single phase dry-thermophilic AD of OFMSW was used as inoculum. The food waste used in this

wileyonlinelibrary.com/jctb

S Zahedi et al.

Table 1. Physical-chemical and microbiological characterization of the feed Parameter

Average

pH CODD (g L− ) TVFAs (g acetic L− ) Acetate (g L− ) Propionate (g L− ) Butyrate (g L− ) TVS (g kg− ) Sulfate (g L− ) Ratio CODD /SO4 -2 Total population (107 cells mL− ) Eubacteria (107 cells mL− ) HABs (107 cells mL− ) SRBs (107 cells mL− ) Acetogens (107 cells mL− ) PUAs (107 cells mL− ) BUAs (107 cells mL− ) Archaea (107 cells mL− ) AUMs (107 cells mL− ) HUMs (107 cells mL− )

5.3 ± 0.6 22.3 ± 4 2.4 ± 0.8 1.7 ± 0.5 0.2 ± 0.2 0.5 ± 0.2 72 ± 9 1.0 ± 0.0 22.3 29.5 ± 3.5 21.1 ± 2.0 6.9 ± 0.7 5.7 ± 0.6 14.2 ± 2.6 7.7 ± 1.3 6.5 ± 1.3 8.4 ± 1.5 4.4 ± 2.0 4.1 ± 2.0

assay was collected from the Municipal Treatment Plant Calandrias located in Jerez de la Frontera (Spain). The OFMSW was stored in drums of 25 kg at –4◦ C to avoid AD by microorganisms in the solid waste itself. The total solids concentration of the feeding was adjusted to 20% (dry AD) by adding tap water. The characteristics of feeding are shown in Table 1. The volume of feed supplied to the reactor was 0.77 L day− throughout the period of the study. During the first phase of the experiment (0–45 days) no inhibitor was used. After the startup period (0–20 days) the reactor reached the stationary phase (21–45 days). On day 46, 2.6 g of sodium molybdate (MW = 205.92 g mol− ) was added to the digester to give 2.5 mmol L− salt concentration in relation with the total digester volume of 5 L. Molybdate concentration of 2.5 mmol L− was adopted because previous studies10 indicated that this value cause inhibition of sulfate reduction. From next day onwards, i.e. from day 47, based on the daily wash out, a predetermined quantity of inhibitor (0.4 g per 770 mL of daily feed) was added every day to maintain the 2.5 mmol L− sodium molybdate concentration in the digester. Analytical methods The analytical methods carried out in this study can be grouped into two categories: physico-chemical analysis and microbiological analysis. Physico-chemical analysis The parameters analysed for the physico-chemical characterization of effluents were as follows: the volume and composition of the biogas, CODD , TVS, pH, total volatile acids (TVFAs), acetic, butyric, propionic and sulfate. The analytical techniques were assessed according to Standard Methods.13 The sulfates were analysed from the filtrate supernatant obtained by means effluent sample lixiviation (10 g of digested waste in 100 mL of Milli-Q water for 20 min). Samples of sulfates were further filtered using a 1 μm pore size glass fiber filter.

c 2013 Society of Chemical Industry 

J Chem Technol Biotechnol 2014; 89: 1379–1387

Effect of molybdate on microbial consortium

www.soci.org

Table 2. Oligonucleotide probes used in this study Probe sequences (from 5´ a 3´)

Probe

Target

S-D-Bact-0338-a-A-18 S-D-Bact-0338-a-S-18 S-D-Arch-0915-a-A-20 S-F-Mbac-1174-a-A-22 S-F-Msae-0825-a-A-23

GCTGCCTCCCGTAGGAGT ACTCCTACGGGAGGCAGC GTGCTCCCCCGCCAATTCCT TACCGTCGTCCACTCCTTCCTC TCGCACCGTGGCCGACACCTAGC

S-*-Srb-0385-a-A-18 SYNBAC824

CGGCGTCGCTGCGTCAGG GTACCCGCTACACCTAGT

S-F-SYNM-0700-a-A-23

ACTGGTXTTCCTCCTGATTTCTA

a b

Eubacteria None (negative control) Archaea Methanobacteriaceae (H2 -utilising) Methanosaetaceae (acetate-utilising) SRBs Syntrophobacter sp.(propionate-utilising) Syntrophomonadaceae (butyrate-utilising)

Formamide (%)

Timea (h)

T b(◦ C)

Reference

20 20 35 35 20

1.5 1.5 1.5 1.5 1.5

46 46 46 46 46

17 18 19 20, 21 20

30 10

2 2

46 46

22 23, 24

30

2

52

23–25

Hybridization time. Incubated temperature.

Figure 1. Evolution of pH, TVFAs and individual VFAs.

This parameter was measured using a commercial kit (Merck Ref. 1.14791.0001) Gas chromatography was used for the analysis of the different biogas components.14 The gases analysed were H2 , N2 , CH4 , CO2 and H2 S(GC-2010 Shimadzu).

The cellular concentration and percentages of Eubacteria, Archaea, BUAs, PUAs, SRBs, HUMs and AUMs were obtained by FISH. The total population was calculated as the sum of the relative amounts of Eubacteria and Archaea, because the main anaerobic groups in the anaerobic reactors are contained within these two domains.26 Acetogens were calculated as the sum of the relative amounts of PUAs and BUAs. HABs were calculated as the difference in the relative amounts of Eubacteria and acetogens. Percentage drop in bacteria growth was calculated as the quotient between the difference in microbial concentration before molybdate addition (day 45) and at the end of the test (day 50), and the initial microbial concentration (day 45). The net growth parameter has been used to evaluate biochemical activity in AD. Considering a spatially homogeneous population of cells growing in a semi-continuous stirred tank reactor, mass balance was used to determinate the net growth, because even though cell growth is a complex process, it follows the laws of conservation of mass and energy.27 The net growth parameter for the different populations was calculated following the equation: r = Xe × Ve − Xa × Va

J Chem Technol Biotechnol 2014; 89: 1379–1387

(1)

where r is the net growth, Va is the daily volume of feeding, Ve is the daily volume of effluent, Xa is the cell concentration of feeding and Xe is the cell concentration of effluent. Growth inhibition was estimated as the ratio between the difference in the net growth prior to molybdate addition (day 45) and at the end of the trial (day 50), and the initial net growth (day 45). The parameter used to assess the performance of the anaerobic digester was the F:M ratio. The ratio F:M was calculated dividing amount of TVS supplied between the amount of total microorganisms in the reactor.

RESULTS AND DISCUSSION Addition of inhibitor to the digester on day 46 caused immediate inhibition of sulfate reduction, resulting in a total absence of H2 S in the biogas. Following this reaction, a general inhibition of all the steps of AD was observed. The test ended when CH4 production was practically zero; the trial lasted 50 days. The values represented on day 45 (Tables and Figures) are mean values of all HRT periods tested (6.5 days) before inhibitor addition.

c 2013 Society of Chemical Industry 

wileyonlinelibrary.com/jctb

1381

Microbiological analysis An epifluorescence method (FISH) was used to count the microorganisms contained in the reactor. The samples were collected from the dry-thermophilic anaerobic reactor into sterile universal bottles. Absolute ethanol was added to the bottles in a volume ratio of 1 sample:1 ethanol. The samples were stored at –20◦ C until they were analysed (within 2 months). Prior to FISH analysis, the samples were pre-treated. The pre-treatment applied for microbiological count of high solids content samples was the addition of Tween 80 and 120 s of shaking.15,16 The main steps of FISH for whole cells using 16S rRNA-targeted oligonucleotide probes are cell fixation, permeabilization and hybridization with the desired probe(s). These determinations were performed according to Montero et al.15,16 The 16S rRNAtargeted oligonucleotide probes, the incubation temperature and the hybridization time (isotonic moisture chamber) are shown in Table 2. All probes were labelled with 6-FAM at the 5´ terminal. The samples were examined visually and cells counted using an Axio Imager Upright epifluorescence microscope (Zeiss) with a 100 W mercury lamp and a × 100 oil objective.

www.soci.org

S Zahedi et al.

Table 3. Characterization of reactor effluent and yield of the process Day Flow (L day− ) MoO4 -2 (mmol L− ) Yields of the process % CH4 % H2 S % H2 mL H2 S L− day− L CH4 L− day− L H2 L− day− Characterization of the reactor SO4 -2 Sulfate loading rate (kg SO4 -2 m-3 day− ) SO4 -2 effa (g L− ) SO4 -2 removal (g day− ) SO4 -2 removal (%) CODD OLR (kg CODD m-3 day− ) CODD effa (g L− ) CODD removal (g day− ) COD removal (%) TVS OLR (kg TVS m-3 day− ) TVS effa (g kg− ) TVSremoval (g day− ) TVS removal (%) a

1–45 0.77 -

46 0.77 2.5

47 0.77 2.5

48 0.77 2.5

49 0.77 2.5

50 0.77 2.5

62 0.7 0 36 2.84 0.00

60 0.0 0 0 1.99 0.00

55 0.0 0 0 1.28 0.00

55 0.0 0 0 0.36 0.00

44 0.0 0 0 0.08 0.00

16 0.0 22 0 0.03 0.05

0.15 0.5 0.4 50

0.15 0.8 0.2 20

0.15 0.9 0.1 10

0.15 1.0 0.0 0

0.15 1.0 0.0 0

0.15 1.0 0.0 0

3.4 14 6.4 37

3.4 16 4.9 28

3.4 19 2.5 15

3.4 23 –0.5 -3

3.4 23 –0.5 -3

3.4 27 -3.6 -21

11 13 45 82

11 21 39 71

11 23 38 68

11 25 36 65

11 26 35 64

11 28 34 61

Effluent.

pH The evolution of pH is shown in Fig. 1. Before inhibitor addition the pH was around 7.5, after that, on day 49, this value did not exceed 6.0. The decrease in pH could be explained by an increase in VFAs production, especially butyric and acetic. The increased acidity in the system was due to the inhibition of acid-consuming microorganisms, particularly methanogens, BUAs and SRBs.

1382

Biogas production The daily CH4 , H2 S and H2 productions are summarized in Table 3. Addition of inhibitor caused cessation of H2 S production on the first day; however, CH4 production also reduced gradually. These results coincide with those obtained by Ranade et al.4 during anaerobic treatment of sulfate-containing waste; the daily addition of 3 mmol L− molybdate also resulted in a continual decline in the CH4 yield and in biogas production. Before inhibitor addition the percentage of H2 S in the biogas was 0.7%, and CH4 and H2 S productions were around 2.84 L CH4 L− day− and 36 mL H2 S L− day− , respectively. After inhibitor addition, on day 48, these productions did not exceed 0.36 L CH4 day− and 0 mL H2 S L− day− , respectively. The decrease in CH4 production is related with the increase in CODD (Fig. 2(a)) and VFAs concentration (Fig. 2(b)), especially butyric and acetic (Fig. 2(c), 2(d)). The linear correlation between VFAs concentration and CH4 production was calculated using the Pearson correlation. All Pearson correlation coefficients were greater than 0.8 indicating a good fit to the experimental data. The relations obtained between VFAs and CH4 production and CODD and CH4 production were negative. This is due to the inhibition of acid-consuming microorganisms by inhibitor

wileyonlinelibrary.com/jctb

addition. The production of H2 at the end of the trial was a consequence of HUMs and SRBs inhibition. Sulfate (SO4-2 ) The reactor evolution is shown in Table 3. Addition of inhibitor in the digester on day 46 caused immediate and complete inhibition of sulfate reduction, thereby resulting in a total absence of H2 S in the biogas (Table 3). Simultaneously, there was a progressive increase in the sulfate content in the effluent. The sharp increase in sulfate (reduction in SO4 -2 removal efficiency) was caused by a strong inhibition of SRBs by molybdate, according to Patidar and Tare.9 Before molybdate addition, the sulfate content in the effluent was around 0.5 g L− , at the end of the assay, on day 50, this value had risen to 1.1 g L− . The efficiency of sulfate removal decreased from 50 to 0. The negative relation obtained between SO4 -2 and L CH4 L− d− (Fig. 2(e)) indicates that both methanogens and SRBs develop within the system as a syntrophism relationship.28 CODD Table 3 shows the evolution of CODD in the effluent. Increase in CODD indicates that microorganisms were strongly affected by the inhibitor, according to microbial results. Before inhibitor addition the CODD levels in the effluent were around 14 g L− , on day 50 this value was around 27 g L− . The efficiency of COD removal dropped from 37 to –21%. Negative values are indicative of solubilization, not of consumption. This result, combined with the inhibition and decrease of HUMs and the low pH values, explains the production of H2 at the end of the trial (Table 3).

c 2013 Society of Chemical Industry 

J Chem Technol Biotechnol 2014; 89: 1379–1387

Effect of molybdate on microbial consortium

www.soci.org

(a)

(b)

(d)

(c)

(e)

(f)

Figure 2. (a) Relation between l CH4 L –1 day –1 and CODD (g L –1 ). (b) Relation between l CH4 L –1 day –1 and TVFAs (mg L –1 ). (c) Relation between l CH4 L –1 day –1 and Butyric (mg L –1 ). (d) Relation between l CH4 L –1 day –1 and Acetic (mg L –1 ). (e) Relation between l CH4 L –1 day and SO4 -2 (g L –1 ). (f) Relation between l CH4 L –1 day and TVS (g kg –1 ).

TVS At the beginning of the assay, before inhibitor addition, the TVS

(Fig. 2(f)). The same happened with the CODD levels (Fig. 2(a)). All Pearson correlation coefficients were greater than 0.9.

were around 13 g kg− , while on day 50 this value had increased to 28 g kg− (Table 3). The efficiency of TVS removal went down from 82 to 61% (i.e. a reduction of almost 20%) and this was due to inhibition of the microorganisms. An increase in TVS indicates that all the steps of the AD were strongly affected by molybdate

VFAs Figure 1 shows the evolution of TVFAs (g acetic L− ) and of individual VFAs (acetic, butyric and propionic) in the effluent over the period of study. The effect of the inhibitor produced acidification in the reactor. Thus, before molybdate addition the concentrations of

Table 4. Microorganism percentage composition on days 45–50 Day 45 46 47 48 49 50

80 82 89 85 83 83

HAB (%)

Acetogens (%)

55 59 60 58 58 56

25 23 29 28 25 28

SRBsa (%) 17 13 15 14 11 11

PUAs (%)

BUAs (%)

14 18 23 21 18 20

11 6 7 7 7 8

Archaea (%) 20 18 11 15 17 17

AUMs (%) 11 10 6 8 9 9

HUM (%) 9 8 5 7 8 8

Percentages compared with total Eubacteria.

J Chem Technol Biotechnol 2014; 89: 1379–1387

1383

a

Eubacteria (%)

c 2013 Society of Chemical Industry 

wileyonlinelibrary.com/jctb

www.soci.org

(a)

S Zahedi et al.

(b)

(d)

(c)

(e)

(f)

(g)

(h)

Figure 3. Microbial population evolution during days 45–50 for total population (a) Eubacteria (b) Archaea (c) AUMs (d) HUMs (e) PUAs (f) BUAs (g) SRBs (h).

1384

VFAs were in the order propionic > acetic > butyric. Molybdate addition caused butyric acid and acetic acid to dominate over propionic acid. The composition of VFAs changed, with a sharp increase in acetic acid until day 48 and dominating over butyric acid. At the end of the trial, both concentrations were similar. The presence of acetic acid in the reactors shows that the absence of methanogenic substrate was not the underlying problem for the inhibition of CH4 production in these studies, but chronic inhibition of AUMs due to the inhibitor. A butyric increment in the effluent

wileyonlinelibrary.com/jctb

resulted in the non-availability of HUMs to consume the H2 . Butyric rather than acetic acid tends to be the main fermentation product when there is an accumulation of H2 in the system.10 Dynamics of microbial communities, net growth and ratio F:M The evolution of the main microbial group involved in the anaerobic process is described in this section. In the present research, concentrations of different microbial groups were evaluated before and after inhibitor addition. All the results

c 2013 Society of Chemical Industry 

J Chem Technol Biotechnol 2014; 89: 1379–1387

Effect of molybdate on microbial consortium

www.soci.org

(a)

(b)

(c)

(d)

Figure 4. (a) Relation between l CH4 L –1 day –1 and total population (cells mL –1 ). (b) Relation between SO4 -2 (g L –1 ) and SRBs (cells mL –1 ). (c) Relation between TVS and Eubacteria (cells mL –1 ). (d) Relation between CODD and Eubacteria (cells mL –1 ).

shown are average values. The percentages of the main microbial groups involved in the process of AD are shown in Table 4. Before molybdate addition, the ratio of Eubacteria:Archaea was 80:20. The Archaea population values are higher than those obtained by Griffin et al.26 (12.1%) and McMahon et al.29 (4.5%).The SRBs population values (17–11%) were in line with those (14%) obtained by Mohan et al.30 and lower than those obtained by Zhang et al.31 (28.6%). After inhibitor addition the microbial population decreased. The decline rate of Eubacteria, Archaea, HABs, acetogens, AUMs, HUMs, SRBs, BUAs and PUAs was 35%, 48%, 38%, 30%, 18%, 14%, 60%, 55% and 11%, respectively. This indicates an inhibitory effect by molybdate addition in all analysed populations. These results do not coincide with those obtained by Ranade et al.4 since they found that the molybdate did not inhibit HABs. The weakest groups were SRBs (60%) and BUAs (55%) and the most resistant group was PUAs (11%); according to physico-chemical studies (increase in sulfate and butyric acid and no increase in the concentration of propionic). Propionate levels did not increase during the research period,

suggesting that the PUA levels may have been sufficient to achieve low propionate concentrations. In fact, they are the population least affected, with the lowest removal percentage (11%). During AD of sulfate-containing wastes, SRBs are said to be more responsible for propionate utilization than PUAs.32,33 After inhibition of SRBs by molybdate, PUAs percentage increased as a consequence of not having to compete with SRBs. Initially, the relation PUA:BUA was 14:11. At the end of assay the relation was 20:8, as a consequence of a sharper decrease of BUAs population compared with PUAs. Archaea population decreased, according to the amount of H2 detected in the biogas and the increase of acetic acid concentration in the effluent. The evolution of microbial populations are represented in Fig. 3. As shown above, all populations decreased after addition of inhibitor. However, after a pronounced drop during the first 2 days of trial, the majority of populations remained constant until the end of the experiment. This means that the amount of microorganisms that daily left the reactor through the effluent must be compensated with the

Table 5. Net growth Day

Total population

Eubacteria

Archaea

HABs

Acetogens

PUAs

BUAs

SRBs

AUMs

HUMs



18.8 14.8 12.5 11.9 12.1 11.6

4.4 2.9 1.1 1.7 2.1 2.0

13.6 11.2 8.9 8.6 9.0 8.3

5.2 3.6 3.5 3.3 3.1 3.3

3.0 2.9 3.0 2.7 2.3 2.6

2.2 0.6 0.6 0.6 0.7 0.7

3.8 2.2 2.0 1.8 1.3 1.3

2.6 1.8 1.8 1.8 2.0 2.0

2.3 1.4 1.5 1.5 1.9 1.9

38.5

55.3

39.2

36.7

13.7

67.2

67.1

23.1

18.1

J Chem Technol Biotechnol 2014; 89: 1379–1387

c 2013 Society of Chemical Industry 

wileyonlinelibrary.com/jctb

1385

Net growth (cells day ) 45 23.2 46 17.7 47 13.6 48 13.6 49 14.2 50 13.5 Growth inhibition (%) 41.7

www.soci.org

S Zahedi et al.

CONCLUSION This research showed that molybdate is not a bactericide for all members of each bacterial group, but only for a few, and the rest of the population studied can remain constant over time. Those most affected were SRBs and BUAs. Behavioural differences between two groups of acetogens were observed: BUAs were the most affected group whereas PUAs were the most resistant. The Archaea population also decreased. All steps of the AD were inhibited and an increase in F:M ratio was observed. The microorganism removal rates and growth inhibition are indicative of the inhibitory effect of molybdate in all populations analysed. Figure 5. Evolution of F:M ratio (g TVS cell –1 day –1 ).

1386

amount of feed introduced and the amount of microorganisms generated during growth. All the microbial’s community structure was affected as well as to physico-chemical parameters. Some correlations between microorganisms and physico-chemical parameters were obtained (Fig. 4). These results show a positive linear correlation between total population and volume of CH4 generated (Fig. 4(a)).The relation between the amount of SRBs and sulfate concentration (Fig. 4(b)) was negative indicating that a decrease in SRBs population produces an accumulation of SRBs substrate inside the reactor. The relation between the amount of Eubacteria and organic matter (TVS and CODD ) was also calculated; the results show a Pearson’s correlation coefficient higher than 0.7 (Fig. 4(c) and 4(d)). The negative relations indicate that a decrease in microbial population produce a reduction in organic matter degradation. To evaluate biochemical activity during the assay, the net growth parameter has been considered. The influence of the inhibitor on net growth parameter was evaluated for all populations. Table 5 displays results for net growth and growth inhibition in the different populations studied. After inhibitor addition a decrease in all population´s net growth was observed, so all the populations were strongly affected by the inhibitor. Those most affected were SRBs and BUAs and the most resistant group was PUAs; this result was based on physico-chemical studies (increase in sulfate and butyric acid and no increase in the concentration to propionic). Nevertheless, the daily net growth of each bacterial group is maintained from the second day until the end of the trial. This is due to the fact that the molybdate is not a bactericide for all members of each population, but only for a few, so the rest of the microbial population remains constant over time. The destabilization of the AD process was evaluated using the F:M ratio.11,12 Figure 5 shows the evolution of F:M ratios observed in our assay: before inhibitor addition the value was 0.33 × 10-8 g TVS cell− day− ; after, it increased to 0.54 × 10-8 g TVS cell− day− . An increase in F:M ratio is indicative of destabilization of the AD process, however, this was stabilized from the third day of assay. Inhibitor addition produced a decrease in the total net growth causing wash out of populations and a high amount of organic matter in the reactor and consequently an increase in the F:M ratio. The stabilization of this parameter and the net growth parameter during the assay is consequence of the stabilization of populations. This paper provides useful information to achieve in-depth knowledge of the changes on microbial consortia responsible for biomethanization from sulfate-containing municipal solid waste. Future studies of molybdate inhibition on microbial consortia are necessary to provide more information.

wileyonlinelibrary.com/jctb

ACKNOWLEDGEMENTS This work was supported by the Spanish Ministry of Science and Innovation (MICINN)(CTM2007-62164) and the Innovation, Science and Enterprise Department (Andalusian Regional Government) (P07-TEP-02472), both projects co-financed by the European Regional Development Fund (ERDF). S. Zahedi acknowledges funding from the Spanish Ministry of Science and Innovation (MICINN) (AP2008-01213).

REFERENCES 1 Demirel B and Yenigun ¨ O, Two-phase anaerobic digestion processes a review. J Chem Technol Biotechnol 77:743–755 (2002). 2 Lee PH, Bae J, Kim J and Chen W, Mesophilic anaerobic digestion of corn thin stillage a technical and energetic assessment of the cornto-ethanol industry integrated with anaerobic digestion. J Chem Technol Biotechnol 86:1514–1520 (2011). 3 Han MJ, Behera SK and Park HS, Anaerobic co-digestion of food waste leachate and piggery wastewater for methane production: statistical optimization of key process parameters. J Chem Technol Biotechnol 87:1541–1550 (2012). 4 Ranade DR, Dighe AS, Bhirangi SS, Panhalka VS and Yeole TY, Evaluation of the use of sodium molybdate to inhibit sulphate reduction during anaerobic digestion of distillery waste. Bioresource Technol 68:287–291 (1999). 5 Laanbroek JH, Geerlings H, Sitjtsma L and Veldkamp H, Competition for sulphate and ethanol among Desulfobacter Desulfobulbus and Desulfovibrio species isolated from intertidal sediments. Appl Environ Microbiol 47:329–334 (1984). 6 McCartney DM and Oleszkiewicz JA, Sulfide inhibition of anaerobic degradation of lactate and acetate. Water Res 2:203–209 (1991). 7 Colleran E, Finnegan S and Lens P, Anaerobic treatment of sulphatecontaining waste streams. Antonie van Leeuwenhoek 67:29–46 (1995). 8 Chen Y, Cheng JJ and Creamer KS, Inhibition of anaerobic digestion process a review. Bioresource Technol 99:4044–4064 (2008). 9 Patidar SK and Tare V, Effect of molybdate on methanogenic and sulfidogenic activity of biomass. Bioresource Technol 96:1215–1222 (2005). 10 Isa MH and Anderson GK, Molybdate inhibition of sulphate reduction in two-phase anaerobic digestion. Process Biochem 40:2079–2089 (2005). 11 Chen SJ, Li TC and Shieh WK, Anaerobic fluidized bed treatment of an industrial wastewater. J Water Pollut Control Fed 60:1826–1832 (1988). 12 Perez M, Romero LI and Sales D, Anaerobic thermophilic fluidized bed treatment of industrial wastewater: effect of F:M relationship. Chemosphere 38:3443–3461 (1999). 13 APHA, Standard Methods for the Examination of Water and Wastewater, 19th edn. American Public Health Association, Washington, DC (1995). 14 Zahedi S, Sales D, Romero LI and Solera, R, Hydrogen production from the organic fraction of municipal solid waste in anaerobic thermophilic acidogenesis: influence of organic loading rate and microbial content of the solid waste. Bioresource Technol 129:85–91 (2013).

c 2013 Society of Chemical Industry 

J Chem Technol Biotechnol 2014; 89: 1379–1387

Effect of molybdate on microbial consortium

www.soci.org

15 Montero B, Garc´ıa-Morales JL, Sales D and Solera R, Evolution of microorganisms in thermophilic-dry anaerobic digestion. Bioresource Technol 99:3233–3243 (2008). 16 Montero B, Garc´ıa-Morales JL, Sales D and Solera R, Analysis of methanogenic activity in a thermophilic-dry anaerobic reactor: use of fluorescent in situ hybridization. Waste Manage 29:1144–115 (2009). 17 Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R and Stahl DA, Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925 (1990). 18 Amann RI, Krumholz L and Stahl DA, Fluorescent-oligonucleotide probing of whole cells for determinative phylogenetic and environmental studies in microbiology. J Bacteriol 172:762–770 (1990). 19 Stahl DA and Amann R, Development and application of nucleic acid probes, in Nucleic Acid Techniques in Bacterial System, ed by Stackebrandt E and Goodfellow M. John Wiley & Sons Ltd, Chichester, 205–248 (1991). 20 Raskin L, Stromley JM, Rittmann BE and Stahl DA, Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens. Appl Environ Microbiol 60:1232–1240 (1994). 21 Sekiguchi Y, Kamagata Y, Nakamura K, Ohashi A and Harada H, Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules. Appl Environ Microbiol 65:1280–1288 (1999). 22 Icgen B, Moosa S and Harrison STL, A study of the relative dominance of selected anaerobic sulfate-reducing bacteria in a continuous bioreactor by fluorescence in situ hybridization. Microbial Ecol 53:43–52 (2006). 23 Okabe S, Satoh H and Watanabe Y, Insitu analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes. Appl Environ Microbiol 65:3182–3191 (1999). 24 Ariesyady HD, Ito T and Okabe S, Funtional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge digester. Water Res 41:1554–1568 (2007).

25 Hansen KH, Ahring BK and Raskin L, Quantification of syntrophic fatty acid-ß-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization. Appl Environ Microbiol 11:4676–4774 (1999). 26 Griffin ME, McMahon KD, Mackie RI and Raskin L, Methanogenic population dynamics during start-up of anaerobic digesters treating municipal solid waste and biosolids. Biotechnol and Bioeng 57:342–355 (1998). 27 Najafpour GD, Material and elemental balance, in Biochemical Engineering and Biotechnology, 1st edn. Elsevier, Iran, 228–251 (2006). 28 Vossoughi M, Shakeri M and Alemzadeh I, Performance of anaerobic baffled reactor treating synthetic wastewater influenced by decreasing COD/SO-2 ratios. Chem Eng Proc: Process Intensification 42:811–816 (2003). 29 McMahon KD, Stroot PG, Mackie RI and Raskin L, Anaerobic codigestion of municipal solid waste and biosolids under various mixing conditions-II. Microbial population dynamics. Water Res 7:1817–1827 (2001). 30 Mohan SV, Rao NC, Prasad KK and Sarma PN, Bioaugmentation of an anaerobic sequencing batch biofilm reactor (AnSBBR) with immobilized sulphate reducing bacteria (SRB) for the treatment of sulphate bearing chemical wastewater. ProcessBiochem 40:2849–2857 (2005). 31 Zhang J, Zhang Y, Quan X, Liu Y, An X, Chen S and Zhao H, Bioaugmentation and functional partitioning in a zero valent iron-anaerobic reactor for sulfate-containing wastewater treatment. Chem Eng J 174:59-165 (2011). 32 Ueki K, Ueki A, Itoh K, Tanaka T and Satoh A, Removal of sulphate and heavy metals from acid mine water by anaerobic treatment with cattle waste: effects of heavy metals on sulphate reduction. J Environ Sci Health A 26:1471–1489 (1991). 33 Ueki K, Ueki A, Takahashi K and Iwatsu M, The role of sulphate reduction in methanogenic digestion of municipal sewage sludge. J Gen Appl Microbiol 38:195–207 (1992).

1387

J Chem Technol Biotechnol 2014; 89: 1379–1387

c 2013 Society of Chemical Industry 

wileyonlinelibrary.com/jctb