Mine wastewater treatment using Phalaris arundinacea plant material hydrolyzate as substrate for sulfate-reducing bioreactor

Bioresource Technology 101 (2010) 3931–3939

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Mine wastewater treatment using Phalaris arundinacea plant material hydrolyzate as substrate for sulfate-reducing bioreactor Aino-Maija Lakaniemi, Laura M. Nevatalo *, Anna H. Kaksonen 1, Jaakko A. Puhakka Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FIN-33101 Tampere, Finland

a r t i c l e

i n f o

Article history: Received 1 October 2009 Received in revised form 7 January 2010 Accepted 9 January 2010 Available online 4 February 2010 Keywords: Canary Grass Hydrolyzation Sulfate reduction Fluidized-bed bioreactor Mine wastewater

a b s t r a c t A low-cost substrate, Phalaris arundinacea was acid hydrolyzed (Reed Canary Grass hydrolyzate, RCGH) and used to support sulfate reduction. The experiments included batch bottle assays (35 °C) and a fluidized-bed bioreactor (FBR) experiment (35 °C) treating synthetic mine wastewater. Dry plant material was also tested as substrate in batch bottle assays. The batch assays showed sulfate reduction with the studied substrates, producing 540 and 350 mg L1 dissolved sulfide with RCGH and dry plant material, respectively. The soluble sugars of the RCGH presumably fermented into volatile fatty acids and hydrogen, which served as electron donors for sulfate reducing bacteria. A sulfate reduction rate of 2.2– 3.3 g L1 d1 was obtained in the FBR experiment. The acidic influent was neutralized and the highest metal precipitation rates were 0.84 g Fe L1 d1 and 15 mg Zn L1 d1. The sulfate reduction rate in the FBR was limited by the acetate oxidation rate of the sulfate-reducing bacteria. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Treatment of acidic, metal- and sulfate containing mine wastewaters with sulfate-reducing bioreactors is a feasible alternative for chemical treatment (Kaksonen et al., 2004a; Buisman et al., 2007; Huisman et al., 2006; Kaksonen and Puhakka, 2007). The anaerobic sulfate reducing bacteria (SRB) reduce sulfate to hydrogen sulfide while oxidizing an electron donor (Widdel, 1988):  2 CH2 O þ SO2 4 ! H2 S þ 2 HCO3 :

ð1Þ

The produced hydrogen sulfide precipitates metals, and the alkalinity generated from the electron donor oxidation neutralizes the acidity (Dvorak et al., 1992). Biological treatment requires an electron donor for SRB, which is the highest operational cost (Buisman et al., 2007). At present, hydrogen (van Houten et al., 1994) and ethanol (Kaksonen et al., 2004a) are used, but a low-cost electron donors, such as fermentation industry wastewater (Buisman et al., 2007), are preferred. The ability to directly utilize cellulose and hemicelluloses of the plant cell wall is not a common feature of SRB (Hansen, 1994). The anaerobic degradation of organic matter, such as plant material, proceeds via hydrolyzation of the polymeric material (cellulose, lignin, etc.) into smaller molecules (sugars, fatty acids, etc.), which are then fermented to carboxylic

* Corresponding author. Tel.: +358 3 3115 11; fax: +358 3 3115 2869. E-mail address: laura.nevatalo@tut.fi (L.M. Nevatalo). 1 Present address: CSIRO Land and Water, Underwood Avenue, Floreat, WA 6014, Australia. 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.01.020

acids, alcohols and hydrogen by fermentative bacteria (Visser, 1995). These molecules serve as electron donors for SRB (Visser, 1995). Plant material as a low-cost substrate for SRB has been reported for only a few sulfidogenic bioreactors types (Table 1), and usually these reactors are batch- or column type reactors used for bioremediation of acid mine drainage. Examples of tested substrates in bioreactors are molasses (Maree and Strydom, 1987), primary sewage sludge (Whiteley et al., 2003), manure (Tsukamoto et al., 2004), compost (Zagury et al., 2006), wood chips (Zagury et al., 2006), cheese whey (Drury, 1999) and biodiesel manufacturing waste (Zamsov et al., 2006). In passive AMD treatment with permeable reactive barriers various low-cost substrates, such as sewage sludge, leaf mulch, wood chips, manure and sawdust have been used successfully (Waybrant et al., 1998). The solid organic materials are not suitable for treatment of mine wastewater in reactors types like fluidized-bed bioreactor (FBR), up-flow anaerobic sludge blanket reactor (UASB) and gas-lift bioreactor (GLB). The Phalaris arundinacea is a relatively high-yielding plant that grows naturally in Finnish climate conditions, and is used for fiber and energy production (Mäkinen et al., 2006). We chose to chemically hydrolyze plant material, and use this solution as substrate for SRB. The amenability of P. arundinacea Canary Grass plant material hydrolyzate (Reed Canary Grass Hydrolyzate, RCGH) and dry plant material as substrates for SRB was first tested with batch bottle assays. A fluidized-bed bioreactor fed with the RCGH solution was used for sulfidogenic mine wastewater treatment for 100 days (35 °C). The use of plant material hydrolyzate as substrate for SRB in bioreactor treating acidic, metal- and sulfate containing mine wastewater has not been reported before.

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Table 1 Comparison of the sulfate reduction rates reported for sulfate-reducing bioreactors utilizing low-cost substrates as electron donor for SRB. Reactor type

Substrate

pH

T (°C)

SRR (g L1 d1)

Reference

Up-flow packed bed reactor Stirred tank Anaerobic filter Anaerobic filter Batch (bottle) Column Column Fluidized-bed bioreactor

Molasses Primary sewage sludge Ethanol and spent manure Methanol and spent manure Mixture of wood chips, leaf compost and poultry manure Mixture of cheese whey, sawdust and cow manure Biodiesel manufacturing waste (glycerol) Soluble reed canary grass hydrolyzate

8.1 7.2 6.5 6.5 7.5 6.5–7.5 6–8 7

25 20–22 5 5 22 14–24 NR 35

2.2 0.8 1.1–1.4 1.0–1.3 0.01 11.5–24 0.5 2.2–3.3

Maree and Strydom (1987) Whiteley et al. (2003) Tsukamoto et al. (2004) Tsukamoto et al. (2004) Zagury et al. (2006) Drury (1999) Zamsov et al. (2006) This study

SRR, sulfate reduction rate; NR, not reported.

2. Methods 2.1. Hydrolyzation of plant material Dry P. arundinacea plant material was hydrolyzed using acid hydrolysis. The RCGH solution was prepared as follows: 7% or 10% w/v of dried and chopped plant material was hydrolyzed by autoclaving (90 min, 121 °C) in 3% H2SO4 solution (Sun and Cheng, 2005), the solution was decanted, the solid material was discarded and the liquid phase was used in the experiments. The organic content of the RCGH was analyzed for soluble sugars, chemical oxygen demand of soluble compounds (CODs), carboxylic acids (volatile fatty acids, VFA) and alcohols and dissolved organic carbon (DOC). The RCGH solution also contained variable concentrations of partially hydrolyzed plant material, which was not characterized or quantified.

centrations were 0.25, 0.27, 0.29, 0.41 and 0.69 g L1. Also solid materials were tested, with 8 g L1 dry, untreated plant material and 8 g L1 hydrolyzed canary grass. In case the medium pH decreased below 7, NaHCO3 buffer solution was added to the bottles after each sampling. The added buffer corresponded to 1–2.5 g L1 CaCO3 alkalinity. The batch bottles were incubated at 35 °C for 35 days. The bottles were sampled weekly for dissolved sulfide (DS), pH, sulfate, soluble sugars, CODs, DOC, carboxylic acids (VFA) and alcohols. A second batch bottle assay was made with the dry plant material with solid material concentrations of 8 and 16 g L1. The dry plant material was autoclaved with the growth medium. In the beginning of the experiment the batch bottles were buffered with 1–2.5 g L1 CaCO3 alkalinity to avoid pH decrease during the experiment, and no further supplementation was needed. The bottles were incubated at 35 °C for 42 days and sampled weekly as described above.

2.2. Batch bottle assays with RCGH and dry plant material 2.3. Reactor experiment The suitability of RCGH and dry plant material for sulfate reduction substrates were assessed with batch bottle assays. The assays were performed in 0.6 L serum bottles containing 0.25 L of modified Postgate medium (Table 2). All the batch bottles were inoculated with 5 mL of enrichment culture from a sulfidogenic, ethanol-lactate fed fluidized-bed bioreactor (Nevatalo et al., 2010) and pre-grown in the Postgate medium with the RCGH. The biological controls contained the inoculum with no substrate. In the first experiment with RCGH the tested soluble sugars con-

The sulfate-reducing FBR was previously used for mine wastewater treatment at 35 °C with ethanol and lactate as electron donors for 540 days as described by Nevatalo et al. (2010). The reactor set-up was as shown in Fig. 1. The FBR was fed with diluted RCGH solution and synthetic, acidic wastewater containing sulfate, metals and nutrients (Table 2). The loading of the soluble sugars, sulfate and iron were stepwise increased during the 100 day experiment, and the influent pH was simultaneously decreased. The FBR

Table 2 The composition of the modified Postgate medium used in the batch bottle assays and the influent solutions of the FBR. The concentrations are given as g L1 except for the pH, dry plant material, vitamin and trace element solutions (DSMZ medium 141). The concentration of the reed canary grass hydrolyzate (RCGH) is presented as total soluble sugars. The sulfate, RCGH and iron loading to the FBR were increased stepwise.

a b

Compound

Batch bottle assay with RCGH

Batch bottle assay with dry plant material

FBR feed solution

Na2SO4  10 H2O Yeast extract Resazurin Vitamin solution Trace element solution Ascorbic acid Sodium thioglycolate MgSO4  7 H2O FeSO4  7 H2O NH4Cl ZnCl2 KH2PO4 Na2SO4 RCGH Dry plant material pH

1.45 0.40 0.50  103 1 ml L1 1 ml L1 0.10 0.10 1.12 0.40 0.11 0.03 0.06 – 0.25–0.69 8 7.5

5.70 0.40 0.50  103 1 ml L1 1 ml L1 0.10 0.10 3.10 0.30 0.11 0.03 0.06 – – 8–16 7.5

– – – 1 ml L1a 1 ml L1a 5.50  103 5.50  103 0.25–9.0 0.25–1.90 5.50  102 1.50–2.10  102 2.8  102 0.18–2.80 0.15–1.78 – 7.8–4.4b

The FBR feed solution was supplemented with vitamin- and trace element solutions for the first 43 days of the reactor run. The pH of the feed solution was decreased stepwise.

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Fig. 3. The hydraulic retention time (HRT, ––) and the inoculation points (d) of the FBR. The arrow indicates the point where the FBR heated to 50–65 °C (day no 590) during malfunction of the equipment.

2.4. Physico-chemical analyses Fig. 1. The fluidized-bed bioreactor set-up used in this study (Nevatalo et al., 2010).

experiment was started with neutral influent to support the activity of SRB with the new substrate in the beginning of the experiment to avoid the inhibitory effect of the low pH influent. There was a good correlation of the measured FBR CODs loading and the theoretical COD load (Fig. 2) calculated from the soluble sugars load as described by Isa et al. (1986). The hydraulic retention time (HRT) of the FBR was as shown in Fig. 3. The FBR was re-inoculated during 3 periods as shown in Fig. 3. The re-inoculation was done with 20–50 mL of FBR-culture grown in batch bottles in Postgate medium at 35 °C with RCGH and acetate as electron donors. The FBR unintentionally heated to 50–65 °C on day 590. After this, the neutralization capacity of the FBR decreased, and NaHCO3 buffer was added daily to the FBR after the sampling, and the added buffer corresponded to 0.5–2.3 g L1 CaCO3 alkalinity. The buffering was continued until the end of the experiment. The FBR liquid was sampled daily for pH and DS, and twice a week for sulfate, DOC, CODs, dissolved metals (Fe and Zn), total alkalinity, organic acids (VFAs) and alcohols. With the exception of DS, these analyses were performed weekly for the FBR feed solution, and the influent acidity or alkalinity and soluble sugars content were also analyzed. All the analyses were performed on filtrated samples (0.45 lm polyethersulfone membrane syringe filter, Whatman, Kent, UK) except for pH, alkalinity and acidity.

The analysis of DS, pH, sulfate, DOC, total alkalinity and acidity were performed as described by Auvinen et al. (2009). The presence of carboxylic acids and alcohols (ethanol, butanol, acetate, propionate, butyrate, iso-butyrate, valerate and caproate) was determined with a gas chromatograph as described by Koskinen et al. (2007). The dissolved Fe and Zn were analyzed with an atomic absorption spectrometer (AAS, Perkin-Elmer 1100B, USA) according to standards SFS 3044 (SFS, 1980a) and SFS 3047 (SFS, 1980b). Chemical oxygen demand of soluble compounds (CODs) was analyzed from filtrates (0.45 lm) with dichromate method according to standard SFS 5504 (SFS, 1988). The hydrogen sulfide interfering with the CODs was removed from the samples prior to analysis as described in SFS (1988) by purging the acidified samples with N2 for 30 min. The RCGH solution samples for the soluble sugars analysis were filtrated through 0.45 lm polyethersulfone membrane syringe filter (Whatman, Kent, UK). The soluble sugars were analyzed with a colorimetric anthrone-method by Hansen and Møller (1975) and Shimadzu UV-1601 spectrophotometer. The biomass accumulation in the FBR liquid was measured as volatile suspended solids (VSS) and accumulation of the inorganic compounds as total suspended solids (TSS) as described by Kaksonen et al. (2004a). The carrier material bound biomass was analyzed as volatile solids (VS) according to the standard SFS 3008 (SFS, 1990). 2.5. Calculations The proportions of HS- and H2S of the DS at the corresponding pH were calculated using sulfide pKa 6.89 at 35 °C (Kawazuishi and Prausniz, 1987). The precipitation of the metals in the reactor was estimated using the OLI chemical modeling program (OLI stream analyzer 2.0, OLI systems Inc. Morris Plains, NJ, USA). 3. Results 3.1. Batch bottle assays

Fig. 2. The organic loading rate of the FBR: the soluble sugars load (h), the soluble sugars load calculated as theoretical COD load (s) according to Isa et al. (1986) and the measured chemical oxygen demand of the soluble compounds (CODs) loading rate (d).

The results of the two batch bottle assays were as presented in Fig. 4A–G. The pH in the batch bottles with RCGH decreased from the initial pH of 8 to 6.1–7.0 during the first week (Fig. 4A). This resulted from the degradation of RCGH and plant material to volatile fatty acids (VFAs) (Fig. 4B). Ethanol and butanol were not detected (data not shown). The total VFA concentration was at the maximum after the first 7 days, and varied between 300 and 1300 mg L1. The total VFAs constituted of acetate (84%),

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Fig. 4. The results of the batch bottle assays: (A) pH, (B) total VFAs (acetate, butyrate and propionate), (C) dissolved sulfide (DS), (D) sulfate concentration, (E) dissolved organic carbon (DOC), and (F) chemical oxygen demand of the soluble compounds (CODs). Biological control (+) that contained no substrate is shown with a dashed line, 0.26 g L1 soluble sugars (d), 0.35 g L1 soluble sugars (s), 0.69 g L1 soluble sugars (), 8 g L1 solid dry CG (h), 16 g L1 solid dry CG (j), and 8 g L1 solid hydrolyzed CG (N). The error bars indicate the standard deviation between the replicates.

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propionate (6%) and butyrate (10%). The solution pH of the bottles containing RCGH was resumed with bicarbonate buffer, and the total VFA concentration started to decrease after 21 days (Fig. 4B). For the solid dry plant material the buffering capacity was sufficient, and no bicarbonate supplementation was required. Also, the acetate production rate in the bottles with dry plant material remained at a lower level than with RCGH. Sulfide production started during the first 7 days in all the bottles with RCGH and dry plant material (Fig. 4C). The highest DS concentration in the batch bottles with RCGH was 530 mg L1 and with dry plant mate-

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rial 350 mg L1. The sulfate concentration decreased steadily (Fig. 4D). All the soluble sugars were consumed during the first 7 days (Fig. 4E). The initial DOC concentration with RCGH was 600–1300 mg L1 and with dry plant material 270–480 mg L1 (Fig. 4F). The DOC concentration started to decrease simultaneously with the VFA oxidation, resulting in final DOC concentrations of 390–1000 mg L1 and 150–270 mg L1 with RCGH and dry plant material, respectively. The degradation of the dry plant material was monitored with CODs. Fig. 4G shows a CODs decrease from 780–1200 to 610–990 mg L1 during the experiment.

Fig. 5. Results of the fluidized-bed bioreactor experiment: (A) sulfate loading and reduction rate, (B) percent sulfate reduction, (C) chemical oxygen demand of soluble compounds (CODs) loading and removal rate, (D) percent CODs removal, (E) dissolved organic carbon (DOC) loading and oxidation rate, (F) percent DOC oxidation, (G) iron loading and precipitation rate, and (H) percent iron precipitation. The loading rate is indicated with (s), the reduction/oxidation/precipitation rate with (d) and percent reduction/oxidation/precipitation with ().

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3.2. Reactor experiment 3.2.1. Sulfate and sulfide Sulfate reduction rate (SRR) increased from 0.6 to 3.3– 5.8 g L1 d1 during the experiment with mean SRR of 2.2 g L1 d1 between days 566 and 640 (Fig. 5A). The mean percent sulfate reduction was 31% (Fig. 5B). The low percent sulfate reduction resulted from excess sulfate in the medium (the RCGH contained sulfate from sulfuric acid used in the hydrolysis). The DS concentration in the FBR was 50–340 mg L1, and on average 79% of the DS was in the H2S form due to low FBR pH (Fig. 6A). 3.2.2. Substrate utilization In the FBR, the CODs removal increased from 0.2 to 1.2– 2.1 g L1 d1 (Fig. 5C). The mean CODs removal rate during days 566–640 was 1.3 g L1 d1. The mean percent CODs removal was 75% (Fig. 5D). The DOC oxidation rate increased from 0.2 to 0.8 g L1 d1 (Fig. 5E). The mean percent DOC oxidation was 80% (Fig. 5F). The CODs removal rate and DOC oxidation rates remained unstable after the FBR heated on day 590. The percent CODs and DOC removal were high, although VFAs accumulated in the FBR after day 590 (Fig. 6B). The total VFA concentration in the influent was on average 54 mg L1, and was

mainly acetate (Fig. 6B). Acetate constituted 30–99% of the total VFAs in the FBR effluent, the rest being mainly propionate and some butyrate. Ethanol and butanol were not detected in the influent and effluent (data not shown). The proportion of acetate of the total effluent VFAs increased towards the end of the experiment. The VFA oxidation capacity of the FBR decreased after the FBR heated on day 590. The VFA oxidation was partially resumed due to regular re-inoculation of the FBR and bicarbonate buffer addition, as the VFA concentration in the FBR effluent started to decrease after day 618. The RCGH contained also some solid material, which may have become hydrolyzed and further served as electron donor. 3.2.3. Iron and zinc precipitation The iron loading to the FBR was gradually increased and the iron precipitation rate increased from 0.1 to 0.84 g L1 d1 (Fig. 5G). The mean percent iron precipitation was 98% (Fig. 5H). The iron feeding controlled the DS concentration in the FBR, therefore decreasing sulfide toxicity. Zn precipitation rate was on average 16 ± 5 mg L1 d1 with 99% precipitation (data not shown). The precipitation of iron and zinc in the FBR was estimated using OLI stream analyzer program, and iron precipitated as hexagonal iron(II)sulfide (FeS) and zinc as cubic zinc sulfide (ZnS).

Fig. 6. The results of the fluidized-bed bioreactor experiment: (A) dissolved sulfide (DS) concentration (d) and H2S concentration, (B) total VFA concentration in the influent (–) and effect (+), (C) pH: influent (h) and effluent (j), (D) alkalinity production rate(*), (E) carrier material bound biomass (VS) (N), and (F) FBR liquid biomass (VSS) (h) and solids (TSS) (j) concentrations.

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3.2.4. Neutralization capacity The influent pH (7.8–4.5) was gradually decreased during the experiment. The acidic influent was neutralized in the FBR to pH 7 (Fig. 6C). The alkalinity production (Fig. 6D) includes the neutralization of the influent acidity. The influent used for the first 30 days was neutral, and this alkalinity was taken into account. The measured total alkalinity production increased from 20 to 60 m mol L1 d1 during the experiment (corresponding to increase from 1 to 3 g L1 CaCO3 alkalinity). The precipitation of metals produces acidity to the FBR liquid, and this combined with VFA accumulation in the FBR caused decrease of the pH towards the end of the experiment. This was compensated with regular addition of bicarbonate buffer on days 590–640. Therefore, during this period, only part of the measured alkalinity was due to sulfidogenic electron donor oxidation.

4.2. RCGH consumption in the FBR Only a few SRB species are able to use sugars, and there are a few reports on the use of carbohydrates by SRB. The ability to directly use fructose as electron donor for sulfate reduction has been reported for Desulfotomaculum nigrificans (Klemps et al., 1985), Desulfovibrio fructosovorans (Ollivier et al., 1988) and Desulfovibrio salexigens (Zellner et al., 1989). These species are also able to ferment fructose in the absence of sulfate (Klemps et al., 1985; Ollivier et al., 1988; Zellner et al., 1989). Also Desulfurispora thermophila was able to use mannose, glucose and fructose as electron donors for sulfate reduction (Kaksonen et al., 2007). The sum equation for sulfidogenic glucose oxidation is (Thauer et al., 1977):  þ C6 H12 O6 þ SO2 4 þ H ! 2CH3 COOH þ 2CO2 þ HS þ 4H2 O 0

ðDG0  358:2 kJ=molÞ: 3.2.5. Biomass The carrier bound biomass, VS, decreased from 41 to 25 mg g1 (carrier material) (Fig. 6E) and suspended biomass, VSS, decreased from 0.7 to 0.1 g L1 during the experiment (Fig. 6F). The total solids concentration in the FBR liquid, TSS, decreased from 2.5 to 1.6 g L1 (Fig. 6F). The regular inoculation combined with the high iron loading and bicarbonate buffer addition caused increase of VSS and TSS after day 590, but the majority of the precipitates, bicarbonate and added biomass were washed-out from the FBR during the experiment. The solid fraction of the RCGH might also have been retained in the FBR, but was presumably degraded in the FBR and not washed-out. 4. Discussion 4.1. RCGH and dry plant material as sulfate reduction substrates The batch bottle assays showed that RCGH containing soluble sugars is a suitable substrate for sulfate reduction. The sugars in the RCGH were presumably first fermented to VFAs by non-sulfate reducing microorganisms. Most of the known SRB are not able to utilize polymeric substrates, and carbohydrates are rarely utilized (Widdel and Pfenning, 1984). There is also possibility that the dry cellulosic material was hydrolyzed directly by bacteria and the hydrolysis products were utilized by SRB, and there is no fermentative step included as shown by Zavarin et al. (2008). In the present study the accumulation of the VFAs indicates that the cellulose degradation products and soluble sugars were fermented, and the SRB were utilizing the fermentation products. The major VFA produced was acetate. Sulfidogenic acetate oxidation requires neutral pH, as the proportion of the undissociated, toxic acetic acid increases as the pH decreases (Thauer et al., 1977). The acetate oxidizing SRB grow slowly due to low energy yield from acetate oxidation (Eq. (4)) (Thauer et al., 1977). Because the sulfidogenic acetate oxidation produces most of the sulfide and alkalinity, the use of RCGH as sulfate reduction substrate requires careful balancing of the RCGH load with the VFA oxidation capacity of the SRB. In the batch bottle assay with the solid, dry plant material, the inoculum efficiently utilized the plant material degradation products for sulfate reduction. Moreover, the release rate of the VFAs was balanced with the oxidation capacity of the SRB. For these reasons, the pH remained neutral and the acetate concentration remained at lower level than with RCGH. The results in Table 3 indicate that the hydrolyzation improved the ability of the SRB to utilize the plant material as substrate, as the DS yield per used dry plant material was higher (6.26 m mol H2S g1 plant material) for RCGH than for dry plant material (0.83 m mol H2S g1 plant material). Also higher total DS concentrations were obtained when RCGH was used (Fig. 4C).

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ð2Þ

Unidentified SRB species from anaerobic digester were reported to have saccharolytic activity (Joubert and Britz, 1987). These SRB fermented carbohydrates to acetate, ethanol, H2 and CO2, and sulfide was produced in the presence of sulfate (Joubert and Britz, 1987). Also Desulfovibrio termitidis isolated from termite gut has been shown to degrade carbohydrates (Trinkerl et al., 1990). The mean ratio of consumed CODs to consumed sulfate was 1.0 in the FBR experiment. For stoichiometric sulfate reduction, this value is 0.67 (Choi and Rim, 1991). Therefore, the soluble sugars in the RCGH were presumably degraded to VFAs by fermentative bacteria, and the fermentation products served as electron donors for SRB. The major degradation product in the FBR was acetate. The microbial community of the FBR inoculum has been shown to be diverse, and it also contained fermentative bacteria (Kaksonen et al., 2004b,c). The fermentation of the RCGH soluble sugars to acetate and hydrogen proceed according to equation (Thauer et al., 1977):

C6 H12 O6 þ 4H2 O ! 2CH3 COO þ 4H2 þ 2HCO3 þ 4Hþ ðDG00  206:3 kJ=reactionÞ:

ð3Þ

The produced hydrogen and acetate were then oxidized by SRB (Thauer et al., 1977):  4H2 þ SO2 4 ! H2 S þ 2H2 O þ 2OH

ðDG00  151:9 kJ=reactionÞ;  00  CH3 COO þ SO2 4 ! 2CHO3 þ HS ðDG  47:6 kJ=reactionÞ:

ð4Þ ð5Þ

The fermentation of the RCGH soluble sugars to lactate and ethanol is also possible. Ethanol and lactate were the previous electron donors for the FBR microbial community (Nevatalo et al., 2010) used in this study. These compounds become quickly oxidized to acetate in the FBR. The RCGH contained some solid material, which may have also been hydrolyzed in the FBR and therefore served as substrate. Moreover, the inoculae added to FBR contained some DOC, but this was not quantified in this study. 4.3. Mine waste water treatment in the FBR In the FBR experiment with RCGH, the sulfate reduction started well. The SRR increased with the increasing organic loading rate. The SRR results of the present study are compared to results from reactor studies with low-cost sulfate reduction substrates in Table 1. The highest metal precipitation rates were 0.84 g Fe L1 d1 and 16 mg Zn L1 d1. The increase of the iron loading did not remarkably improve the SRR, although the precipitation of iron sulfide decreases the concentration of the toxic H2S. The major factor influencing the SRR in the FBR was the limited substrate oxidation capacity rather than the inhibitory effects of H2S,

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Table 3 Summary of the sulfide production in the batch bottle assays with RCGH and dry plant material. Substrate

Production as mmol per L culture medium

Yield as mmol per g added CODsa

Yield as mmol per g removed CODsa

Yield as mmol per g plant material usedb

Dry plant material RCGH

8.99 14.7

16.4 16.8

99.5 9.9

0.83 6.26

a

Hydrogen sulfide was removed from the sample before analysis. The amount of dry plant material added to the batch bottle. With RCGH the amount of dry plant material needed to produce the amount of soluble sugars added to the bottle. b

undissociated acetic acid or the metal precipitates. The suspended biomass (VSS) in the FBR increased only slightly due to repeated inoculation, and the TSS and VSS were mainly being washed-out from the FBR. 4.4. The effect of the heating on reactor performance The SRR became unstable after day 590 due to FBR heating, but the SRR resumed as the FBR loading was decreased and the FBR was regularly re-inoculated. The acetate oxidizing SRB were inhibited by the FBR heating on day 590, decreasing the VFA oxidation and neutralization capacity. The re-inoculation with enrichment cultures containing acetate oxidizing SRB resumed the acetate oxidation capacity partially. Due to the length of the experiment, the acetate oxidizing SRB did not grow at the same rate with the increasing organic loading and, therefore, acetate accumulated in the FBR. Because sulfidogenic acetate oxidation produces majority of the alkalinity, the FBR pH decreased, and this in turn caused the increase of toxic H2S form of DS. The addition of bicarbonate buffer from day 590 onwards to the FBR had limited effect on VFA oxidation capacity, unlike in the batch bottle assays. 4.5. Amenability of the RCGH for full-scale AMD treatment The amenability of the RCGH as substrate for a full-scale treatment plant was estimated by calculating the required reactor size with influent AMD flow of 2000 m3 h1 and the assumption that 65% of the CODs is consumed with H2S production rate of 0.4 g H2S L1 d1. Then 205 kg h1 H2S would be produced and 370 kg h1 iron could be precipitated, requiring the supplementation of 532 kg h1 soluble sugars. The efficiency of the hydrolyzation of dry plant material to soluble sugars was 10–17%, thus the requirement for solid plant material is 5450 tons per year. In Finnish conditions, this would require a cultivation area of 970–1300 hectares, as the dry matter yield of P. arundinacea is 6–8 tons per hectare (including 30% harvest loss) (Pahkala et al., 2005). In 2005 the cultivation area of P. arundinacea in Finland was 9000 hectares (Finnish Ministry of Forestry and Agriculture, 2005). The acid hydrolyzation used in this study would result in acidified plant material that requires neutralization prior to disposal, which increases operational costs. 5. Conclusion The hydrolyzed P. arundinacea plant material (RCGH) was a suitable substrate for SRB. The soluble sugars of the RCGH were presumably fermented by fermentative bacteria to volatile fatty acids and hydrogen, which served as electron donors for SRB. In the FBR fed with RCGH and synthetic mine waste water, sulfate reduction rate was 2.2–3.3 g L1 d1. The acidic, synthetic mine wastewater was neutralized in the FBR, and Fe and Zn were 99% precipitated, the maximum iron precipitation rate being 0.84 g Fe L1 d1. Acetate accumulation indicated that the process was limited by the acetate oxidation rate by the SRB.

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