w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 5 4 5 e5 5 3

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Biological oxidation of Mn(II) coupled with nitrification for removal and recovery of minor metals by downflow hanging sponge reactor Linh Thi Thuy Cao a, Hiroya Kodera a, Kenichi Abe b, Hiroyuki Imachi c, Yoshiteru Aoi d, Tomonori Kindaichi a, Noriatsu Ozaki a, Akiyoshi Ohashi a,* a

Department of Civil and Environmental Engineering, Graduate School of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8527, Japan b Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan c Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan d Institute for Sustainable Science and Development, Hiroshima University, 2-313 Kagamiyama, VBL403, Higashi-Hiroshima, Hiroshima 739-8527, Japan

article info

abstract

Article history:

Biogenic manganese oxides (bio-MnO2) have been shown to absorb minor metals. Biore-

Received 4 August 2014

actor cultivation of heterotrophic manganese oxidizing bacteria (MnOB), which produce

Received in revised form

bio-MnO2 via oxidation of Mn (II), can be expected to be involved in a promising system for

30 September 2014

removal and recovery of minor metals from wastewater. However, MnOB enrichment in

Accepted 2 October 2014

wastewater treatment is difficult. This study investigated whether MnOB can be cultivated

Available online 23 October 2014

when coupled with nitrification in a system in which soluble microbial products (SMP) from nitrifiers are provided to MnOB as a substrate. A downflow hanging sponge (DHS)

Keywords:

reactor was applied for MnOB cultivation with ammonium (NHþ 4 ) and Mn (II) continuously

Biogenic manganese oxides

supplied. During long-term operation, Mn (II) oxidation was successfully established at a

Biological manganese oxidation

rate of 48 g Mn m3 d1 and bio-MnO2 that formed on the sponges were recovered from the

Downflow hanging sponge (DHS)

bottom of the reactor. The results also revealed that Ni and Co added to the influent were

reactor

simultaneously removed. Microbial 16S rRNA gene clone analysis identified nitrifiers

Nitrification

supporting MnOB growth and showed that only one clone of Bacillus subtilis, which was

Minor metals removal and recovery

affiliated with a known MnOB cluster, was present, suggesting the existence of other novel bacteria with the ability to oxidize Mn (II). © 2014 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel./fax: þ81 82 424 5718. E-mail address: [email protected] (A. Ohashi). http://dx.doi.org/10.1016/j.watres.2014.10.002 0043-1354/© 2014 Elsevier Ltd. All rights reserved.

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1.

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Introduction

Minor metals are essential to modern technologies; however, their use and manufacture inevitably result in a portion of the metal being discharged into the environment. Heavy metals have negative effects on aquatic systems, even when present at low concentrations; accordingly, the removal and recovery of minor metals from wastewater streams is necessary. Recently, biosorption technology for the removal and recovery
s et al., of metals has received a great deal of attention (Andre 2011). Biomineralization has also been investigated as a promising method. Specifically, the application of biogenic manganese oxides (bio-MnO2), which are products of biologically mediated Mn(II) oxidation processes, is very attractive because these compounds adsorb significant amounts of minor metals owing to their structural features (Tebo et al., 2004, 2010; Miyata et al., 2007; Hennebel et al., 2009). The key points for bio-MnO2 application are to facilitate enrichment of manganese oxidizing bacteria (MnOB) and clarify how and why MnOB oxidize Mn(II) to produce bio-MnO2 (Tebo et al., 2005, 2010). To date, no autotrophic bacteria with the ability to oxidize Mn(II) have been identified. Ali and Stokes (1971) observed slow, but appreciable growth of Leptothrix discophorus (formerly Sphaerotilus discophora) under autotrophic conditions with Mn(II) as the sole available source of energy. They also reported that Mn(II) stimulated the heterotrophic growth of L. discophorus based on increases in total nitrogen, protein, and DNA of the cells in flask cultures. However, the conclusions of their report have been questioned by van Veen (1972), who found that the presence of Mn(II) contributed to harvested cellular protein, affecting estimation of cell growth. Hajj and Makemson (1976) demonstrated that Mn(II) oxidation was not concomitant with heterotrophic growth of L. discophorus, but occurred late in growth based on elimination of the manganese effect on biomass measurement. All currently known MnOB are heterotrophs (Ehrlich and Newman, 2008; Tebo et al., 2010). As a result, organic carbon is supplied during cultivation of MnOB. However, cultivation is not easily accomplished by simply adding substrate to open mixed cultures owing to competition with other fast-growing heterotrophs. Accordingly, a rich medium might not be favorable for enrichment of MnOB in engineering processes, and a better approach may be the use of very low concentrations of organic substrate. Despite its potential, this method may not enable organic loading rates to reach levels sufficient to enhance MnOB without interfering with the overall wastewater treatment processes. Therefore, we have conceived a unique strategy to continuously provide low concentrations of organic substrate during cultivation of MnOB. During autotrophic nitrification, a small amount of organic carbon is excreted (Rittmann et al., 1994). Heterotrophs were often found with nitrifiers in autotrophic nitrifying biofilms (Kindaichi et al., 2004; Okabe et al., 1999, 2005; Matsumoto et al., 2010) and also with nitrite oxidizing bacteria (NOB) in nitrite oxidizing granules (Ni et al., 2011a,b) without any external organic substrate. This phenomenon can be attributed to the fact that soluble microbial products (SMP) produced by

autotrophs can support heterotroph growth (Kindaichi et al., 2004; Merkey et al., 2009; Matsumoto et al., 2010; Ni et al., 2011a, b; Kang et al., 2014). Accordingly, cultivation of nitrifiers on ammonium (NHþ 4 ) without any added organic substances might result in the generation of SMP that could serve as a substrate for heterotrophic MnOB. Even if the NHþ 4 loading rate were increased, by decreasing hydraulic retention time (HRT) with maintaining low NHþ 4 concentration, a low concentration of SMP would be kept in the reactor (Ni et al., 2011a). Indeed, nitrification activity was shown to stimulate Mn(II) oxidation in a rapid sand filter system (Vandenabeele et al., 1995). Because nitrifiers are slow-growing bacteria, coupled growth of MnOB with nitrifiers would be very slow. Biofilm type reactors may be favorable to the retention of slowgrowing bacteria. The downflow hanging sponge (DHS) reactor, which is a kind of trickling filter, has been developed for cost-effective wastewater treatment without imposing external aeration. Such a DHS reactor has the ability to enrich even low growth rate bacteria such as nitrifiers (Chuang et al., 2007a, b; Imachi et al., 2011). Another distinguishing feature of the DHS reactor is that production of bio-MnO2 on substratum carriers of sponge is expected to cause particulates of bio-MnO2 to fall to the bottom, where they can be easily collected for minor metals recovery (Fig. 1). The present study was conducted to determine whether heterotrophic MnOB could be cultivated in combination with nitrification, and investigate the removal rates for Mn(II) and minor metals by DHS reactors using synthetic wastewater.

Fig. 1 e Schematic diagram of a DHS reactor used to enrich MnOB for removal and recovery of minor metals. Artificial wastewater containing Mn(II) and minor metals was fed to the reactor with a substrate of NHþ 4 and minerals. Air was supplied to generate an aerobic environment.

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2.2.

2.

Materials and methods

2.1.

Experimental set-up and operational conditions

A DHS reactor with a 4.2 L column (110 cm in height and 7 cm in diameter) was used for cultivation of MnOB to produce bio-MnO2 (Fig. 1). The column contained a string of 32 polyurethane sponge cubes (each 2  2  2 cm3) connected to each other diagonally in series as the biofilm substratum. The sponges were inoculated by soaking in diluted activated sludge obtained from a municipal sewage treatment plant. A synthetic substrate composed of Mn (II) (MnCl24H2O), ammonium ((NH4)2SO4) and minerals, but no organic carbon, was prepared to enable nitrification. The composition of minerals was 30.4 mg L1 NaNO3 (5 mg N L1), 100 mg L1 KHCO3, 100 mg L1 NaHCO3, 11 mg L1 Na2HPO412H2O, 20 mg L1 MgSO47H2O, 5 mg L1 CaCl22H2O, 0.1 mg L1 FeSO47H2O, 0.025 mg L1 CuSO45H2O, 0.005 mg L1 Na2SeO4, 0.019 mg L1 NiCl26H2O, 0.024 mg L1 CoCl26H2O, 0.022 mg L1 Na2MoO42H2O, 0.001 mg L1 H3BO3, and 0.043 mg L1 ZnSO4. The concentrations of Mn (II) and NHþ 4 eN were varied from 5 to 20 mg L1 during 449 days of operation (phases 1e9) to identify appropriate conditions for Mn(II) oxidation. In phases 7 and 8, Ni(II) and Co(II) were added to the substrate at 2.5 mg L1 to investigate the adsorption removal efficiency of Mn oxides produced. The DHS reactor was operated in a temperature-controlled room at 25  C. The substrate was fed to the top of the reactor at a HRT in the range of 1.5e12 h based on sponge volume (Table 1). The air was supplied to the reactor at an air retention time (ART) of 8 or 4 days based on column volume, and the off gas was collected to evaluate oxygen consumption. In addition to the above DHS operation, two more operations were conducted to investigate whether autotrophic Mn(II) oxidation can occur and MnOB can be enriched under eutrophic conditions. One was operated using the same synthetic substrate of 5 mg Mn(II) L 1 except for adding ammonium. The other one was performed with a modified K medium (Krumbein and Altmann, 1973) of 100 mg COD L 1 containing Mn(II) at 5 mg L 1 at a HRT of 12 h.

Analytical methods

The influent and effluent were routinely sampled twice a week to investigate the performance of Mn(II) oxidation. Prior to analysis, the samples were filtered using PTFE 0.2-mm poresize membranes (Advantec Co., Ltd., Tokyo, Japan). The dissolved Mn(II), Ni(II), and Co(II) were determined by the periodate oxidation method using a Hach water quality analyzer (DR-2800; Hach Co., Loveland, CO, USA). The concentrations of NHþ 4 eN and NO3eN were measured by ion chromatography (Shimadzu HPLC 10A), while the concentration of oxygen in the off gas was determined by gas chromatography (Shimadzu GC-8APT). The particulates produced in the reactor were checked whether they were MnO2 using Leucoberbeline Blue I (LBB; SigmaeAldrich) according to Krumbein and Altmann (1973) and Tebo et al. (2007).

2.3.

Microbial community analysis

Three samples of sludge on the sponges were collected by squeezing from the upper, middle, and lower parts of the reactor on day 444, after which they were washed with phosphate buffer. DNA extraction was then carried out using a Fast DNA spin kit for soil (MP Biomedicals, Irvine, CA, USA) according to the manufacturer's instructions. The three extracted DNA samples were mixed in equal amounts after being adjusted to approximately the same concentration. Polymerase chain reaction (PCR) amplification of the 16S rRNA gene for the mixed DNA was performed using a ONE Shot LA PCR MIX (Takara Bio, Otsu, Japan) with the EUB338Fmix (50 ACTCCTACGGGAGGCAGC-30 ; 50 -ACACCTACGGGTGGCTGC-30 ; 50 -ACACCTACGGGTGGCAGC-30 ) - BAC1389Rm17mer (50 ACGGGCGGTGTGTACAA-30 ) primer set. The PCR conditions were as follows: 5 min of initial denaturation at 94  C followed by 20 cycles of 30 s at 94  C, 30 s at 50  C, and 80 s at 72  C and then final extension at 72  C for 4 min. The PCR product was subsequently purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and then cloned using a TOPO XL PCR Cloning Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The cloned 16S rRNA gene fragments were sequenced from both ends at the Dragon Genomics Center, after which they were classified using the

Table 1 e Operational conditions of DHS reactor during nitrification coupled with Mn(II) oxidation. Phase

1 2 3 4 5 6 7 8 9 a b

Day

0e77 78e136 137e157 158e211 212e234 235e359 360e379 380e399 400e449 HRT: hydraulic retention time. ART: air retention time.

Mn2þ

Ni2þ

Co2þ

NHþ 4

NO-3

HRTa

ARTb

(mg/L)

(mg/L)

(mg/L)

(mg-N/L)

(mg-N/L)

(hour)

(day)

20 20 10 5 5 5 5 5 5

0 0 0 0 0 0 2.5 2.5 0

0 0 0 0 0 0 2.5 2.5 0

5 10 20 5 5 5 5 5 5

5 5 5 5 5 5 5 5 5

12 12 12 3 1.5 1.5 1.5 3 3

8 8 8 8 8 4 4 4 4

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FastGroupII (Yu et al., 2006) program. Sequences showing 97% identity were grouped into operational taxonomic units (OTUs). The 16S rRNA gene sequence data were deposited in the GenBank, EMBL, and DDBJ databases under accession numbers AB980115 to AB980196.

3.

Results

3.1.

Mn(II) oxidation coupled with nitrification

We initially operated the reactor with influent containing 1 at a hydraulic retention 20 mg Mn(II) L1 and 5 mg NHþ 4 eN L time (HRT) of 12 h in Phase 1 (from day 0e77) because there was little information regarding the appropriate cultivation conditions available (Table 1). Soon after startup, the Mn(II) concentration of effluent decreased, while no change was observed after 18.8 mg L1 was reached throughout Phase 1 (Figs. 2A, B), suggesting that 5% of the Mn(II) removal might have occurred via adsorption onto microbial cell surfaces. Conversely, NHþ 4 was completely oxidized to NO3 (Fig. 2C). These findings indicate that MnOB growth was not initially coupled with nitrification. However, the failure to enrich MnOB might have been due to the low level of SMP produced by nitrifiers at approximately 10 g N m3 d1 of nitrification rate, which was very slow for wastewater treatment (Fig. 2D). To improve Mn(II) oxidation by producing more SMP, the 1 in NHþ 4 eN concentration was increased from 5 to 10 mg L Phase 2 (day 78e136). Even though the nitrification rate was increased to 100% NHþ 4 oxidation, no change in Mn(II) removal was observed for 58 days, indicating that this nitrification rate was insufficient for MnOB cultivation. Ghiorse (1984) and Adam et al. (1985) reported that high concentrations of Mn(II) were toxic to heterotrophic MnOB, which may also explain the lack of Mn(II) removal. For Phase 3 (day 137e157), the reactor was operated at an 1 accompanied with a NHþ 4 eN concentration of up to 20 mg L reduction in Mn(II) concentration to 10 mg L1. As expected, the nitrification rate increased by approximately two times even though some NHþ 4 remained in the effluent. However, this condition resulted in no Mn(II) removal because the Mn(II) concentration in the effluent became higher than that of the influent, indicating that some of the Mn(II) that had been adsorbed onto the cells during Phases 1 and 2 was desorbed. The effluent had a pH of 5.6, while that of the influent was maintained at 7.5 (Fig. S1), which was likely caused by high nitrification. This decrease in pH triggered Mn(II) desorption. Neutral or higher pH is favorable to chemical and biological Mn(II) oxidation (Kothari, 1988; Mouchet, 1992; Vandenabeele et al., 1992; Hallberg and Johnson, 2005). To prevent a decrease in pH by nitrification, the NHþ 4 eN concentration in the influent was decreased back to 5 mg L1 in Phase 4 (day 158e211) by maintaining a high NHþ 4 loading rate via a decrease in HRT from 12 to 3 h. For Mn(II), the concentration was decreased to 5 mg L1 to mitigate the toxicity of Mn(II) to MnOB. As expected, a high nitrification rate was obtained without a decrease in pH. Under these conditions, Mn(II) removal was again observed, and the removal steadily increased with cultivation time until its complete removal. Specifically, there was a Mn(II) removal rate of 40.5 g m3 d1 (based on sponge

Fig. 2 e Nitrification and Mn oxidation performance of the DHS reactor. Time course of influent and effluent concentrations of Mn(II) (A), Mn(II) loading and removal rates (B), influent and effluent concentrations of NHþ 4 eN, NO-3eN (C), nitrification rate and oxygen consumption rate (D).

volume) at the end of Phase 4 (on day 211; Figs. 2A, B). Around day 200, the color of the sponges had changed from light brown to black (Fig. 3), and black fine particles were deposited at the bottom of the DHS reactor. The black particulates on sponges were confirmed to be Mn oxides by LBB method (Fig. S2). These findings indicate that Mn(II) oxidation successfully produced Mn oxides in the DHS reactor when coupled with nitrification. Microscopic observation of DAPI-

w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 5 4 5 e5 5 3

549

oxidation was successfully established at a maximum rate of 48 g Mn m3 d1.

3.2.

Ni and Co removal with bio-MnO2 production

To investigate the ability of biogenic Mn oxides (bio-MnO2) produced in the DHS reactor to adsorb minor metals, Ni(II) and Co(II) were added in the influent during Phase 7 (day 360e379, almost 3 weeks) at a concentration of 2.5 mg L1 with Mn(II) at 5 mg L1. Soon after the addition, neither Ni(II) nor Co(II) were detected in the effluent, while Mn(II) was immediately increased to 6.4 mg L1, indicating complete Ni(II) and Co(II) removal and negative Mn(II) removal (Figs. 2A and 4). These results indicate that not all Mn(II) in the influent was oxidized, but a portion was adsorbed on the produced bio-MnO2, and some of the adsorbed Mn(II) was exchanged for Ni(II) and Co(II). However, Mn(II) removal subsequently recovered while deterioration of Ni(II) and Co(II) removal was observed to form a plateau. The stable simultaneous removal of Mn(II), Ni(II), and Co(II) suggests that Mn(II) was oxidized to bio-MnO2, on which Ni(II) and Co(II) were adsorbed in the reactor. Even though the minor metals loading rate was reduced by increasing the HRT to 3 h in Phase 8, the removal performance was maintained at 16.4, 1.7, and 7.9 g m3 d1 for Mn(II), Ni(II), and Co(II), respectively. The removal molar ratios of Ni(II) and Co(II) to Mn(II) were 9 and 45%, respectively, which are comparable to the values reported by Tani et al. (2004), who investigated bio-MnO2 produced by the fungal strain KR21-2. These results indicate that the adsorption affinity of Co(II) to bio-MnO2 was higher than that of Ni(II). The characteristics of bio-MnO2 obtained in this study agree with those reported by Kay et al. (2001) and Tani et al. (2004). The performance in this experiment demonstrated the possibility of continuous minor metals removal from wastewater for recovery by the DHS reactor via MnOB cultivation coupled with nitrifiers.

3.3.

Fig. 3 e Black sponges covered with bio-MnO2 in the DHS reactor on day 203.

stained biomass samples showed microorganisms embedded in the black fine particles of Mn oxide (Fig. S3). Reactor operation was continued to investigate the performance of Mn oxidation. In Phase 5 (day 212e234), the NHþ 4 loading rate was doubled by decreasing the HRT from 3 to 1.5 h. Additionally, to increase the oxygen loading, the air flow rate was increased in Phase 6 (day 235e359) to maintain the oxygen concentration at a high level in the reactor because high oxygen consumption was observed in Phase 5 (Figs. 2D and Fig. S1). Even though a higher nitrification rate was observed at the same oxygen concentration as in Phase 4, the Mn(II) removal rate was not enhanced, and deteriorated slightly during Phase 6. After long-term operation, Mn (II)

Microbial community

To reveal the bacterial community of biomass retained in the DHS reactor, we constructed a 16S rRNA gene clone library for a DNA sample extracted on day 444 (Phase 9). Out of 82 clones, 3 and 16 were affiliated with the genera Nitrosomonadaceae and Nitrospira, which are ammonia and nitrite oxidizers, respectively (Table 2). Nitrifiers accounted for 23% of the total clones,

Fig. 4 e Loading and removal rates of Ni and Co by the DHS reactor.

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while the remaining 77% primarily consisted of unknown bacteria. These remaining clones were likely heterotrophs that could use SMP from the nitrifiers as the sole organic material because no organic substrate was fed to the reactor. The presence of Pedomicrobium, Hyphomicrobium or Aurantimonas in the Alphaproteobacteria, Leptothrix in the Betaproteobacteria, or Pseudomonas in the Gammaproteobacteria, which are known to be Mn(II) oxidizers and detected in oligotrophic environments (Tyler, 1970; Gebers, 1981; Ghiorse, 1984; Sly et al., 1988; Anderson et al., 2009), was not confirmed in the clone library. Only one clone affiliated with Bacillus subtilis (100% similarity) was identified as MnOB (van Waasbergen et al., 1993; Hullo et al., 2001). One clone likely does not account for the relatively high Mn(II) oxidation; accordingly, some of the unknown bacteria should play a role in biological Mn(II) oxidation in the reactor. Based on these findings, further studies to investigate the microbial community are warranted.

3.4. Mn(II) oxidation under autotrophic or eutrophic conditions We attempted to cultivate MnOB under autotrophic conditions by continuously providing 5 mg L1 of Mn(II) using the

DHS reactor. Even after 200 days of operation, no Mn(II) oxidation was observed (data not shown), indicating the failure of autotrophic cultivation. Existence of autotrophic MnOB remains unclear. In addition to the attempt to cultivate autotrophic MnOB, we attempted to enrich heterotrophic MnOB under open mixed culture conditions using the DHS reactor with K medium. Under the conditions, almost 81 mg COD L1 was removed by microorganisms grown on the sponge media after a short time. However, Mn(II) oxidation was not detected through operation for 207 days (data not shown), suggesting that the biomass was dominated by heterotrophic bacteria other than MnOB under eutrophic conditions.

4.

Discussion

During cultivation coupled with autotrophic nitrification, Mn(II) oxidation was successful (Fig. 5). SMP derived from nitrifiers are commonly provided to heterotrophic bacteria as substrate in nitrogen removal processes (Rittmann et al., 1994). Okabe et al. (2005) demonstrated that cross-feeding of SMP occurred in a nitrifying biofilm using 14C-labeled

Table 2 e Phylogenetic affiliations and clone numbers of bacterial 16S rRNA genes of the biomass and references of nitrification without Mn(II). Phylogenetic group

This study Number of clones

AOB (Betaproteobacteria) Nitrosomonas spp. NOB (Alphaproteobacteria) Nitrobacter sp. NOB (Nitrospira) Nitrospira spp. MnOB (Firmicutes) Bacillus sp. Other alphaproteobacteria Other betaproteobacteria Gammaproteobacteria

Percentage

3

4%

37%

25%

5

6%

1%

0%

13%

0%

39%

1% 12% 10% 7%

0% 2% 9% 3%

0% 3% 0% 9%

2% 2% 9% 7%

0% 1% 6% 24%

0% 0% 0% 9%

1% 4% 4% 4% 9%

0% 4% 0% 0% 0%

0% 0% 14% 0% 0%

2% 1% 1%

0% 0% 0% 13% 1% 100%

1% 0% 0% 0% 0% 100%

11 1 10 8 6

Deltaproteobacteria Actinobacteria Acidobacteria Bacteroidetes

2 2 7 6

Chamydiae Chlorobi Chloroflexi Gemmatimonadetes Planctomycetes

1 3 3 3 7

Verrucomicribia WS3 OD1 OP10 Spirochaetes Total

2 1 1

a

Remarks

Chuang et al., 2007b Kindaichi et al., 2004

82

( ) Represent the clone number detected.

Sublineage-I(8)a, Sublineage-II(3)

Burkholderiales(4), MND1(4) Xanthomonadales(4), Legionellales(2)

Gp1(1), Gp3(2), Gp4(4) b-17BO(1), PHOS-HE21(4), Saprospirales(1) OPB56(3) Anaerolineae(1), Chloroflexi(1) Pirellulales(1), Planctomycetales(4), Gemmatales(1), Other(1) Gp3(1), Opitutus(1)

100%

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Fig. 5 e Mn(II) removal rate in relation to the nitrification rate based on the sponge volume.

bicarbonate and microautoradiography combined with fluorescence in situ hybridization. Heterotrophic bacteria were reported to comprise about 50% of all bacteria in a biofilm grown using NHþ 4 as the only energy source (Kindaichi et al., 2004). In the present study, heterotrophs accounted for 73% of microbes grown in the reactor. In nitrification and nitritation processes similar to those employed in the present study (Kindaichi et al., 2004; Chuang et al., 2007b), bacteria belonging to the phyla Gammaproteobacteria and Bacteroidetes were commonly detected in the nitrifying microbial consortia (Table 2). These bacteria may have a high affinity for organic substances because the COD concentration of SMP must be very low in nitrifying consortia. Rittmann et al. (1994) estimated that amounts of SMP accompanied with nitrification were 0.073 mg COD/mg NHþ 4 eN from ammonia oxidizing bacteria (AOB) and 0.025 mg COD/mg NO-2eN from nitrite oxidizing bacteria (NOB). The small amount of SMP was then used by heterotrophic bacteria, resulting in its becoming smaller. Indeed, very low total organic carbon (TOC) levels were observed in the effluent of our experiment (data not shown). The present study demonstrated that some MnOB co-existed with other heterotrophs, suggesting that MnOB cultivated in this reactor should also have a high affinity for organic substances. Although MnOB failed to use organic substrates when in competition with commonly observed heterotrophic bacteria under eutrophic environments, oligotrophic conditions would be favorable for both MnOB and oligotrophic bacteria, allowing co-existence within nitrifying consortia. Indeed, this would explain why MnOB have been detected and isolated from oligotrophic environments such as drinking water treatment biofilms and riverbeds, where nitrifiers also exist (Vandenabeele et al., 1995; Burger et al., 2008). The Michaelis constant (Km) characterizes the substrate affinity of bacteria. The comparison of Km values of MnOB and other heterotrophic bacteria can reveal whether MnOB preferably survive in oligotrophic environments. However, Km values of MnOB have not yet been reported to our knowledge. Accordingly, future studies of Km should be conducted to understand the

551

characteristics of MnOB and identify promising methods for their enrichment. The members of Chloroflexi, which were detected at 4% in this reactor, are reported to preferentially use microbial products derived primarily from biomass decay (Okabe et al., 2005). Surprisingly, in the present nitrification process with Mn(II) oxidation, Planctomycetes (9%) and Gemmatimonadetes (4%) phyla were detected, while they were not found in nitrification process without Mn(II) oxidation (Table 2). Planctomycetes contains anaerobic ammonium oxidation (anammox) groups. Gemmatimonadetes consists of polyphosphate-accumulating bacteria (Zhang et al., 2003); however, there are no reports of these bacteria oxidizing Mn(II), and the role they played in Mn(II) oxidation coupled with nitrification is unclear. Microbial community analysis based on the 16S rRNA gene could not definitively identify which members were responsible for Mn(II) oxidation, and only indicated that Bacillus sp. were present, in the nitrifying consortia. Bio-MnO2 consists of stacked hexagonal sheets of MnO6 octahedra, and these particulates have numerous structural defects, particularly cation vacancies, which provide coordinated sites for binding of exogenous metal ions (Tebo et al., 2004; Villalobos et al., 2003). The bio-MnO2 produced by Pseudomonas putida strain MnB1/GB-1 reportedly comprise 17% of the vacant sites (Villalobos et al., 2003), while Leptothrix discophora SP6 occupied 12% (Saratovsky et al., 2006), and A. strictum KR21-2 22e30% (Grangeon et al., 2010). Ni(II) and Co(II) adsorbed on bio-MnO2 produced by fungal strain KR21-2 were reported to be present at molar ratios of 12% Ni/Mn and 30% Co/Mn, respectively (Tani et al., 2004). Although this study did not reveal which bacteria produced bio-MnO2, the absorbed molar ratios of 9% Ni/Mn and 45% Co/Mn were comparable to these previously reported values. The structure and adsorptive properties of bio-MnO2 likely do not differ greatly among organisms.

5.

Conclusions

An attempt to enrich MnOB to produce bio-MnO2 failed under high organic substrate loading conditions, but was effective when the strategy coupled to nitrification was employed. This is the first report of such enrichment under these conditions, and the method described herein will be effective for wastewater contaminated with bacteria. The continuous cultivation of MnOB with nitrifiers in a DHS reactor amended with ammonium holds great potential for the removal and the recovery of minor metals such as Ni and Co from wastewater.

Acknowledgments This research was supported by the Japan Society for the Promotion of Science (JSPS) as a Grant-in-Aid for Scientific Research (A), JSPS Fellows, and the Environment Research and Technology Development Fund (2-3K133004) of the Ministry of the Environment, Japan.

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Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.watres.2014.10.002.

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