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. 2022 Jul 21;12(9):187. doi: 10.1007/s13205-022-03250-y

Manganese oxidation and prokaryotic community analysis in a polycaprolactone-packed aerated biofilm reactor operated under seawater conditions

Masataka Aoki 1,2,✉, Yukina Miyashita 2, Toru Miwa 3, Takahiro Watari 4, Takashi Yamaguchi 3,4, Kazuaki Syutsubo 1,5, Kazuyuki Hayashi 2
PMCID: PMC9304527  PMID: 35875177

Abstract

Biogenic manganese oxides (BioMnOx) have been receiving increasing attention for the removal of environmental contaminants and recovery of minor metals from water environments. However, the enrichment of heterotrophic Mn(II)-oxidizing microorganisms for BioMnOx production in the presence of fast-growing coexisting heterotrophs is challenging. In our previous work, we revealed that polycaprolactone (PCL), a biodegradable aliphatic polyester, can serve as an effective solid organic substrate to enrich Mn-oxidizing microbial communities under seawater conditions. However, marine BioMnOx-producing bioreactor systems utilizing PCL have not yet been established. Therefore, a laboratory-scale continuous-flow PCL-packed aerated biofilm (PAB) reactor was operated for 238 days to evaluate its feasibility for BioMnOx production under seawater conditions. After the start-up of the reactor, the average dissolved Mn removal rates of 0.4–2.3 mg/L/day, likely caused by Mn(II) oxidation, were confirmed under different influent dissolved Mn concentrations (2.5–14.0 mg/L on average) and theoretical hydraulic retention time (0.19–0.77 day) conditions. The 16S rRNA gene amplicon sequencing analysis suggested the presence of putative Mn(II)-oxidizing and PCL-degrading bacterial lineages in the reactor. Two highly dominant operational units (OTUs) in the packed PCL-associated biofilm were assigned to the genera Marinobacter and Pseudohoeflea, whereas the genus Lewinella and unclassified Alphaproteobacteria OTUs were highly dominant in the MnOx-containing black/dark brown precipitate-associated biofilm formed in the reactor. Excitation-emission matrix fluorescence spectroscopy analysis revealed the production of tyrosine- and tryptophane-like components, which may serve as soluble heterotrophic organic substrates in the reactor. Our findings indicate that PAB reactors are potentially applicable to BioMnOx production under seawater conditions.

Keywords: Biodegradable polymer, Biogenic manganese oxide, Mn(II) oxidation, 16S rRNA gene

Introduction

Manganese (Mn) occurs widely in natural ecosystems in three different oxidation states (+2, +3, and +4), and Mn(III, IV) oxides are ubiquitously found in a variety of geological settings (Post 1999). Abiotic Mn(II) oxidation by molecular oxygen is feasible, but the reaction occurs effectively under highly alkaline pH conditions. Therefore, Mn(II) oxidation in aerobic natural ecosystems with a near-neutral pH (e.g., pH 6–8.5) is thought to be mainly caused by biological activity (Tebo et al. 2005; Zhou and Fu 2020). Phylogenetically diverse fungi and bacteria with the capacity to oxidize Mn(II) to Mn(III, IV) oxides have been reported. Currently, Mn(II)-oxidizing bacteria have been found in five bacterial phyla: Actinomycetota (formerly Actinobacteria and Actinobacteriota), Bacteroidota (formerly Bacteroidetes), Bacillota (formerly Firmicutes), Nitrospirota (formerly Nitrospirae), and Pseudomonadota (formerly Proteobacteria) (Tebo et al. 2005; Yu and Leadbetter 2020; Zhou and Fu 2020). Numerous studies have demonstrated that biogenic manganese oxides (BioMnOx) have excellent adsorption capacities for various heavy metals, including minor metals (e.g., cobalt [Co] and nickel [Ni]), and excellent adsorption and oxidative degradation capacities of recalcitrant compounds (Hennebel et al. 2009; Zhou and Fu 2020). Thus, BioMnOx has received attention for the removal of environmental contaminants and recovery of minor metals from different water environments. However, BioMnOx production using marine continuous-flow reactors has not yet been fully studied (Kato et al. 2017; Aoki et al. 2021).

As most of the currently known microorganisms capable of Mn(II) oxidation are heterotrophic (Yu and Leadbetter 2020), an appropriate supply of organic substrate(s) is required for the biotechnological use of Mn(II)-oxidizing microorganisms (Aoki et al. 2021). However, their cultivation is sometimes not easily accomplished by simply adding soluble organic substrate(s) to non-axenic cultures, possibly because of substrate competition with fast-growing co-existing heterotrophic microorganisms (Cao et al. 2015). Recently, various studies have demonstrated the effectiveness of unique enrichment methods that utilize co-cultured methanotrophic, nitrifying, or polycaprolactone (PCL)-degrading microorganisms for Mn(II)-oxidizing microorganisms (Cao et al. 2015; Kato et al. 2017; Matsushita et al. 2018; Aoki et al. 2021). In these enrichment techniques, certain soluble organic substrates that are naturally and slowly produced by co-cultured microorganisms successfully sustain the robust growth of potentially oligotrophic Mn(II)-oxidizing microorganisms. Cao et al. (2015) and Matsushita et al. (2018) reported the effective simultaneous removal of dissolved Mn, Ni, and Co using ammonium-fed and methane-fed freshwater Mn(II)-oxidizing down-flow hanging sponge (DHS) reactors that utilize a polyurethane sponge as a biofilm carrier. Kato et al. (2017) reported a successful Mn(II) oxidation coupled with methane oxidation using a DHS reactor under seawater conditions. In our previous work, we successfully constructed a Mn-oxidizing microbial enrichment system utilizing co-cultured PCL-degrading microorganisms under seawater conditions (Aoki et al. 2021). PCL-degrading bacteria have been isolated from a wide range of marine environments, and most of the previously isolated bacteria have been taxonomically assigned to the phylum Pseudomonadota (formerly Proteobacteria) (Suzuki et al. 2021). In this novel cultivation technique, 6-hydroxyhexanoic acid, the biodegradation product of PCL (Suzuki et al. 2021), was expected to be utilized as a carbon and energy source by heterotrophic microorganisms with the capacity for Mn(II) oxidation (Aoki et al. 2021). In addition, although we did not characterize the soluble organic substrates produced by PCL-utilizing cultivation techniques, the soluble microbial products (SMPs) produced by PCL-degrading microorganisms may be utilized by Mn(II)-oxidizing microorganisms (Cao et al. 2015; Kato et al. 2017; Matsushita et al. 2018).

In this study, we operated a laboratory-scale PCL-packed aerated biofilm (PAB) reactor (Fig. 1) under seawater conditions to evaluate its feasibility for BioMnOx production, as BioMnOx-producing reactors utilizing PCL have not been established thus far. In addition, the prokaryotic biofilm communities and dissolved organic matters (DOMs) in the PAB reactor were characterized by 16S rRNA gene amplicon sequencing and excitation–emission matrix (EEM) analyses, respectively. In a PAB reactor, PCL can serve as a solid heterotrophic organic substrate and act as an effective biofilm carrier (Boley et al. 2000), thereby allowing easy operation and maintenance of the reactor. Another distinctive advantage of the PAB reactor is that dissolved oxygen can be continuously supplied through aeration under less turbulent conditions when compared with other typical aerobic biofilm reactors (e.g., moving bed biofilm reactors) (Wang et al. 2019, 2022). Aeration-induced shear forces are likely to cause cell membrane destruction, which contributes to the inhibition of microbial growth and productivity (Toma et al. 1991). Therefore, we expected that effective biotic Mn(II) oxidation would be possible using the PAB reactors. Mn(II)-oxidizing microbial communities were successfully enriched in various DHS reactors (Cao et al. 2015; Kato et al. 2017; Matsushita et al. 2018), which can be operated under less turbulent conditions with a sufficient gaseous substrate supply.

Fig. 1.

Fig. 1

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Schematic diagram of the polycaprolactone (PCL)-packed aerated biofilm reactor used in this study

Materials and methods

Operation of a laboratory-scale PAB reactor under seawater conditions

The laboratory-scale PAB reactor used in this study (Fig. 1) was designed based on the previous study by Ruan et al. (2016). The planktonic fraction of a PCL-degrading and Mn-oxidizing enrichment culture established in our previous study (Aoki et al. 2021) was used as an inoculum for the PAB reactor. The enrichment culture used in this study (22.5 mL) was collected at the end of a batch-type enrichment step (i.e., after 139 days of the enrichment procedure) and was stored at  − 80 °C after mixing with 2.5 mL of glycerol. The glycerol-stocked enrichment culture was mixed with 200 g of dried PCL pellets (~ 3 mm pellets, average Mn of 80,000, 1.145 g/mL at 25 °C) purchased from Sigma-Aldrich Co. LLC, and the PCL pellets were randomly packed into a polypropylene-based cultivation vessel (height 12.2 cm, inner diameter 8.8 cm, thickness 0.2 cm). The inner polypropylene tube had a height of 5.9 cm, inner diameter of 2.8 cm, and thickness of 0.1 cm. No holes were observed at the bottom of the inner tube. The working volume (liquid-phase volume) after packing the PCL pellets was 0.238 L. The synthetic seawater feed used in this study was prepared by dissolving 2 g of KNO3, 1 g of KH2PO4, a variable amount of MnCl2·4H2O (as a Mn(II) source), and 20 mL of an SL-10 trace element solution (Widdel et al. 1983) in 20 L of chemically defined artificial seawater (MARINE ART SF-1, Osaka Yakken Co., Ltd.) (Fushimi and Umeda 2016), which did not contain any organic carbon. It should be noted that a seawater medium prepared using the same artificial seawater was used to obtain the inoculum culture, and no obvious dissolved Mn removal caused by Mn(II) oxidation was confirmed in the seawater medium under well-mixed and aerated conditions in the absence of a sufficient amount of Mn-oxidizing biomass (Aoki et al. 2021). In addition, no significant dissolved Mn removal in the influent feed, even after long-term storage under well-mixed and aerobic atmospheric conditions, was confirmed (e.g., over 1 month of storage at the start-up period in this study). The synthetic seawater feed in the influent medium tank was continuously stirred using a magnetic stirrer at 120 rpm. The influent feed was supplied to the cultivation vessel via a peristaltic feeding pump (MP-2010, Tokyo Rikakiki Co., Ltd.) and Viton tubing (Cole-Parmer) to achieve 0.19, 0.33, or 0.77 day of initial working volume-based theoretical hydraulic retention time (HRT). Ambient air was continuously sparged into the cultivation vessel at a flow rate of 0.1 L/min, using an air stone and air pump (e-AIR 1000SB, GEX Co., Ltd.) connected to a gas flowmeter (RK1710-AIR-1L/MIN, KOFLOC Corp.). To evaluate the robustness of the PAB reactor to microbial contaminants under laboratory conditions, the entire reactor system, including the prepared influent feed, was not subjected to sterilization procedures. The PAB reactor was installed in a dark room to prevent the growth of phototrophs and the degradation and deterioration of rarely biodegradable materials (e.g., polypropylene-based cultivation vessel) initiated by ultraviolet exposure. The room temperature was maintained at 25 °C. The PAB reactor was operated under different conditions, as shown in Table 1. To evaluate the Mn(II) oxidation performance of the PAB reactor, the dissolved Mn removal rate (mg/L/day) was calculated using the following equation:

DissolvedMnremovalrate(mg/L/day)=[Cinf]-[Ceff]×QV 1

where [Cinf] is the measured influent dissolved Mn concentration (mg/L), [Ceff] is the measured effluent dissolved Mn concentration (mg/L), Q is the feed flow rate (L/day), and V is the initial working volume of the PAB reactor (0.238 L).

Table 1.

Operational conditions for the polycaprolactone-packed aerated biofilm reactor

Phase (days) Influent dissolved Mn concentration (mg/L) Theoretical hydraulic retention time (day)
Start-up (days 1 − 39) 9.9 ± 0.6a 0.77
1 (days 40 − 114) 10.2 ± 0.4 0.77
2 (days 115 − 149) 2.5 ± 0.4 0.77
3 (days 150 − 179) 13.9 ± 0.9 0.77
4 (days 180 − 207) 13.6 ± 0.6 0.33
5 (days 208 − 238) 14.0 ± 0.4 0.19
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aAverage ± standard deviations of measured dissolved Mn concentration

Measurement of the pH, oxidation–reduction potential (ORP), and dissolved Mn concentrations in the influent and effluent of the PAB reactor

The pH and oxidation–reduction potential (ORP) of the water samples were measured using a pH meter (GPH70, AS ONE CORPORATION) and ORP meter (ORP70, AS ONE CORPORATION), respectively. The dissolved Mn concentrations in the water samples were measured using a spectrophotometer (DR6000, HACH Company) using the periodate oxidation method (HACH method 8034) after filtration using a 0.22 µm pore size polyethersulfone (PES) membrane filter (Membrane Solutions Limited).

Determination of the weight and weight loss of PCL pellets

To determine the weight of the PCL pellets, the biofilms that developed on the PCL pellets were removed after 5 min of treatment in an MCS-2 ultrasonic bath (55 W, 40 kHz; AS ONE CORPORATION), followed by thorough washing with distilled deionized water. Finally, randomly collected fresh and used PCL pellets (n = 30 each) were completely vacuum dried at 40 °C in a desiccator until a constant weight was obtained. The average weight loss (%) was calculated using the following equation:

Averageweightloss(%)=Wf-WuWf×100 2

where Wf and Wu are the average weights of fresh and used PCL pellets (mg), respectively.

EEM analysis

EEM analysis was performed to characterize the DOMs observed in the influent and effluent of the PAB reactor. The water samples that underwent EEM analysis were collected on day 225 and filtered with a 0.22 µm pore size PES membrane filter (Membrane Solutions Limited). EEM spectra were recorded on an FP-8300 spectrofluorometer (JASCO Corporation). The fluorescence intensity was recorded at an excitation wavelength ranging from 200 to 440 nm in 5 nm increments and an emission wavelength ranging from 220 to 550 nm in 5 nm increments. The scan speed was 5000 nm/min and the slit widths for both excitation and emission were 5 nm. The EEM of ultrapure water was subtracted from the obtained EEM to remove the Raman scatter peaks. The EEM data were visualized using Spectra Manager Version 2 software v.2.15.01 (JASCO Corporation).

Leucoberbelin blue I assay for MnOx identification

The black/dark brown precipitates formed in the cultivation vessel of the PAB reactor were checked for their MnOx content using the leucoberbelin blue I (LBB) assay (Krumbein and Altmann 1973; Johnson and Tebo 2008). In brief, the black/dark brown precipitates formed at the gas–liquid interface of the cultivation vessel were collected manually from the PAB reactor at the end of reactor operation (i.e., on day 238) using a sterilized spatula and rinsed four times with distilled deionized water. The precipitate was dried at 60 °C for 24 h. In the LBB assay, 2.5 mL of LBB solution [0.04% (wt./vol.) LBB in 45 mM acetate] was mixed with 1 mg dry weight of the black/dark brown precipitate or 1 mg dry weight of manganese(IV) oxide [99.5% (wt./wt.)] (FUJIFILM Wako Pure Chemical Corporation) and incubated at room temperature. The LBB used in this study was purchased from Sigma-Aldrich Co. LLC.

Total genomic deoxyribonucleic acid (DNA) extraction, polymerase chain reaction (PCR), and 16S rRNA gene amplicon sequencing

The black/dark brown precipitate-associated biofilm sample was collected manually from the PAB reactor using a sterilized spatula, whereas the PCL-associated biofilm sample was obtained by ultrasonic treatment followed by washing with distilled deionized water, as described previously. After biofilm sampling, the collected biofilm samples were immediately stored at  − 20 °C until total genomic deoxyribonucleic acid (DNA) extraction was performed. Total genomic DNA was extracted using an Extrap Soil DNA Kit Plus Ver.2 (NIPPON STEEL & SUMIKIN Eco-Tech Corporation). For the 16S rRNA gene amplicon sequencing, the variable region V4 of the 16S rRNA genes of the extracted DNA samples was amplified via polymerase chain reaction (PCR) using a KAPA3G Plant PCR Kit (Kapa Biosystems, Inc.) and 0.3 µM each of prokaryotic universal primers 515F and 806R (Caporaso et al. 2011), containing the target gene specific and Illumina adapter overhang sequences. The PCR conditions were as follows: initial denaturation at 95 °C for 3 min; 30 cycles of 95 °C for 20 s, 50 °C for 15 s, and 72 °C for 30 s; and a final extension at 72 °C for 1 min. The PCR products were then purified using a MonoFAS DNA Purification Kit I (GL Sciences Inc.), and a sequencing library was prepared using the Nextera XT Index Kit v2 (Illumina, Inc.). 16S rRNA gene amplicon sequencing was performed on a MiSeq System using the MiSeq Reagent Kit v3 (2 × 300 bp; Illumina, Inc.).

Processing and analysis of the 16S rRNA gene amplicon sequence data

The primer sequences at the 5′ end of the raw 16S rRNA gene amplicon reads were trimmed using Cutadapt v.3.5 (Martin, 2011). Then, sequence processing was performed using mothur v.1.44.1 (Schloss et al. 2009) based on MiSeq standard operating procedures (Kozich et al. 2013), as described by Aoki et al. (2022). As a slight modification, the assembly of paired-end amplicon reads was performed using the “make.contigs” command with the option trimoverlap = t. Taxonomic classification of the obtained 16S rRNA gene amplicons was performed using the SILVA reference database v.138.1 (Quast et al. 2013). Finally, obtained amplicon sequences were grouped into operational taxonomic units (OTUs) based on a sequence similarity threshold of 97%. The closest cultured relatives (with validated names) of the dominant OTUs were searched using the representative sequence of each OTU and the EzBioCloud 16S-based ID service with the EzBioCloud 16S database v.20210707 (Yoon et al. 2017). Heat maps were generated using the superheat package (Barter and Yu 2018).

Statistical analysis

Statistical significance was assessed using Welch’s t test. The Bonferroni correction was applied for multiple comparisons. Statistical p value < 0.05 was considered statistically significant. All statistical analyses were conducted using the statistical software R v.4.1.0 (R Core Team 2021).

Results and discussion

Dissolved Mn removal and oxidation by the PAB reactor

In this study, we operated a laboratory-scale continuous-flow PAB reactor for 238 days to evaluate its feasibility for BioMnOx production under seawater conditions. The changes in the influent and effluent dissolved Mn concentrations, dissolved Mn removal rates, influent and effluent pH, and ORP values over time are shown in Fig. 2. The slightly alkaline pH and relatively high ORP conditions of the influent and effluent of the reactor suggest that environmental conditions favorable for biotic Mn(II) oxidation were maintained in the PAB reactor (Hallberg and Johnson 2005; Zhou and Fu 2020). In the present study, the Mn(II) oxidation performance of the PAB reactor was evaluated by measuring the dissolved Mn concentrations in accordance with previous research (Cao et al. 2015; Kato et al. 2017; Matsushita et al. 2018; Aoki et al. 2021). Figure 3 summarizes the dissolved Mn removal rates during the operation of the PAB reactor. During the start-up phase (days 1–39), the dissolved Mn removal rate was 1.1 ± 0.6 mg/L/day [average ± standard deviation (SD)]. In Phase 1 (days 40–114), the PAB reactor was operated under the same conditions (i.e., same average influent dissolved Mn concentration and theoretical HRT) as the start-up phase. The average influent dissolved Mn concentration was 10.2 mg/L, which was not statistically different from that of the start-up phase (p value > 0.05), and the theoretical HRT was 0.77 day. In this Phase 1, the average dissolved Mn removal rate was increased to approximately twofold [i.e., 2.3 ± 0.5 mg/L/day (average ± SD)] compared with the start-up phase. In addition, the maximum dissolved Mn removal rate of 3.2 mg/L/day observed in Phase 1 is also higher than that observed in the start-up phase (i.e., 2.1 mg/L/day). The increase in the average and maximum dissolved Mn removal rates was likely caused by the acclimation of the Mn(II)-oxidizing microorganisms in the PAB reactor. We further operated the PAB reactor under different operational conditions (Table 1), because the changes in influent dissolved Mn concentrations and HRT affect the availability of substrates and microbial growth inhibition by Mn(II). However, a significant enhancement in the average dissolved Mn removal rates was not confirmed under the tested conditions. The dissolved Mn removal rates in Phases 2, 3, 4, and 5 were 0.4 ± 0.5, 1.5 ± 1.2, 2.2 ± 1.5, and 2.1 ± 1.7 mg/L/day (average ± SD), respectively. The maximum dissolved Mn removal rate of 4.7 mg/L/day was observed in Phase 5. As the PAB reactor was operated under non-sterile laboratory conditions, the removal of dissolved Mn, even after 238 days of the PAB reactor operation, indicated the robustness of the PAB reactor to microbial contaminants under laboratory conditions. It should be noted that the black/dark brown precipitates were formed in the cultivation vessel following reactor operation. Precipitates appeared abundantly at the gas–liquid interface on the inner wall of the cultivation vessel (Fig. 4A). The LBB method confirmed the presence of MnOx in the precipitate (Fig. 4B). These results indicate that Mn(II) oxidation occurred successfully in the PAB reactor.

Fig. 2.

Fig. 2

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Changes over time in the influent and effluent dissolved Mn concentrations, dissolved Mn removal rate (mg/L/day), influent and effluent pH, and influent and effluent oxidation–reduction potential (ORP) values of the polycaprolactone-packed aerated biofilm reactor

Fig. 3.

Fig. 3

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Boxplot of dissolved Mn removal rates of the polycaprolactone-packed aerated biofilm reactor. The asterisk (*) indicates a statistical difference (p value < 0.05) using Welch’s t test with Bonferroni’s correction

Fig. 4.

Fig. 4

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Black/dark brown precipitates formed in the polycaprolactone (PCL)-packed aerated biofilm (PAB) reactor and the result of leucoberbelin blue I (LBB) assay for manganese oxide identification. A Photograph showing the analyzed black/dark brown precipitates formed in the cultivation vessel of the PAB reactor. B Photograph showing the result of LBB assay: (a) LBB solution (0.04% [wt./wt.] LBB in 45 mM acetic acid) reacted with 1 mg black/dark brown-precipitate formed in the polycaprolactone-packed aerated biofilm reactor, (b) LBB solution reacted with 1 mg manganese(IV) oxide [99.5% (wt/wt.)], and (c) fresh LBB solution

The average and maximum dissolved Mn removal rates that were observed in the PAB reactor were lower than those using a methane-fed Mn(II)-oxidizing DHS reactor operated under seawater conditions (average 5.4 mg/L/day; maximum 11.4 mg/L/day) (Kato et al. 2017). On the contrary, the dissolved Mn removal performance of the PAB reactor was similar to or higher than the removal performance of moving bed biofilm reactors treating synthetic sewage (2.30–18 mg/L/day) (Wang et al. 2022) or synthetic phenol wastewater (0.014–0.017 mg/L/day) (Wang et al. 2019). It should be noted that the DHS reactor reported by Kato et al. (2017) achieved the effective dissolved Mn removal rate after over ~ 1.5 years of reactor operation. In addition, once sufficient BioMnOx is produced in the reactor, the organic radicals created by BioMnOx-catalyzed oxidation may enhance Mn(II) oxidation (Learman et al. 2011; Kato et al. 2017). Thus, further long-term operation may enhance the Mn(II) oxidation performance of PAB reactors. Further improvements, including the selection of PCL/other biodegradable polymer blends and recirculation of the reactor effluent, should be investigated to stimulate Mn(II) oxidation more effectively.

PCL degradation in the PAB reactor

The average weight of fresh PCL pellets used in this study was 13.4 ± 1.6 mg/pellet (n = 30), whereas that of used PCL pellets was 13.2 ± 1.5 mg/pellet (n = 30) (average ± SD). Although there was no statistical difference between the average weight of fresh and used PCL pellets (p value > 0.05), the calculated average weight loss of the packed PCL pellets was 2%. These data indicate that limited degradation of the packed PCL pellets occurred in the PAB reactor even after 238 days of reactor operation. The limited degradation of the PCL pellets is to be expected based on a previous study; the mass loss of PCL films after a 1 year incubation in controlled static artificial seawater was only approximately 1% (Bagheri et al. 2017). In contrast, it was noticed that the weight loss of PCL pellets during 139 days of incubation under well-mixed and aerated seawater conditions was 8.982% (Aoki et al. 2021). Thus, the limited PCL degradation confirmed in this study (i.e., 2% weight loss) may have been caused by the relatively static cultivation conditions when compared with previous research (Aoki et al. 2021).

Characterization of DOMs in the influent and effluent of the PAB reactor

The EEM spectra of the influent and effluent of the PAB reactor are shown in Fig. 5. The intensities of the three distinct fluorescence regions (peaks A, B, and C) of the PAB reactor effluent were significantly higher than those of the reactor influent. Peaks A and B were located at excitation/emission wavelengths (Ex/Em) of 275/305 and 230/305 nm, respectively. These peaks likely represent tyrosine-like components (Chen et al. 2003; Hudson et al. 2007). Peak C, located at Ex/Em of 230/335 nm, likely represents tryptophan-like components (Hudson et al. 2007). These results suggest that tyrosine-like and tryptophan-like components were produced by PCL-degrading microorganisms during biomass growth and released from cell lysis during biomass decay in the PAB reactor. Therefore, these DOMs were likely available as soluble organic substrates for Mn(II)-oxidizing microorganisms in addition to 6-hydroxyhexanoic acid, the biodegradation product of PCL. The occurrence of the tyrosine-like component-related fluorescence in the influent indicates the presence of microbial contaminant-derived DOMs in the influent.

Fig. 5.

Fig. 5

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Excitation–emission matrix fluorescence spectra of the influent and effluent of the polycaprolactone-packed aerated biofilm reactor on day 225

Notice that DOMs released into the environment should be avoided as much as possible, since some SMP-like DOMs are slowly biodegradable cellular macromolecules, which might cause effluent quality deterioration (Ni et al. 2011). In fact, the fluorescence peaks observed in the Ex > 230 nm range within the range of Em = 280–330 nm are in fact more characteristic of SMP-like DOMs, whereas Ex < 230 nm is more characteristic of extracellular polymeric substance-like DOMs (Yu et al. 2020). In addition, peaks A, B, and C were assigned to a less readily biodegradable region (Yu et al. 2020). Therefore, the optimization of the operational parameters (e.g., operation temperature and HRT) and post-treatment system installation may be further considered to minimize the water pollution caused by effluent DOMs (Chu and Wang 2013; Wang and Chu 2016; Zhong et al. 2020).

Prokaryotic community analysis based on 16S rRNA gene amplicon sequencing

16S rRNA gene amplicon sequencing analysis was used to investigate the prokaryotic diversity and community composition in the inoculum and biofilm samples derived from the PAB reactor. The number of 16S rRNA gene amplicon sequences, observed OTUs, alpha diversity indices, and Good’s coverage values for the samples analyzed by mothur are shown in Table 2. High Good’s coverage values (> 0.99) indicated that adequate sequencing depth was obtained. In contrast, the richness estimators of Chao1 and abundance-based coverage estimator (ACE) were higher than the number of detected OTUs, which suggested that additional rare prokaryotic OTUs could have been detected by additional sequencing. We cannot exclude the possibility of the involvement of undetected prokaryotic OTUs in PCL degradation and dissolved Mn removal in the PAB reactor. In addition, although similar levels of Chao1 richness estimators have been calculated for the inoculum culture and reactor-derived biofilm samples, the difference in the ACE richness estimators and Shannon and Simpson diversity indices suggested that the prokaryotic diversity in the PAB reactor was higher than that in the inoculum culture.

Table 2.

Number of 16S rRNA gene amplicon sequences and observed operational taxonomic units (OTUs), alpha diversity indices and Good’s coverage values for the polycaprolactone (PCL)-packed aerated reactor samples

Sample Number of sequencesa Number of observed OTUs Chao1 richness estimator ACEc richness estimator Shannon’s diversity index Simpson’s diversity index Good’s coverage
Inoculum culture 87,518 349 531 (463 − 640)b 613 (553 − 691) 2.98 (2.97 − 2.99) 0.0963 (0.0953 − 0.0973) 0.999
Black/dark brown precipitate-associated biofilm 95,733 352 584 (482 − 759) 616 (552 − 699) 3.71 (3.70 − 3.72) 0.0526 (0.0519 − 0.0533) 0.999
PCL-associated biofilm 89,807 355 552 (477 − 672) 777 (695 − 879) 3.43 (3.42 − 3.44) 0.0606 (0.0599 − 0.0613) 0.999
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aNumber of obtained 16S rRNA gene amplicon sequences upon the analysis with mothur

bNumbers in parentheses indicate 95% confidence interval

cAbundance-based coverage estimator

The Venn diagram in Fig. 6 shows the number of shared and uniquely observed OTUs in the inoculum culture and biofilm samples collected from the PAB reactor. A total of 349 OTUs were identified in the inoculum culture. Among the detected OTUs, 50.4% of the OTUs (176/349 OTUs) were also detected in at least one of the black/dark brown precipitate-associated or PCL-associated biofilm samples, indicating that the reactor sustained the growth of diverse bacteria which existed in the inoculum culture. A total of 345 OTUs were detected in at least one of the black/dark brown precipitate-associated or PCL-associated biofilm samples, but these OTUs were not detected in the inoculum culture. These data indicate that the growth of rare OTUs existed in the inoculum and/or the presence of bacterial contaminants in the PAB reactor. The detection of a limited number of shared OTUs (i.e., 54 OTUs) between the black/dark brown precipitate-associated or PCL-associated biofilm samples might have reflected the difference in the MnOx tolerance of microorganisms (Matsushita et al. 2020).

Fig. 6.

Fig. 6

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Venn diagram showing the number of shared and uniquely observed operational taxonomic units in the inoculum and polycaprolactone (PCL)-packed aerated biofilm reactor-derived biofilm samples

In the inoculum culture, Planctomycetota and Proteobacteria were the two dominant groups at the phylum-level (relative abundances: 11.81% and 65.23%, respectively). The phylum Proteobacteria was also most dominant prokaryotic phylum in the black/dark brown precipitate-associated and PCL-associated biofilm samples, with relative abundances of 37.30% and 61.61%, respectively. Other dominant prokaryotic phyla (relative abundance > 10%) found in the black/dark brown precipitate-associated biofilm were Bacteroidota, Chloroflexi, Planctomycetota, with relative abundances of 25.86%, 13.51%, and 12.10%, respectively. Bacteroidota was the other dominant phylum found in the PCL-associated biofilm (relative abundance 26.00%). No 16S rRNA gene sequences related to archaeal phyla or chemolithoautotrophic Mn-oxidizing bacteria within the phylum Nitrospirota (Yu and Leadbetter 2020) were detected in the analyzed samples. In this study, several putative Mn(II)-oxidizing and/or PCL-degrading bacterial genera were defined based on the presence of bacterial isolates with capacity for Mn(II) oxidation and/or PCL degradation (Table 3). The detected putative Mn(II)-oxidizing and/or PCL-degrading bacterial genera were Hyphomicrobium (Tyler 1970; Gregory and Staley 1982), Aurantimonas (Dick et al. 2008; Anderson et al. 2009), Brevundimonas (Cerrato et al. 2010; Nawaz et al. 2015), Caulobacter (Gregory and Staley 1982), Aeromonas (Zhang et al. 2019), Pseudoalteromonas (Templeton et al. 2005), Shewanella (Sekiguchi et al. 2010; Wright et al. 2016), Pseudomonas (Gregory and Staley 1982; Templeton et al. 2005; Cerrato et al. 2010; Sekiguchi et al. 2010; Urbanek et al. 2017; Matsushita et al. 2020), Alcanivorax (Sujith et al. 2014; Zadjelovic et al. 2020), Marinobacter (Templeton et al. 2005; Liao et al. 2013), and Rhodococcus (Templeton et al. 2005; Urbanek et al. 2017; Matsushita et al. 2020). However, their relative abundances, except for the genus Marinobacter, were low (< 1%) in the analyzed biofilms sampled.

Table 3.

List of putative Mn(II)-oxidizing and/or polycaprolactone (PCL)-degrading bacterial genera detected in the polycaprolactone-packed aerated biofilm reactor

Taxonomic classification based on the SILVA 138.1 reference database Relative abundance (%) Putative function Reference
Phylum Family Genus Inoculum culture Black/dark brown precipitate-associated biofilm PCL-associated biofilm
Proteobacteria Hyphomicrobiaceae Hyphomicrobium NDa 0.02 ND Mb

Tyler (1970)

Gregory and Staley (1982)
Rhizobiaceae Aurantimonas ND  < 0.01 ND M

Dick et al. (2008)

Anderson et al. (2009)
Caulobacteraceae Brevundimonas ND ND  < 0.01 M, Pc

Cerrato et al. (2010)

Nawaz et al (2015)
Caulobacter ND  < 0.01 ND M Gregory and Staley (1982)
Aeromonadaceae Aeromonas ND ND  < 0.01 M Zhang et al. (2019)
Pseudoalteromonadaceae Pseudoalteromonas  < 0.01 ND  < 0.01 M Templeton et al. (2005)
Shewanellaceae Shewanella ND ND 0.41 M, P

Sekiguchi et al. (2010)

Wright et al. (2016)
Pseudomonadaceae Pseudomonas ND  < 0.01  < 0.01 M, P

Gregory and Staley (1982)

Templeton et al. (2005)

Cerrato et al. (2010)

Sekiguchi et al. (2010)

Urbanek et al. (2017)

Matsushita et al. (2020)
Alcanivoracaceae 1 Alcanivorax 0.01 0.02 0.05 M, P

Sujith et al. (2014)

Zadjelovic et al. (2020)
Marinobacteraceae Marinobacter 0.01 4.86 15.39 M

Templeton et al. (2005)

Liao et al. (2013)
Actinobacteria Nocardiaceae Rhodococcus  < 0.01 0.50  < 0.01 M, P

Templeton et al. (2005)

Urbanek et al. (2017)

Matsushita et al. (2020)
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aNot detected

bMn(II) oxidation

cPCL degradation

The major OTUs (where the relative abundance was more than 1% in at least one of the analyzed biofilm samples), which might have important roles in the PAB reactor, are shown in Fig. 7. Many dominant OTUs observed in the black/dark brown precipitate- and PCL-associated biofilm samples were rarely detected (relative abundance < 0.1%) or not detected in the inoculum culture. Diverse bacterially mediated Mn(II) oxidation mechanisms have been presented before: multicopper oxidase- and animal heme peroxidase-mediated direct Mn(II) oxidation and non-enzyme-mediated manganese oxidation (e.g., superoxide-mediated Mn(II) oxidation and pH increase caused by microbial metabolism) (Tebo et al. 2005; Zhou and Fu 2020). Therefore, considering the difference in the dominant OTUs between the inoculum and bioreactor-derived biofilm samples, it can be speculated that the dissolved Mn removal mechanism in the PAB reactor differs from that observed in the inoculum culture (Aoki et al. 2021). Most of the dominant OTUs were not assigned to cultured bacterial genera but were assigned to certain bacterial families. These dominant OTUs assigned families included the proteobacterial families Rhizobiaceae, Rhodobacteraceae, and Pseudomonadaceae, which contain several bacterial isolates with the capacity for PCL degradation and/or Mn(II) oxidation (Tebo et al. 2005; Matsushita et al. 2020; Zhou and Fu 2020; Suzuki et al. 2021).

Fig. 7.

Fig. 7

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Relative abundance and consensus taxonomy of the dominant operational taxonomic units (OTUs) observed in the polycaprolactone-packed aerated biofilm reactor. Abbreviations are as follows: INO, inoculum culture; BDP, black/dark brown precipitate-associated biofilm; PCL, polycaprolactone-associated biofilm; ND, not detected. The taxonomic name with prefixes, c_, o_, f_, and g_ indicate the consensus taxonomy of each OTU at class-, order-, family-, and genus-level, respectively

The two highly dominant OTUs in the black/dark brown precipitate-associated biofilm were OTU001 (relative abundance, 14.76%) and OTU003 (relative abundance, 12.48%), assigned to the genus Lewinella and unclassified Alphaproteobacteria, respectively. The closest cultured relatives of OTU001 and OTU003 were Lewinella cohaerens DSM 23179T (GenBank accession number ARBR01000027; 94.1% sequence similarity) and Acidimangrovimonas pyrenivorans PrR001T (GenBank accession number MF774691; 94.1% sequence similarity), respectively. Lewinella spp. can predominantly colonize poly(ethylene terephthalate) drinking bottles submerged in seawater (Oberbeckmann et al. 2016). In addition, they can degrade various polysaccharides (Khan et al. 2007; McIlroy and Nielsen 2014). Extracellular polysaccharides are the major structural components of the biofilm matrix (Limoli et al. 2015) and are likely present in the PAB reactor. These biofilm-forming and polysaccharide-degrading abilities may make them suitable for growth in the black/dark brown precipitate-associated biofilms, where the biofilm matrix is one of the limited heterotrophic substrates. It can be speculated that bacteria with the potential to produce BioMnOx preferentially colonized the black/dark brown precipitate-associated biofilm. However, the ecological roles of these dominant bacterial OTUs in the PAB reactor remain unclear because of the lack of taxonomically close isolates, and should be further investigated.

In the PCL-associated biofilm, OTU005 (relative abundance 14.63%) and OTU007 (relative abundance 12.69%), assigned to the genera Marinobacter and Pseudohoeflea, respectively, were highly dominant in the PCL-associated biofilm. The closest cultured relatives of OTU005 were Marinobacter vinifirmus FB1T (GenBank accession number NEFY01000015) and Marinobacter nauticus ATCC 27132T (GenBank accession number AB021372), with 98.8% sequence similarity, whereas Pseudohoeflea suaedae YC6898T (GenBank accession number HM800935; 99.6% sequence similarity) was the closest cultured relative of OTU007. Notably, the genus Marinobacter was also highly dominant in PCL immersed in seawater (Odobel et al. 2021). Although, to our knowledge, direct evidence of PCL degradation by Marinobacter and Pseudohoeflea isolates has not been reported before, and these data suggest that these dominant OTUs significantly contributed to PCL degradation in the PAB reactor. In contrast, Mn(II)-oxidizing Marinobacter spp. have been isolated from marine environments (Templeton et al. 2005; Liao et al. 2013). Therefore, the involvement of Marinobacter OTU005 in both PCL degradation and dissolved Mn removal in the PAB reactor cannot be excluded.

Overall, our 16S rRNA gene amplicon sequencing analysis revealed prokaryotic diversity and community composition in the PAB reactor used for BioMnOx production. Although the analysis did not allow definitive determination of which detected genera and OTUs were responsible for PCL degradation and Mn(II) oxidation, the obtained data supported the occurrence of bacterially mediated Mn(II) oxidation and PCL degradation in the PAB reactor.

Conclusions

In this study, dissolved Mn removal and MnOx production were successfully achieved using a laboratory-scale PAB reactor operating under seawater conditions. The 16S rRNA gene amplicon sequencing analysis revealed the presence of phylogenetically diverse bacteria, which may have significantly contributed to Mn(II) oxidation and PCL degradation. The genera Marinobacter and Pseudohoeflea were the two highly dominant OTUs in packed PCL-associated biofilms. The two highly dominant OTUs in the MnOx-containing black/dark brown precipitate-associated biofilm formed in the PAB reactor were assigned to the genus Lewinella and unclassified Alphaproteobacteria. The EEM analysis suggested the production of tyrosine- and tryptophane-like components, which might have served as soluble heterotrophic organic substrates for Mn(II)-oxidizing microorganisms, in the PAB reactor. However, the observed dissolved Mn removal, likely resulted by Mn(II) oxidation, had relatively low rates (0.4–2.3 mg/L/day). Therefore, further improvements are required for practical use of PAB reactors. The results of this study provide profound insights into the design and development of bioreactors for producing BioMnOx under seawater conditions.

Accession number

The raw 16S rRNA gene amplicon sequence data were deposited in the DNA Data Bank of Japan Sequence Read Archive (DRA) under the accession number DRA014193.

Acknowledgements

We thank Yoshiharu Okuno and Naoaki Tsuda for their technical assistance with this research. We also thank an anonymous reviewer for constructive comments on the manuscript. This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 20K15222 to MA.

Author contribution

MA: Conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, supervision, visualization, writing—original draft preparation, and writing—review and editing; YM: data curation, formal analysis, investigation, methodology, visualization, and writing—review and editing; TM: data curation, formal analysis, investigation, methodology, visualization, and writing—review and editing; TW: resources, supervision, and writing—review and editing; TY: resources, supervision, and writing—review and editing; KS: resources, supervision, and writing—review and editing; KH: resources, supervision, and writing—review and editing.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Informed consent

Not applicable.

Research involving human participants or animals

This article does not contain any studies involving human participants or animals performed by any of the authors.

Contributor Information

Masataka Aoki, Email: aoki.masataka@nies.go.jp.

Toru Miwa, Email: s173279@stn.nagaokaut.ac.jp.

Takahiro Watari, Email: watari@vos.nagaokaut.ac.jp.

Takashi Yamaguchi, Email: ecoya@vos.nagaokaut.ac.jp.

Kazuaki Syutsubo, Email: stubo@nies.go.jp.

Kazuyuki Hayashi, Email: k-hayashi@wakayama-nct.ac.jp.

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