PH: S0273-1223(98)00095-X

~ Pergamon War. Sci. Tech. Vol. 37. No. 4-5. pp. 131-138. 1998.=1998 IAWQ. Published by Elsevier Science LtdPrinted in Great Britain.

0273-J223/98 $J9'00 + 0'00

SULFATE REDUCTION AND SULFIDEOXIDATION IN AEROBIC MIXEDPOPULATION BIOFILMS

Satoshi Okabe*, Takayuki Matsuda**, Hisashi Satoh*,Tsukasa Itoh* and Yoshimasa Watanabe*

*Department ofUrban & Environmental Engineering. Faculty ofEngineering,Hokkaido University, North 13, West 8, Kita-ku, Sapporo 060. Japan** The Ministry ofHealth and Welfare. Chiyoda-ku, Tokyo 100-45, Japan

ABSTRACf

The microzonation of 02 respiration, H2S oxidation and S042- reduction in aerobic biofilms grown onrotatlDg disk reactors of a sewage treatment plant was studied by measuring concentration profiles withmicroelectrodes for 02' S2- and pH. The vertical distribution of sulfate-reducing bacteria (SRB) in thehiolilms was also determined by the conventional culture-dependent MPN method and fluorescently labeled16S rRNA-targeted oligonucleotide probes for SRB. The SRB probe stained cells were distributedthroughout the biofilm with a distinct higher fluorescence intensity ID the middle part of the biofilm. Thisresult corresponded well with 02 and S2. concentration gradients measured by microelectrodes. showingsulfate reducing activity was largely restricted to a narrow anaerobic zone located in the middle of thehiofilm. Measurements of accumulation of reduced sulfur compounds (FeS. FeS2 and sOJ in the biofilmIDdicated that the H2S produced by SRB became oxidized by 02 and other oxidants. probably ferric/ferroushydrates. and precipitated as FeS and SO just above the sulfate reduction zone. © 1998 IAWQ. Published byElseVier Science Ltd

KEYWORDS

Aerobic biofilm; microelectrodes; 16S rRNA-targeted oligonucleotide probes; fluorescent in situhybridization; (FISH); confocal laser scanning microscope (CSLM); sulfate reduction; sulfide oxidation.

INTRODUCTION

It is thought that a successive vertical zonations of respiratory processes can be found in aerobic wastewaterbiofilms with a typical thickness of only a few millimetres. Sulfate reduction can be anticipated to take placein the deeper anaerobic biofilm strata. Reoxidation of sulfide with oxygen and/or nitrate accordingly wouldtake place in a stratum close to the sulfate reduction zone, dependent on the oxygen and nitrate penetrationdepths. Sulfate reduction is an important mineralization process in aerobic wastewater biofilms and aconsiderable removal of organic compounds may be due to sulfate reduction. Internal sulfide reoxidation isexpected to account for a substantial part of oxygen consumption (KUhl and Jorgensen, 1992; Norsker et al.,1995). However, the sulfur cycle in aerobic biofilms is very complex and is presently not well described.because mass balance of sulfide flux across a biofilm-water interface cannot describe sulfur transformationwithin biofilms due to an internal sulfur cycle.

131

132 S. OKABE et al.

Therefore, we have used oxygen, pH and sulfide microelectrodes to study the Microzonation of 02respiration, H2S oxidation and S042- reduction in aerobic wastewater biofilms. One-dimensional spatialdistributions of potential sulfate reduction rate and sulfide oxidation rate in the biofilm were also measuredby slicing the biofilm with a Microslicer™ followed by a standard batch experiment. To investigate thedistribution of sulfate-reducing bacteria in the biofilm, fluorescent in situ hybridization with 16S rRNA•targeted oligonucleotide probes were used together with the conventional culture-dependent NTN method.This technique has been used successfully in a variety of complex environmental microbial communitiessuch as activated sludges (Wagner et ai., I994a,b) and biofilms (Amann et ai., 1992; Ramsing et ai.,1993; Schramm et ai., 1996). In addition, the spatial distributions of reduced sulfur compounds (FeS, FeS2and So) in the biofilm were measured to evaluate the occurrence of an internal iron-sulfur cycle. Becausethe produced H2S quickly reacts with iron, it forms insoluble metal sulfides (Le. FeS, FeS2) and elementalsulfur (So), and accumulates in the biofilm. The goals of this research were (I) to demonstrate theoccurrence of sulfate reduction and sulfide reoxidation in aerobic wastewater biofilms and (2) to relate thedistribution of SRB populations to their potential and in situ metabolic activity profiles and chemicalconcentration profiles (Le., 02 and H2S). A combined study of the microbial spatial distribution by theoligonucleotide probe technique and microelectrode measurements can explain the coupling between sulfatereduction, sulfide reoxidation and oxygen respiration in aerobic biofilms.

EXPERIMENTAL MATERIALS AND METHODS

Bjofilm samples

Aerobic mixed population biofilms were grown in rotating disk reactors (RDR) on domestic wastewaterfrom a municipal wastewater treatment plant at Sapporo, Japan. Dissolved oxygen concentrations in the bulkliquid were in the range 31-156 vM during, the experiment.

SRB enumeratjon and actjyjty measurements

The biofilms collected from the rotating disk reactor were cut into 50-IOOmm thick slices parallel to thesubstratum without any pretreatment by use of the Microslicer™ as described previously (Okabe et ai.,1996), and then apportioned into samples representing 4-6 layers. The apportioned samples werehomogenized and subjected to the enumeration of SRB and the measurement of potential metabolic activity.

The SRB were enumerated by the five tube multiple dilution most probable number (NPN) method usingHangate type test tubes after homogenization of the sectioned biofilm samples. Postcate medium B(Postgate, 1984) containing sodium lactate (50% solution) (4000 mg I-I) and sodium acetate (1000 mg 1-1) ascarbon and energy source was used for the MPN. Black cultures were counted at least 21 days afterincubation.

Sulfate removal rate (SRR) was determined from a standard batch experiment. The homogenized biofilmsections (variable volume) were transferred to 100 ml of the anaerobic Postgate medium B in a serum vial(130 ml). The vials were sparged with N2 gas for at least 5 minutes and subsequently sealed with butylrubber stoppers and aluminium seals. The vials were incubated on a rotary shaker at 30°C in the dark. Atregular time intervals, samples were withdrawn with a sterilized syringe and immediately added into 1%zinc acetate solution for total sulfide analysis.

Sulfide oxidation rate (SOR) was also determined using a growth medium containing (mM): NH4CI (3.6),KH2P04 (0.9), MgS04.7H20 (1.6), yeast extract (30 mgll), NaHC03 (104), Na2S203.5H20 (1.7) and traceof metals (pH =7.0-7.5). The biofilm samples were inoculated into 100 ml of the medium under aerobicconditions and incubated on a rotary shaker at 30°C in the dark. At regular time intervals, samples werewithdrawn for S042-measurement. The concentration of S042-was analyzed with an ion chromatograph.

Measurement of sulfide compounds

Aerobic mixed population biofilms 133

Elemental sulfur (SO), acid-volatile sulfides (AVS; H2S and FeS) and chromium reducible sulfide (CRS;FeS2) in biofilms were determined by the method described originally by Fossing and Jorgensen (1989) andmodified by Nielsen et al. (1993). The biofilm samples were immediately fixed in 1% zinc acetate solution,sliced into 50-IOOJlm thick slices parallel to the substratum in 1% zinc acetate solution without anyprefixation by use of the Microslicer™, and then apportioned into samples representing 4-6 layers. Thebiofilm samples were sequentially analyzed for elemental sulfur (SO) by extraction in ethanol, for AVS (H§and FeS) by distillation with 2N HCI, and then for FeS2 by distillation with I M Cr2+ in 0.5 N HCI. Thesamples extracted in ethanol were analyzed by HPLC using a UV detector at 254 mm. A reversed phasecolumn (Partisil 5, ODS-3 (Whatman» was used with 100% HPLC grade methanol as eluent and flow rateof I ml min-I. Sulfide was measured colorimetrically by the methylene blue method. Recovery of FeS andFeS2 was determined previously to be 87±7% (n=3) and 73±24% (n=3) for FeS and FeS2' respectively. Themeasured AVS by distillation with 2N HCl will be designated FeS in the rest of the paper.

Microelectrode measurements

Concentration gradients of oxygen, pH, and sulfide in the biofilms were measured by microelectrodesmanufactured in our laboratory as described previously (Revsbech and Jorgensen, 1986). The oxygenconcentration was measured with a Cathord-type microelectrode using an AglAgCI reference electrode(HORIBA, 20 lOA). Linear calibration was done in air-saturated medium above the biofilm and in theanaerobic zone of the biofilm. pH and sulfide were measured by microelectrodes connected to a pH/mYmeter (HORIBA, F-23) with a pinhole type AgCVAg reference electrode. The pH microelectrode wascalibrated in standard pH buffers. The sulfide electrode was calibrated as described by KUhl and Jorgensen(1992). The position of the biofilm surface was determined visually under a dissection microscope. Allmeasurements were performed at 20±IoC in a chamber containing I litre of synthetic medium with a knowncomposition. Flow velocity (2-3 cm s-I) above the biofilm was provided by a Pasteur pipette blowing airand/or N2 gas onto the water surface. Bulk DO concentration was varied by changing the mixing ratio of airand N2 gas. The total amount of dissolved H2S, HS- and S2- will be designated H2S in the rest of the paper.

The specific reaction rate for the production and consumption of a solute was determined by manually fittingparabolic functions of the steady state Fick's second law equation of diffusion to the measured concentrationprorle~ as described by Nielsen et al. (1990). We used the molecular diffusion coefficients of 2.1 x 10-5cm S- for oxygen and 1.39 x 10-5 cm2 S-I for H2S, respectively at 20°C.

Fluorescent in situ hybridizatjon

Biofilm samples were fixed in 4% paraformaldehyde solution immediately after the microelectrodemeasurements and embedded in OCT compound (Tissue-Tek). Thin sections of the fixed biofilm wereprepared as described in detail by Ramsing et al. (1993) and Schramm et at. (1996). In situ hybridization ofbiofilm sections was performed using the following 16S rRNA-targeted oligonucleotide probes: (I)EUB338, 5'-GCTGCCTCCCGTAGGAGT-3' complementary to domain Bacteria (Amann et al.. 1990) and(2) SRB385, 5'-CGGCGTCGCTGCGTCAGG-3'), complementary to general sulfate-reducing bacteria(Amann et at.. 1992). The probe of EUB338 was labelled with fluorescein isothiocyanate (FITC) and theprobe for SRB385 was labelled with tetramethylrhodamine-5-isothiocyanate (TRITC).

The hybridization procedure followed in principle the method described by Amann (1995). The biofilmsections were hybridized in 8 JlI of hybridization buffer (0.9 M NACI, 20 mM Tris hydrochloride (pH = 7.2),0.01% sodium dodecyl sulfate (SDS), 20% formamide) with I JlI of probe solution for 2 h at 46°C in anequilibrated sealed moisture chamber. After hybridization, the slide was flashed with about 5 ml ofprewarmed washing solution (180 mM NACI, 20 mm Tris hydrochloride (pH = 7.2), 0.01 % SDS), andquickly immersed in 50 ml of prewarmed washing, solution at 48°C for 20 min. The slides were then rinsedwith ddH20, allowed to air dry, and mounted in antifading solution (PermafluorTM Immunon).

134 S. OICABE el 01.

An LSM 4/0 inverted scanning confocal laser microscope (Carl Zeiss) equipped with an Ar-ion laser (488nm) and a HeNe-laser (543 nm) was used to record optical sections of the biofilm specimen. Objectivelenses (20x. 40 x and 63 x oil) were used.

RESULTS AND DISCUSSION

Biofilm archjtecture

A conspicuous grey filamentous biofilm was found at the surface of the biofilm. which was most likely thefilamentous sulfur-oxidizing Beggiatoa mat. The color of the upper base biofilm was yellowish-gray andgradually turned to dark brown with depth in the middle of the biofilm. But the yellowish color appearedagain in the innermost part of the biofilm. The biofilm was generally very porous and consisted of voids andpores. 0 creating a biomass network.

Spatial djstributions of SRB and potentjal actiyjty

Figure I depicts the spatial distribution of SRB population enumerated by the MPN method and potentialsulfate reduction rate (SRR) and sulfide oxidation rate (SOR) with 02 as electron acceptor in a 65 days oldbiofilm. Although the surface biofilm contained the highest number of SRB (4 x lOs MPN cm-3). the lowest,but significant. potential SRR of about 5 Ilmol cm-3 h-I was found in the surface layer. The density of SRBdecreased with depth. whereas the potential sulfate reduction rate increased. The highest SRR of 29 Ilmolcm-3 h-I was found about 300 11m below the surface. However, the SRR decreased at the bottom of thebiofilm due probably to transport limitation of electron donors. The potential SOR with 02 was slightlydecreased from 131lmol cm-3 h-I at the surface layer to 51lmol cm-3 h-I in the anoxic region below 350mm.

~100 200 300 400 500

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o 100 200 300 400 SOODistance from Surface (\lm)

Figure I. Spalial distribulions of SRB density. potential sulfate reduction rate (SRR) and sulfide oxidation rate(SOR) in the biofilm.

Similar observations of sulfate reduction in oxic environments have been reported previously in theliterature. The highest density of SRB population was measured by the MPN technique in the oxic zone just

Aerobic mixed population biofilms 135

below the surface of near-shore marine sediments, at which the concurrently measured sulfate reduction ratewas the lowest (Jorgensen and Bak, 1991).

In Situ detection of SRB

Immediately after the microelectrode measurements (the results are shown in Fig. 4(B», the biofilm wassubjected to in situ hybridization with the SRB 3 8 5 probe. The fluorescent signal derived from SRB 385probe stained cells was found at all depths. The fluorescent intensity and abundance was higher in themiddle part of the biofilm as compared with the uppermost and innermost part of the biofilm. Very denseclusters of SRB probe stained cells were found in the middle of biofilm (about 800-1000 ~m below thesurface), which corresponded well with the sulfate reduction zone as shown in Fig. 4(B). A close-up view ofthis section is presented in Figure 2. The SRB were unevenly distributed in the biofilm in all states fromsingle scattered cells to dense clustered cells. It should be also noted that the SRB probe stained cells werefound more at the biomasslliquid interface than the inside. Fewer SRB probe stained cells and their clusterswere found in the uppermost part of the biofilm even though the maximum population density of SRB wasobserved by the NPN technique. This is probably because the SRB present in the surface of the biofilm werenot active, or probably dormant with low ribosomal RNA content. However, slow growing cells, or evendormant cells became active after the microbial environment became suitable for the SRB growth bytransferring to the MPN test tubes, consequently displaying positive counts. Alternatively, a versatilemetabolism of SRB with nitrate or even oxygen could help to explain the presence of the maximumpopulation density of SRB in the oxidized uppermost. The results of in situ hybridization corresponded wellwith the microelectrode measurements and activity profiles determined by the batch experiments, showingthat the SRB activity was restricted in a narrow zone (approx. 200-300 ~m) in the middle part of the biofilm.

Figure 2. In silu hybridizalion of a vertical biofilm section. phase conlrllsl (A) and proJccllon Image of z sequencesoblamed from 12 oplical seclioning acquired al 2 lUll inlervals by !he confocal scanning laser microscope (Carl

ZeiSS 410). (B). Slaining was carried oul wi!h TRITC-Iabelled SRB385 probe. Signals are displayed as rgb Images.The bars corresponds 10 50!!m.

Accumulation of reduced sulfur compounds

Figure 3 represents the spatial distribution of FeS, FeS2' and SO in a 600 ~m thick biofilm. Although FeSwa~ not detectable in the surface and the bottom of the biofilm, about 40 ~mol cm·3 of FeS wa~ found atabout 250 ~m from the surface, in which the H2S produced by SRB in the reduced deeper zone wa~

expected to encounter oxygen (the 021H2S interface) (Fig. 4A). Elemental sulfur seemed to be the dominantsulfur type at all depth of the aerobic biofilm. T-he concentration of SO at about 150 ~m from the surface wasthe highest (about 30 ~mol cm·3) and gradually decreased with depth. FeS2 concentration was below 10~mol cm·3 throughout the biofilm, constituting a relatively small fraction of the total sulfur.

136 S. OKABE ~I al.

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"c: 108

o

___ FeS

o 100 200 300 400 500 600

Distance from Surface (IJm)

Figure 3 ConcenlrallOn profile~ of FeS. FeSZ and Sa In the hlOfilm.

The results of detennination of the reduced sulfur compounds in the biofilm suggested that the oxidizediron~ may play an important role in an internal sulfur cycle. Reductive and oxidative pathways of sulfurcycle are. however. very complex and a further quantitative study is apparently needed to understandoxidant~ for sulfide oxidation in aerobic biofilms.

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1000

1500

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Specific rates ijJmol/cm3/h)

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o 10 20 30 40 50 60

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o 50 100 150 200 250Concentrations (IJM)

F'l!urc 4 Avcrage conccnlrallon profile~ 01 Oz. HZS and pH In lhe mlcroaerohlc hlOf,lm~ with dllferenllhlckne"e\~'l .I~prox,malely600 jjm (AI and ISIXI jjm (BI. The hlOf,lms were Incubaled In a ~ynlhel'c medIUm containing

S04°' (I 111M I. acetate (0 K3 mMI. lactale (O.K3 mM) and no NOf The specific react"," rales of 0z resp,ratlOn.S042- reduct"," and HZS OXidatIOn were calculaled from the' measured profile~ and indicated hy hoxe~.

02 resplrallnn (open box). S04Z- reducllon (dark box) and HZS oxidallon (hghl-dark hox)

Typical concentration profiles of O2 and HzS in aerobic biofilms are shown in Figure 4. Oxygen penetratedonly about 300-400 flm from the surface in a 600 flm thick biofilm. when bulk DO concentration was 61flM. corresponding to a typical DO level during the reactor operation (Fig. 4A). At this bulk DO level.sulfide was slightly produced In the bottom of the blofilm at a specific rate of 0.15 H2S flmol cm- 3h- l. A

Aerobic mixed population biofilms 137

significant increase in sulfide production rate (3.4 H2S Jlmol cm-3h- l) was observed 400-520 Jlm below thesurface when the bulk DO was reduced to 20 JlM. However, no free H2S escaped from the biofilm becauseof a complete internal oxidation within the biofilm at the specific rate of 3.3 H~ Jlmol cm-3tI- J.

Por a 1500 Jlm thick biofilm sulfide production became more evident 600-900 Jlm below the surface even ifthe bulk DO concentration was 154 JlM (Fig. 4B) compared with that in the 600 Jlm thick biofilm. Thespecific H2S production rate was 16.4 H2S Jlmol cm-3h- l . Below the sulfate reduction zone, constant H2Sconcentration (approx. 200 JlM) was observed, indicating no net sulfide production. The produced H2S wasdepleted at 380 Jlm from the surface, while 02 penetrated down to 500 Jlm from the surface, where 02 andH2S coexist. A narrow sulfide oxidation zone (380-630 Jlm from the surface) was found just above thesulfate reduction zone with a specific H2S oxidation rate of 16.3 H2S Jlmol cm-3h- I , giving a total H~oxidation rate of 0.41 Jlmol cm-2h- l • The specific 02 reaction rate was, however, 5.8 Jlmol cm-3h- I, giving atotal consumption rate of 0.29 Jlmol cm-2h- l . Taking into account that oxidation of I mol of H~ to sol•requires 2 mol of °2, it clearly indicated that H2S became oxidized by 02 and other oxidants, probablyferric/ferrous hydrates, and precipitated as PeS and SO in this narrow zone. This speculation was supportedby the fact that the biofilm became completely black due to accumulation of FeS after a few days ofincubation. Continuous sulfide production uses up available sulfide oxidation capacity (e.g. oxidized ironand manganese) in the biofilm, consequently, free H2S will escape from the biofilm.

The average turnover times of 02 and H2S in this H2S-oxidizing zone were calculated to be 9.0 and 2.6 s,respectively, and the ratio of the average concentration to the average reaction rate was as described by KUhland Jorgensen (1992). These turnover times extremely fast compared with the possible spontaneouschemical reaction of 02 and H2S. The time scale of the 02-H2S reaction at wastewater temperature hasbeen reported to be in the range of minutes to several hours (Chen and Morris, 1972; Eary and Schramke,1990). Thus, the observed oxidation of H2S in the biofilm was attributed to microbial activity asdemonstrated by the distribution of potential sulfide oxidation rate in the biofilm (Fig. I).

The sulfide concentration profile in the biofilm corresponded well with the concentration profiles of reducedsulfur compounds as shown in Fig. 3. A distinct peak of FeS about 250 Jlm below the surface was probablyresponsible for the sharp decline of sulfide concentration in this region.

CONCLUDING REMARKS

~se .of ~icroelectrodes toge~er with specific oligonucleotide probes made it possible to relate the spatialdlstnbutlon of SRB populatIons to their activities. The abundance of the SRB detected by fluorescentlyI~beled 16S. rRNA-targeted probes ~as the highest in a restricted zone located in the middle part of theblofilm, whIch corresponded well wtth the sulfate reduction zone detected with microelectrodes. However,there was a negative correlation between the SRB numbers determined by the culture-dependent MPNtechnique and the potential sulfate reduction rate measured by the batch experiment. Furthermore, thehighest accumulation of total sulfur compounds (PeS, FeS2 and SO) observed just above the anoxic sulfatereduction zone indicated that oxidized iron may play an important role in an internal sulfur cycle in aerobicwastewater biofilms.

ACKNOWLEDGEMENT

We are indebted to Dr R. Funada, Department of Forest Sciences, Faculty of Agriculture, HokkaidoUniversity for generously providing the CSLM equipment and for various discussions. This research hasbeen supported by the CREST (Core Research for Evolutional Science and Technology) of Japan Scienceand Technology Corporation (JST).

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