Vol. 59, No. 10APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1993, p. 3406-34100099-2240/93/103406-05$02.00/0Copyright X) 1993, American Society for Microbiology

Near-Bottom Pelagic Bacteria at a Deep-Water SewageSludge Disposal Sitet

M. TAKIZAWA,t W. L. STRAUBE, R. T. HILL, AND R. R. COLWELL*Center ofMarine Biotechnology, Maryland Biotechnology Institute, University ofMaryland,

600 East Lombard Street, Baltimore, Maryland 21202

Received 10 February 1993/Accepted 28 July 1993

The epibenthic bacterial community at deep-ocean sewage sludge disposal site DWD-106, located approxi-mately 106 miles (ca. 196 km) off the coast of New Jersey, was assessed for changes associated with theintroduction of large amounts of sewage sludge. Mixed cultures and bacterial isolates obtained from wateroverlying sediment core samples collected at the deep-water (2,500 m) municipal sewage disposal site weretested for the ability to grow under in situ conditions of temperature and pressure. The responses of culturescollected at a DWD-106 station heavily impacted by sewage sludge were compared with those of samplescollected from a station at the same depth which was not contaminated by sewage sludge. Significant differenceswere observed in the ability of mixed bacterial cultures and isolates from the two sites to grow under deep-seapressure and temperature conditions. The levels of sludge contamination were established by enumeratingClostridium perfringens, a sewage indicator bacterium, in sediment samples from the two sites. The results ofhybridization experiments in which DNAs extracted directly from the water overlying sediment core sampleswere used indicate that the reference site epibenthic community, the disposal site epibenthic community, andthe community in a surface sludge plume share many members. Decreased culturability of reference site mixedcultures in the presence of sewage sludge was observed. Thus, the culturable portions of both theautochthonous and allochthonous bacterial communities at the disposal site may be inhibited in situ, the formerby sewage sludge and the latter by high pressure and low temperature.

Disposal of sewage sludge from waste treatment plants inNew York and northern New Jersey at a site approximately106 nautical miles (ca. 196 km) southwest of New YorkHarbor began in 1986 (15). In 1988, this site, designatedDWD-106, received ca. 8 x 106 wet metric tons of sewagesludge (11). The volume of sludge discharged at the siterepresents approximately one-half of the world's oceansludge disposal volume (12) and as much as 10% of the totalsewage sludge generated in the United States in 1988 (14).The study reported here was one part of a multidisciplinarystudy to assess the fate of sewage sludge released at theDWD-106 site and the effect of the sludge dumping onbenthic and epibenthic communities in the vicinity of thedisposal site.The DWD-106 site is located over the continental slope,

with water depths at the site ranging from 2,340 to 2,740 m(2). It was believed that sewage sludge released into verydeep water would not reach the ocean floor in significantquantities; it was thought that it would either be dispersed byocean currents or be consumed by ocean life. However,there is now ample evidence that sewage sludge materialdoes reach the ocean floor in relatively high concentrations,not only directly below the actual DWD-106 site, but also ata significant distance to the southwest of the site (2, 5).Our objective was to investigate whether the epibenthic

bacterial communities at a station designated DS (disposalsite), located in the DWD-106 site (38°49.11'N, 72°08.03'W)and heavily impacted by sewage sludge dumping, weresignificantly different from the epibenthic bacterial commu-nities at a reference station (RS) located ca. 75 nautical miles

* Corresponding author.t Contribution no. 201 from the Center of Marine Biotechnology.t Present address: Discovery Research Laboratories, Takeda

Chemical Industries, Ltd., Yodogawa-ku, Osaka 532, Japan.

(ca. 140 km) northeast of DS (39°20.00'N, 70°39.91'W); thedepth of RS was similar to the depth of DS, but RS wasrelatively free of sludge material. It is assumed that bacterialiving in the deep sea are adapted to the low-temperature(2°C) and high-hydrostatic pressure conditions characteristicof the deep sea (9, 16) (the hydrostatic pressure increasesapproximately 1 atm [ca. 0.1 MPal per 10 m of depth); thatis, autochthonous deep-sea bacteria are assumed to haveadapted to deep-sea conditions, while allochthonous bacte-ria in deep-sea samples are inhibited by these conditions. Byobserving the effects of deep-sea pressure, temperature, andsalinity on mixed bacterial cultures and isolates obtainedfrom the epibenthic environment at the disposal site, wesought to answer the question of whether these microbialcommunities were dominated by autochthonous bacteria orpoorly adapted allochthonous bacteria. In addition, differ-ences in the DS and RS bacterial communities were inves-tigated by counting the total and culturable bacteria at eachsite and by hybridizing DNAs recovered directly from watersamples. The anaerobic, spore-forming bacterium Clostrid-iumperfringens, a reliable indicator of sludge contaminationat the DVWD-106 site (5), was counted at both the DS and RSto measure the levels of sewage sludge contamination at thetwo sites.

MATERIALS AND METHODS

Sample processing. Samples consisted of the water over-lying sediment in tube cores collected by the ROV Jasonfrom 23 June through 3 July 1991. Within 1 h after tube coreswere recovered, the overlying water was aseptically trans-ferred to sterile bottles and placed on ice. Culturable bacte-ria were counted by plating appropriate dilutions of thewater samples onto 1.5% (wt/vol) agar plates prepared frommarine broth 2216 (Difco Laboratories, Detroit, Mich.). For

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each water sample, three plates were incubated at 4°C and atroom temperature (ca. 20°C). The plates incubated at roomtemperature were counted after incubation for ca. 4 days,and the plates incubated at 4°C were counted after incuba-tion for ca. 8 days. Water samples were fixed with filteredformalin (final concentration, 2%, vol/vol) to obtain directmicroscopic counts with acridine orange staining (6).The remaining water from each deep-sea station was

pooled. A particulate fraction was prepared from each of thepooled samples by passing ca. 5 liters of water throughduplicate, barrel type filter cartridges containing 0.22-p,m-pore-size membranes (Sterivex GS; Millipore Corp., Bed-ford, Mass.). One of the cartridges was processed for DNAextraction (13) and frozen. The other cartridge was filledwith cold marine broth 2216, capped, placed in a pressurevessel, and returned to the approximate in situ pressure (250atm [ca. 25.3 MPa]). The pressure vessels were kept at 4°C.

Sediments within the tube cores were sampled for C.perfringens counts by using mCP medium (1), as describedpreviously (5).

Samples of the surface water overlying the RS and DS andof surface water contaminated with a plume of sewagesludge being released from a barge were collected withsterile 8-liter bottles. Plate count, acridine orange directcount, and particulate DNA extracts were prepared fromthese samples as described elsewhere (6, 12).Mixed cultures. After approximately 3 weeks, the pressure

vessels containing the marine broth 2216-filled Sterivex GSfilters containing both DS and RS samples were opened. Thesuspension was carefully withdrawn from each filter unit andused to inoculate fresh tubes of broth, which were subjectedto in situ pressure and temperature conditions for 1 week.The mixed cultures which grew in these tubes were used toprepare growth curves. To minimize changes in the speciescompositions of the mixed cultures caused by repeatedtransfers, subsamples were mixed with sterile glycerol (finalconcentration, 30%) and frozen at -85°C. The resultingfrozen stocks were used as inocula to establish growthparameters for the bacterial communities.

Growth. In general, growth was monitored as follows.Frozen stocks were used to inoculate 200 ml of marine broth2216. Inoculated broth cultures were transferred to a seriesof 1.5-ml microcentrifuge tubes. Care was taken to fill thetubes completely. The snap tops were cut off the tubes, andthe tubes were sealed with Parafilm (American Can Co.,Greenwich, Conn.). For pressure studies, equal numbers oftubes were placed into each of two pressure vessels; one ofthese pressure vessels was pressurized to 250 atm, and theother was kept at 1 atm. When the effects of salt on thegrowth of mixed cultures were tested, a medium withoutadded salt (0.5% Bacto Peptone, 0.1% Bacto Yeast Extract;pH 7.6) was used.Growth was monitored by measuring A6., using a model

Lambda 3A UVIvis spectrophotometer (Perkin-Elmer, Nor-walk, Conn.) and disposable 1-ml cuvettes. Cultures werediscarded after absorbance values were determined. Growthwas also monitored by plating appropriate dilutions in trip-licate onto agar plates prepared with marine broth 2216 andincubated at either 4 or 25°C. Well-isolated colonies from thefirst community growth experiment were streaked onto freshplates, which were the sources of bacterial isolates fromeach site, and subjected to further study. Growth of theseisolates was tested under the following conditions: 4 and24°C in marine broth 2216 at atmospheric pressure; inmedium without added salt and marine broth 2216 at 24°C

and atmospheric pressure; and at 1 atm and 250 atm inmarine broth 2216 at 4°C.Growth inhibition. Inhibition of the growth of mixed

cultures obtained from water collected at the RS by sewagesludge was tested in two ways. In the first method we usedUltrafree-MC filter units (Millipore Corp.), each of whichconsisted of a small plastic cup whose bottom was a 10,000-nominal-molecular-weight-limit membrane. The reservoir fitinto a standard 1.5-ml microcentrifuge tube. Sewage sludgewas placed into the lower chamber, and an RS communityculture in marine broth 2216 was placed into the upperchamber. The entire unit was sealed with Parafilm, placedinto a pressure vessel, and returned to in situ pressure andtemperature conditions.

In the second method, dilutions of sewage sludge inartificial seawater were inoculated with RS cultures andincubated in 1.5-ml microcentrifuge tubes in pressure ves-sels, as described above. The numbers of culturable cells inthe sludge-culture mixtures were determined by performingplate counts on marine broth 2216 incubated at 4°C. Suspen-sions of uninoculated sludge were also incubated, and thebacterial growth in these samples, which served as controls,was monitored as described above.DNA-DNA hybridization. DNA was recovered from

Sterivex GS filters by using procedures described in detailelsewhere (13). DNAs extracted from water samples werecross-hybridized by using a protocol similar to that de-scribed by Lee and Fuhrman (7). DNA samples were dena-tured by adding 0.1 volume of 3 M NaOH and incubating thepreparation at 65°C for 1 h. One volume of 6x SSC (1x SSCis 0.15 M NaCl plus 0.015 M trisodium citrate, pH 7.0) wasadded, and the following amounts of DNA were applied toduplicate nylon membranes (MagnaGraph; MSI, Westboro,Mass.) in 20-,ul volumes: DNAs extracted from DS and RSepibenthic water samples, 5, 50, 200, and 400 ng; DNAsextracted from surface water and sludge-contaminated sur-face water samples, 200 ng; and calf thymus and Bacillussubtilis DNAs (negative controls), 200 ng. Probes wereprepared by labelling 500-ng portions of DNAs extractedfrom the RS and DS epibenthic water samples with[a-32P]dCTP to a specific activity of ca. 1 x 108 dpm/,ug byusing a nick translation kit (Boehringer Mannheim, India-napolis, Ind.) and the manufacturer's protocol. Unincorpo-rated nucleotides were removed by passing the probethrough a Sephadex G-50 spin column (8).

Hybridization. DNA-DNA hybridization reactions werecarried out as recommended by the manufacturer. Mem-branes were prehybridized at 65°C in 6x SSC containing 5 xDenhardt's solution (0.1% Ficoll [Pharmacia Fine Chemi-cals, Piscataway, N.J.], 0.1% polyvinylpyrrolidone, 0.1%bovine serum albumin fraction V), 0.05% sodium dodecylsulfate (SDS), and 10 ,ug of denatured calf thymus DNA perml and then hybridized at 65°C for 18 h in 6x SSC containing1% polyvinylpyrrolidone and 1% bovine serum albumin. Themembranes were washed twice for 15 min in each of thefollowing solutions: 5x SSC-0.5% SDS at room tempera-ture; lx SSC-0.5% SDS at 37°C; and 0.1x SSC-1% SDS at37°C. The membranes were air dried, and circles containingeach DNA sample were punched out. The radioactivity ofeach spot was determined by suspending the circle ofmembrane in 5 ml of scintillation fluid (ScintiVerse BD;Fisher Scientific, Pittsburgh, Pa.) and counting with a scin-tillation counter (model LS1801; Beckman Instruments Inc.,Fullerton, Calif.).

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.3 I 06 24CE Y

0.2°0:12

0.1

0.0;40 1 2 3 4 5

Time of Incubation (days)FIG. 1. Effect of temperature on growth of mixed cultures from

water overlying sediment cores collected at the DS and RS. O.D.(600 nm), optical density at 600 nm.

RESULTS AND DISCUSSIONC. perfiringens counts. The C. perffingens counts in the top

1 cm of sediment were 6.8 x 103 + 1.9 X 103 CFU/g (dryweight) (mean + standard error) for the sediment samplesfrom the DS and 5.4 x 102 ± 1.5 x 102 CFU/g (dry weight)for the sediment samples from the RS. These results confirmthat the DS received far more sludge than the RS.

Bacterial enumeration. Direct microscopic counting of bac-teria in water overlying sediment cores collected at the DSand RS yielded counts of 3.5 x 105 cells per ml for the DSsamples and 9.0 x 104 cells per ml for the RS samples. Platecounting begun immediately after the samples were recoveredindicated that 0.16% of the bacteria present in the DS samplescould be cultured, while only 0.04% of the bacteria in the RSsamples could be cultured on marine broth 2216. In theseinitial plate count experiments, temperature of incubation didnot influence the level of recovery significantly (data forvarious incubation temperatures not shown). The resultsobtained with both enumeration methods revealed that thebacterial population in samples collected at the DS was muchlarger than the bacterial population in samples collected at theRS and that a much larger proportion of the bacteria in the DSsamples than of the bacteria present in water samples col-lected at the RS was able to grow on marine broth 2216.Growth. The growth of mixed cultures obtained from

samples of epibenthic water collected at the DS showed thatthe culturable portion of this community is poorly adapted todeep-sea conditions. Figure 1 shows the effects of tempera-ture on the growth of mixed cultures obtained fromepibenthic water samples collected at the two sites when thecultures were incubated at a pressure of 1 atm. RS mixedcultures grew well at 4°C, the in situ temperature, but moreslowly at 24°C. DS mixed cultures, in comparison, exhibitedlimited growth at 4°C and much more rapid growth at 24°C.

Figure 2 shows that growth of the DS mixed cultures wasinhibited at the in situ temperature and pressure. In contrast,the RS mixed cultures grew almost as well at a pressure of 250atm as at a pressure of 1 atm at 4°C (Fig. 1). Growth wasmonitored by plate counting and spectrophotometric esti-mates. It was found that, even when the optical density of aDS mixed culture was significantly lower than that of an RSmixed culture, if the plates after a given time of incubationwere returned to the incubator for long periods (e.g., 2 weeks)at 4°C and 1 atm, both DS and RS mixed cultures producedlarge numbers of colonies on the plates, indicating thatpressure inhibited but did not kill components of the DSmixed culture.

E 0.4

e 0.3

o 0.2

0.1

0.00 2 4 6 8 10 12 14 16

Time of Incubation (days)at 4 C and 250 atm

FIG. 2. Effect of deep-sea pressure (250 atm) and temperature(4°C) on growth of mixed cultures from water overlying sedimentcores collected at the DS and RS. O.D. (600), optical density at 600nm.

The effects of salinity on the growth of mixed culturesincubated at 4 and 24°C are shown in Fig. 3a and b,respectively. When mixed cultures obtained from epibenthicwater samples collected at the RS were incubated at 4°C,they grew more rapidly in media containing 3.5% NaCl thanin media lacking salt. In contrast, although growth of mixedcultures from the DS was inhibited by the low temperature,nevertheless, growth was significantly better on the salt-free

a

Ec

0

6

b

E

(0

ci6

0 1 2 3 4

Time of Incubation (days) at 4 C

5

0 1 2 3 4 5

Time of Incubation (days) at 24 C

FIG. 3. Effect of salinity on growth of DS and RS mixed culturesincubated at 4'C (a) and 24'C (b). O.D. (600), optical density at 600nm; 2216, marine broth 2216; w/o salts, without salts.

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TABLE 1. Selected characteristics of bacteria isolated fromwater overlying sediment cores collected at the DS and RS'

Growth Growth at

GmGrowth at: presence a pressureIsolate stain Motility of: of

reaction4°C 24°C 0% 3.5% 1 250

NaCI NaCI atm atm

RS-2 Negative + + ++ ++ + ++ +RS-7-1 Negative + ++ - - ++ ++ ++RS-8-1 Negative + ++ + - ++ ++ ++RS-32-1 Negative + ++ - - ++ ++ ++RS-35-1 Negative + ++ - - ++ ++ ++

DS-2 Negative + + ++ ++ + + -

DS-3-2 Negative + + ++ ++ + + -

DS-4 Negative + + ++ ++ + + -

DS-11 Negative - ++ - ++ + ++ +a Isolates designated RS and DS were collected at RS and DS, respectively.

Growth was assessed as no growth (-), moderate (+), and good (+ +).

medium. When mixed cultures from epibenthic water sam-ples collected at both sites were incubated at 24°C, they grewmore rapidly in the absence of NaCl, suggesting that thebacterial communities at both sites contained species capa-ble of growth at the high temperature and in the absence ofsalt (i.e., bacteria likely to be allochthonous species).

Characterization of isolates. Nine cultures isolated fromepibenthic water samples collected at the two sites werecharacterized with respect to the effects that temperature,salinity, and pressure had on their growth (Table 1). Of fivepure cultures isolated from samples collected at the RS, fourgrew more rapidly under deep-sea environmental conditions(low temperature, 3.5% NaCl, and high pressure) than atroom temperature with no salt. In contrast, three of fourcultures isolated from samples collected at the DS grewmore rapidly at the high temperature in the absence of saltthan under simulated deep-sea environmental conditions.The fourth isolate from the sample collected at the DS didnot grow at 24°C or in the presence of NaCl and dominatedenumeration plates after prolonged incubation of broth cul-tures at 4°C and high pressure.

Inhibition of growth by sewage sludge. No inhibition of thegrowth of mixed cultures obtained from samples collected atthe RS was detected when these cultures were exposed tothe low-molecular-weight components of the sewage sludgein Ultrafree-MC filter units incubated at 4°C and at 1 and 250atm of pressure (data not shown). The results of experimentsin which various concentrations of sewage sludge wereadded to RS mixed cultures are shown in Fig. 4. Whencultures were incubated at 4°C and 1 atm (Fig. 4a), thesewage sludge enhanced growth slightly. However, aftervery long incubation (i.e., after incubation for 15 weeks) at4°C and 250 atm (Fig. 4b), the numbers of culturable bacteriain sludge-treated tubes were significantly lower than thenumbers in tubes containing RS mixed cultures in artificialseawater without added sewage sludge.

Southern hybridizations. Table 2 shows the results of prelim-inary DNA-DNA hybridization experiments. Some of theresults, such as the values that were higher than self-bindingvalues between the probe prepared from DNA extracted fromwater overlying RS sediment cores and target DNA extractedfrom water overlying DS sediment cores, revealed some incon-sistencies in these experiments. However, the results do sug-

a 108E

7107 iU-

i60

5 10 -05 Control (ASW)008 250 ppm

104 * 2500 ppm-U-sI* 25000 ppm

103 __

0 5 10 15Time of Incubation (Weeks)

at 4°C and 1 atm

b 108 ---- Control (ASW)

E0 5 p

107

M~~~a 4C ad 25 at

grwt ofR2ixdcltrsinuaea 0tm()an 5 am()

00

0

0

10

0 5 1 0 1 5

Time of Incubation (Weeks)

at 4C and 250 atm

FIG. 4. Effect of increasing concentrations of sewage sludge on

growth of RS mixed cultures incubated at 1 atm (a) and 250 atm (b).ASW, artificial seawater.

gest that DNA recovered directly from RS epibenthic watersamples hybridized significantly with DNA recovered directlyfrom DS epibenthic water samples. In contrast, probes pre-pared from the DS epibenthic water samples produced a lowersignal with the DNA extracted from RS epibenthic watersamples. The DS and RS probes also exhibited strong homol-ogy to DNA recovered from surface water samples collected inthe sludge plume, but significantly less homology to DNAextracted from samples of surface water collected at the RSand at the DS outside the sludge plume.A comparison of the growth of bacterial communities in

water overlying sediment cores collected at the sewage DSand RS indicated that these bacterial communities have

TABLE 2. Results of hybridizations between DNAs extractedfrom water samples collected at the RS and DS

% Hybridization witha:Source of DNA

RS probe DS probe

RS water overlying sediment core lOOb 54RS surface water 12 27DS water overlying sediment core 125 1OObDS surface sludge plume 82 91DS surface water 1 <1

a The background levels of hybridization were as follows: RS, 40%; DS,32%.

b Self-hybridization.

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significantly different growth characteristics. The physicalparameters of the RS were similar to those of the DS (2, 5).However, C. perfringens counts for sediment samples col-lected from the DS and RS clearly indicated that the DS washeavily impacted by sewage sludge, whereas the RS wasrelatively uncontaminated. It is, therefore, reasonable toconclude that the bacterial communities in samples collectedat the DS were affected by the extraordinary amount ofsewage sludge material released at the DS. This conclusionis supported by reports that the DS communities at highertrophic levels are significantly different from the communi-ties at clean sites at similar depths (4).Enumeration plates inoculated immediately after the sam-

ples were recovered indicated that the bacteria in epibenthicwater at the DS were more numerous and more easilycultured than the bacteria at the RS. The high degree ofhomology which was evident between probes prepared fromDNA extracted from water samples collected at the DS andDNA extracted from sludge-contaminated surface watersuggests that many bacterial species associated with sewagesludge arrive at the epibenthic environment at depths of 2.5km. These findings also suggest that at least some of theseallochthonous bacteria retain, perhaps temporarily, theirviability and culturability in the deep sea.

Subsequent trials performed with mixed cultures obtainedfrom DS epibenthic water samples which had been incubatedfor 3 weeks at the in situ pressure and temperature indicatedthat growth of these populations was strongly inhibited by insitu temperature, salinity, and pressure conditions. Manybacteria quickly enter a nonculturable state (10) when theyare exposed to the deep-sea environment. The model of thedistribution of sewage sludge at the disposal site (3) predictsthat only the fastest-settling sludge particles reach the seafloor in the area of the disposal site sampled in this study, withthe more slowly settling particles carried to the south and eastby the prevailing ocean currents. The Fry-Butman model (3)predicts that 11% of the sludge particles that reach the bottomin the area have settling rates greater than 1 cm s-1. Thus,transit times of 3 days or less for particles moving from thesurface to the bottom in this area of the disposal site should becommon, and the numerous colonies appearing on the initialenumeration plates may have arisen from bacteria newlyarrived from the surface which later became nonculturable.The results of the DNA-DNA hybridization experiments

(Table 2), although preliminary, indicate that many of thebacteria present at the RS are also present at the DS, whileother bacteria are present only at the DS. Mixed culturesobtained from the RS grew well under in situ conditions.These findings suggest that at the DS the autochthonousbacterial communities were inhibited by some factor presentat the DS but not at the RS. No inhibition of growth of mixedcultures obtained from RS samples was observed when thesecultures were exposed to sludge components which wereable to pass through a 10,000-molecular-weight membrane.However, long incubation of RS mixed cultures mixed withsludge under in situ pressure and temperature conditions didresult in decreased numbers of culturable bacteria (Fig. 4b).Thus, autochthonous (RS) bacteria present in the epibenthicwater of the DS may be inhibited by the sewage sludgepresent at that site and, therefore, do not grow in DS mixedcultures incubated under in situ conditions.

If this paradigm is accurate, the rate at which sewagesludge is degraded in the epibenthic environment is pro-foundly affected. If autochthonous bacteria are inhibited bysewage sludge present at the disposal site and allochthonousbacteria are inhibited by deep-sea low-temperature and

high-pressure conditions, it may take many years before thedispersed sludge is fully degraded. In addition, the alloch-thonous bacteria may persist for many years in a noncultur-able state in which they are difficult to detect.

ACKNOWLEDGMENTS

We acknowledge the leadership of Fred Grassle, who coordinatedthe efforts of the diverse participants in the DWD-106 project.

This study was supported by NOAAINURP contract NA16RU0217-01 through IMCS, Rutgers University, and by cooperativeagreement CR817791-01 (RRC) between the Environmental Protec-tion Agency and the University of Maryland.

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