APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1986, p. 915-9230099-2240/86/100915-09$02.00/0Copyright C 1986, American Society for Microbiology

Alteration of a Salt Marsh Bacterial Community by Fertilizationwith Sewage Sludge

NANCY V. HAMLETTDepartment ofBiology, Swarthmore College, Swarthmore, Pennsylvania 19081, and Marine Biological Laboratory,

Woods Hole, Massachusetts 02543

Received 30 May 1986/Accepted 30 July 1986

The effects of long-term fertilization with sewage sludge on the aerobic, chemoheterotrophic portion of a saltmarsh bacterial community were examined. The study site in the Great Sippewissett Marsh, Cape Cod, Mass.,consisted of experimental plots that were treated with different amounts of commercial sewage sludge fertilizeror with urea and phosphate. The number of CFUs, percentage of mercury- and cadmium-resistant bacteria,and percentage of antibiotic-resistant bacteria were all increased in the sludge-fertilized plots. Preliminarytaxonomic characterization showed that sludge fertilization markedly altered the taxonomic distribution andreduced diversity within both the total heterotrophic and the mercury-resistant communities. In control plots,the total heterotrophic community was fairly evenly distributed among taxa and the mercury-resistantcommunity was dominated by Pseudomonas spp. In sludge-fertilized plots, both the total and mercury-resistantcommunities were dominated by a single Cytophaga sp.

I took advantage of a long-term study on the impact ofsewage sludge on salt marsh ecology to investigate the effecton the bacterial community. In this program, conducted bythe Boston University Marine Program and the Woods HoleOceanographic Institution, commercial sludge-based fertil-izer was experimentally applied to the Great SippewissettMarsh, Cape Cod, Mass.The Sippewissett program was undertaken not only be-

cause salt marshes are frequently subject to unintentionalsewage pollution but also because coastal wetlands are beingconsidered (and in some places used) as sludge disposalsites. Marshes have attracted interest as disposal sites be-cause of their ability to act as filters, adsorbing both excessnutrients and toxic substances from the sludge. The limits tothis sorptive capacity and the effects of long-term accumu-lation of sludge components are not, however, understood.The intensive study of the Sippewissett system provides a

unique opportunity to relate the effects of sludge fertilizationon the microbial community to the effects on other aspects ofecosystem functioning. Sludge fertilization has had mixedeffects on the marsh macrobiota. Retention of sludge fertil-izer nutrients in the marsh has increased above-groundproduction of the salt marsh cordgrass Spartina alterniflora(21, 53), which is normally nitrogen limited (49). The numberand diversity of herbivorous insects have increased (57).Growth of mussels (Modiolus demissus) has been stimu-lated, whereas oysters (Crassostrea virginica) and clams(Mercenaria mercenaria) are unaffected (48).

In contrast, accumulation of toxic substances in the sludgefertilizer has had more detrimental effects. Fiddler crab (Ucapugnax) populations initially suffered drastic reductions dueto aldrin and dieldrin in the sludge fertilizer (29, 30) but haverecovered since these compounds disappeared from thesludge fertilizer after their prohibition in the United States(45). Unidentified toxic materials have adversely affectedlarval populations of the biting flies Tabanus nigrovittatusand Chrysops fulginosus (35). Toxic heavy metals have alsoaccumulated in the marsh sediment, and some of these,particularly Cr and Cd, have been transferred to plants andanimals (3, 21, 23, 48).

Less is known about the effects of sludge fertilization onthe Sippewissett microbiota, which plays a critical role insalt marsh nutrient cycling. Diatom diversity is reduced (55),and cyanobacterial nitrogen fixation is inhibited (11, 54).Both of these effects probably result from sludge fertilizernitrogen, since addition of a similar amount of nitrogen asurea produces similar effects (11, 54, 55). Other measuredmicrobial activities are not impaired. Epibenthic algal pro-duction is slightly increased (56), sulfate reduction is notaffected (24), and sediment oxygen consumption is increased(24) by sludge fertilization. Decomposition of litter in situ, inlitter bags in the field (7, 50), or in laboratory percolators (31)is as fast as or faster than controls.These results are perhaps surprising in view of the known

toxicity of metals to bacteria (16). Several explanations arepossible. One is that metals might be sequestered by thesediments in a form that is not biologically available. Theobserved transfer of metals through plants and animals,however, argues against this possibility.A second possibility is that a metal-resistant microbial

community has been selected. A variety of metal tolerancemechanisms in bacteria have been described (16). The best-studied of these are plasmid encoded (19). Since metalresistance and antibiotic resistance genes are often carriedon the same plasmid (38, 40, 44), the frequent correlation ofantibiotic and metal resistance in bacteria from pollutedenvironments (2, 14, 34, 46) has invited speculation on therole of metal resistance plasmids in adaptation to theseenvironments. Alternatively, metal tolerance may developthrough physiological adaptation (1, 37) or by selection oftypes of bacteria that are inherently metal resistant (17, 47).Development of metal tolerance in the Sippewissett mi-

crobes is supported by the observation that aerobic respira-tion of the microbial community from sludge-fertilized marshsediment is less sensitive to metal inhibition than controlcommunity respiration (22). Except for one preliminaryaccount (F. Mollura, Biol. Bull. [Woods Hole] 159:463,1980), metal resistance in individual bacterial isolates fromsludge-fertilized plots has not, however, been reported.

Consequently, I investigated the effect of sludge fertiliza-tion on the structure of the sediment bacterial community. I

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FIG. 1. Location of experimental plots in the Great Sippewissett Marsh, Cape Cod, Mass.

specifically asked (i) whether bacteria resistant to mnetals hadbeen selected for in sludge-fertilized sediments, (ii) whethermetal resistance was associated with antibiotic resistance,(iii) whether sludge fertilization had selected for specificbacterial taxa, and (iv) whether metal resistance was asso-ciated with specific taxa.

I restricted my study to aerobic, heterotrophic bacteriathat would grow on a standard marine medium. Since theexcess metals have accumulated almost entirely at the marshsurface (0 to 2 cm; 3, 8, 23), I expected that aerobic bacteriawould be affected. The study focused primarily on mercuryfor three reasons. First, although this metal is present infairly small amounts, it is one of the most toxic to bacteria(15). Second, other studies (39, 46) reported an increase inmercury-resistant bacteria in sludge-polluted marine envi-ronments, and I wanted to determine whether a similar effectoccurred in this system. Third, since it appears that strongresistance to mercury is invariably determined by plasmids(60), the study of mercury-resistant bacteria in this habitatmight reveal a useful system for future investigation of therole of plasmids in adaptative evolution of bacterial commu-nities.

MATERIALS AND METHODS

Sample sites. Experimental plots in the Great SippewissettMarsh, Cape Cod, Mass. (Fig. 1), were established and are

maintained by the Woods Hole Oceanographic Institutionand the Boston University Marine Program. Seawater fromBuzzards Bay floods the marsh twice daily with a mean tidalrange of approximately 1.3 m (11). Other than precipitation,the only input of fresh water is groundwater. Each circularplot is 10 m in radius and is drained by a single creek. Theplots contain mostly low marsh (flooded by approximately50% of the high tides) dominated by a short form of the saltmarsh cordgrass S. alterniflora Loisel, except for within 1 to2 m of the creek, where S. alterniflora grows in a tall form(51). The plots also contain some areas of high marshdominated by Spartina patens and Distichlis spicata. Thesediments underlying both high and low marsh consist ofhighly organic peat containing living and dead roots andrhizomes with only a minor component of sand or silt (8, 9,26).

Duplicate plots with four experimental treatments werestudied. Control (C) plots were untreated. The other plotshave been fertilized every 2 weeks from late April to earlyNovember since 1971 (high-fertilizer [HF] and urea andphosphate [UP] plots) or 1974 (extra-high-fertilizer [XF]plots). HF plots received 50.4 g/m2 per 2 weeks, and XFplots received 151.2 g/m2 per 2 weeks of a commercialfertilizer prepared from dried or composted, sterilized sew-age sludge (Chicagro [Kerr-McGee Corp.] from 1971 through1976, Turf and Tree [Kerr-McGee Corp.] for 1977 and 1978,and Milorganite [Milwaukee Metropolitan Sewerage Corp.]

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TABLE 1. Metal contents of sludge fertilizers

Range of metal content (,pg/g)aFertilizer brand (yr applied)

Cd Pb Cu Zn Mn Fe Cr HgChicagro (1974-1976) 56.9-68.3 254-261 329-343 1,008-1,191 168-185 15,775-16,439 1,389-1,515 0.94bTurf and Tree (1977-1978) 3.53-4.85 27.8-261 15.4-30.4 37.9-62.6 118-181 2,242-2,967 672-869 0.03bMilorganite (1979-present)c 113-156 586-610 390-489 1,019-1,203 136-357 46,413-59,811 7,810-8,308 0.94b

a All data except Hg from Giblin, Ph.D. dissertation.b Data from reference 8.c Actual analysis of metals was performed only in 1979 and 1980.

from 1979 to the present). The fertilizer contained 10% N (orwas supplemented to 10% N with urea), 6% P205, and 4%K20 (52; Anne Giblin, personal communication). The or-

ganic content was 48.8% (8). The metal contents of thesludge fertilizers are given in Table 1. UP plots were

fertilized with urea and phosphate to the same levels of Nand P as the HF plots to evaluate separately the effects ofadditional nutrients and of contaminants in the sludge fertil-izer. Urea was used because it was readily available as a

commercial fertilizer and is not directly susceptible todenitrification (49). Physical characteristics and metal con-tents of the sediments in the experimental plots are summa-

rized in Tables 2 and 3. The marsh sediment apparently hasa limited capacity to bind most metals (23), and the metalloading in the HF and XF plots has largely saturated thiscapacity. Consequently, sediment metal content does notreflect the relative rates of metal loading. Metal remobiliza-tion may also be enhanced by the increased Eh of thefertilized plots (23). Details of the fate and retention of theadded metals can be found in the extensive published studiesof the marsh metal budgets (3, 8, 21, 23).Sampling procedure. Samples were taken at low tide in

June 1981 and July 1983. In each plot, sediment cores (2-cmdiameter, 2 cm deep) were removed from three sites at a 5-mradius separated by 120°. The cores were immediately placedin weighed sterile vials. Samples were processed the sameday they were obtained. For each gram of sediment, 5 ml of75% sterile charcoal-treated, filtered seawater (Marine Bio-logical Laboratory, Woods Hole, Mass.) was added, and thesamples were suspended by mixing for 1 min at highestspeed on a Vortex mixer. After the samples had settled for 5min, the liquid portion was diluted in 75% sterile seawaterand plated by spreading. Marine CFUs were enumerated onZoBell medium 2216E (42) modified to a final pH of 6.5instead of 7.6. Metal-resistant CFUs were enumerated onthe same medium to which freshly prepared sterile solutionsof HgCl2 or CdCl2 in distilled water were added just beforethe plates were poured. The final metal concentrations were23 ,uM HgCl2 (6 mg/liter) and 438 p.M CdCl2 (100 mg ofCdCl2. 2.5H20 per liter). Plates were incubated for 5 days at20°C before CFUs were counted.

Isolation and maintenance of stock cultures. Representativemarine bacteria were chosen by picking all colonies on a

plate or a sector of a plate from a high dilution. Eightcolonies on unamended ZoBell medium and eight colonieson Hg-amended medium were picked from each samplecore. Colonies were purified by streaking. Stock cultureswere grown in ZoBell medium without agar for 24 to 48 h at25°C with shaking. To each culture was added 0.5 ml ofsterile 30% (vol/vol) glycerol in the same medium. Thecultures were immediately frozen and stored at -70°C.

Antibiotic and metal resistance. Antibiotic and metal resist-ance patterns were determined by replica plating (36) onZoBell medium containing 100 mg of ampicillin per liter, 20mg of kanamycin sulfate per liter, 12.5 mg of tetracycline perliter, 15 mg of chloramphenicol per liter, 15 mg of strepto-mycin sulfate per liter, 438 ,uM CdCl2, or 23 p.M HgCl2 andon control plates with no addition. Concentrations of antibi-otics and metals were chosen to discriminate between resist-ant plasmid-bearing and sensitive plasmidless strains (13, 38,40) and are comparable to those used in other environmentalstudies (2, 34, 39, 46). Antibiotics and metals were freshlyprepared as lOx solutions in distilled water, except fortetracycline which was prepared in 50% (vol/vol) ethanol.Antibiotic solutions were sterilized by filtration, and metalsolutions were sterilized by autoclaving; they were added tothe medium just before pouring. Analysis by atomic absorp-tion spectroscopy showed no detectable loss of mercuryfrom HgCl2 solutions autoclaved in either screw-cap tubes orplugged flasks. Isolates were scored as resistant if uniformgrowth occurred on the supplemented medium, even if it wasslightly weaker than growth on the unsupplemented control.Isolates were scored as sensitive if there was no growth oronly isolated colonies on the supplemented medium. Mer-cury resistance was confirmed by streaking on plates ofZoBell medium with 23 FM HgC12.

Diagnostic tests. Gram stain (Hucker's modification), cat-alase (slide test), oxidase (filter paper method), oxidation/fermentation of glucose in Leifson modified 0-F medium,and determination of percent G+C were performed bypreviously described methods (20). Growth without sea-water was tested on ZoBell medium prepared with distilledwater. Cell morphology, swimming motility, and the pres-ence of spores were determined by phase-contrast micros-copy of suspensions of agar-grown cultures. Gliding motilitywas observed on a medium containing 0.5 g of yeast extract,

TABLE 2. Physicochemical characteristics of sediment in experimental salt marsh plots

Type of Organic matter pH at 1 cm Pore H20 salinity Eh at 2 cm H20 content Nitrogen C/Ndplot (% dry wt + SD)a (yearly range)" (%C)b (mV) c (% vol ± SD)d (% dry wt + SD)d

C 42.0 ± 10.1 5.5-6.5 32.0 +10 71 ± 11 1.06 ± 0.1 13.5XF 45.2 ± 1.8 6.0-6.8 32.4 +350 80 ± 9 3.50 ± 0.3 6.6a Data from reference 9.b Giblin, Ph.D. dissertation.c Data from references 25 and 26. Eh in UP and HF plots is not significantly different from that in XF plots (25, 26).d Data from reference 22.

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918 HAMLETA

TABLE 3. Metal content of sediments of fertilized plots

Type of Amt of metal in top 0-2 cm (,,g/g)aplot Cu Fe Pb Cd Mn Zn Cr Hg

C 10 12,320 33 1.4 52 46 3 0. lOObHF 150C 11.8c 514c 0.506bXF 285 31,200 446 13.7 118 450 3,465 0.575b

a All data from reference 23 unless otherwise indicated.b Reference 9.c Reference 3.

0.1 g of peptone, 0.01 g of FePO4, and 12 to 20 g of agar perliter of 75% seawater. Portions of the agar medium contain-ing isolated colonies were excised and observed by phase-contrast microscopy. The arrangement of flagella was deter-mined either by a modified Leifson procedure (33) or byelectron microscopy of whole cells negatively stained withphosphotungstic acid. Bacteria were identified by standardprocedures (10, 43).DNA hybridization. DNA was extracted by the Marmur

method (32) as described by Johnson (28) and was furtherpurified by equilibrium density centrifugation in CsCl. TheDNA was labeled with [a-35S]dATP by nick translation(Bethesda Research Laboratories kit). Colony blots were

prepared and hybridization was conducted essentially as

described by Davis et al. (13).Statistical analysis. CFUs and the percentage of metal-

resistant CFUs from different years and different treatmentswere compared by two-way analysis of variance (ANOVA)(one-way ANOVA for data collected in only 1 year) with a

correction for unequal cell size (62). Significant differencesamong treatment means from data analyzed by ANOVAwere evaluated by the Newman-Keuls test (62) and by theFisher least significant difference program included in theStatistics with Finesse microcomputer statistics package(James Bolding). Both methods gave the same results.Analysis of CFUs was performed on logarithmically trans-formed data, and analysis of percentages was perfermed onarcsine-transformed data. Distributions of taxonomlc groupswere tested for homogeneity by the chi-square test (12).Shannon's diversity indices for different communities werecompared by the method of Hutcheson (27). In all cases, P <0.05 was considered significant.

RESULTSPlate counts of total and mercury-resistant marine bacteria.

Total and mercury-resistant aerobic chemoheterotrophicmarine bacteria in the upper 2 cm of sediment from theexperimental salt marsh plots were enumerated for twosummers (1981 and 1983) by viable plate counts on ZoBellmedium (Table 4). Two-way ANOVA (years versus treat-ments) indicated there was no significant difference betweenyears for either total CFUs or for the percentage of mercury-

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FIG. 2. Antibiotic and metal resistance patterns of mercury-sensitive and mercury-resistant bacterial isolates from experimentalsalt marsh plots. Isolates were tested for resistance to ampicillin,kanamycin, tetracycline, streptomycin, chloramphenicol, andcadmium.

resistant CFUs. Cadmium-resistant bacteria were also enu-merated in 1983. The numbers of CFUs were significantlyhigher in all treated plots than in C plots. The numbers ofCFUs in UP and HF plots, which received equal amounts ofnitrogen and phosphorus, were not significantly different.XF plots, which received three times as much sludge fertil-izer as HF plots, had the greatest numbers of CFUs.The percentages of mercury-resistant CFUs were signifi-

cantly greater in both types of sludge-fertilized plots than inC plots. The percentage of mercury-resistant bacteria in UPplots, however, was not significantly different than in Cplots. The percentage of cadmium-resistant bacteria in 1983was significantly increased only in XF plots.

Antibiotic resistance patterns of mercury-sensitive and -re-sistant marine bacteria. To determine whether mercury re-sistance was associated with antibiotic resistance in the saltmarsh sediment bacteria, 308 representative isolates ob-tained in 1981 from ZoBell plates both with and withoutmercury were screened by replica plating for resistance tofive antibiotics and cadmium (Fig. 2).

TABLE 4. CFUs and percentage of metal-resistant CFUs in salt marsh sediment samples plated on ZoBell medium

Type of plot Mean CFUs (106/g [wet Wt])a Mean Hgr CFUs (%)a Mean Cdr CFUs (%b)

C 0.70 (0.53-0.92) [12]C 8.5 (5.6-11.9) [121f 6.7 (4.5-9.4) [6]hUP 9.8 (6.7-14.5) [12]d 9.7 (6.0-14.2) [91f 6.3 (3.6-9.4) [6]hHF 15.7 (12.1-20.2) [12]d 28.8 (25.2-32.4) [11]9 5.6 (3.5-8.0) [5]hXF 93.9 (57.5-153) [11]1 37.5 (28.6-46.9) [11] 31.6 (18.0-46.3) [5Y

a Data from 1981 and 1983. Values in parentheses represent mean - SE to mean + SE (68% confidence interval). Values in brackets are numbers of samples.b Data from 1983 only.i Means with the same letter are not significantly different.

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EFFECT OF SEWAGE SLUDGE ON MARSH BACTERIA 919

TABLE 5. Antibiotic and metal resistance of salt marsh isolates

Response Type of No. of % of isolates resistant toa:to mercury plot isolates None Amp Kan Tet Str Cam Cd

Sensitive C 23 17 43 39 70 17 0 0UP 17 29 24 28 65 12 0 0HF 48 5 67 81 88 49 0 1XF 37 3 60 84 78 49 0 3

Resistant C 48 0 77 96 94 85 21 4UP 48 0 98 96 98 90 19 3HF 39 0 64 90 87 79 0 6XF 50 0 70 100 92 78 0 7

a Amp, Ampicillin; Kan, kanamycin; Tet, tetracycline; Str, streptomycin; Cam, chloramphenicol; Cd, cadmium.

Mercury resistance was clearly associated with antibioticresistance among isolates from C and UP plots. In these twotypes of plots, 95% of the mercury-resistant isolates were

resistant to three or more other metals and antibiotics butonly 17% of the mercury-sensitive isolates were resistant tothree or more.The association of mercury resistance with antibiotic

resistance among isolates from sludge-fertilized plots was

much less pronounced. The fraction of bacteria resistant tothree or more other metals and antibiotics was substantial

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not only among the mercury-resistant isolates (84%) but alsoamong the mercury-sensitive isolates (62%). The frequenciesof resistance to particular antibiotics and metals were alsodifferent in the sludge-fertilized plots (Table 5). For example,chloramphenicol resistance was not observed in any isolatesfrom sludge-fertilized plots but was observed in mercury-resistant isolates from C and UP plots. Cadmium resistanceoccurred in both mercury-sensitive and -resistant isolatesfrom sludge-fertilized plots, but only among mercury-resistant isolates from C and UP plots.

Composition of the heterotrophic bacterial salt marsh com-munity. A possible explanation for the differences in antibi-otic resistance patterns is that the types of bacteria insludge-fertilized plots were different from those in C plots.To test this possibility, the 1981 isolates were characterizedtaxonomically. Cell morphology, Gram reaction, presence ofspores, type of motility, flagellar arrangement, colony mor-phology and pigmentation, reaction on Leifson modified 0-Fglucose, and the oxidase test were used to classify theisolates into taxonomic groups. These particular tests en-abled me in most cases to determine the genus of each group;in a few cases additional tests (including percent G+C andspecial stains) were performed to determine the genus of thegroup. The additional tests did not further subdivide thegroups. Most of the isolates appear to be marine bacteriasince they failed to grow on ZoBell medium prepared withdistilled water instead of seawater. The sole exception wassome of the Bacillus isolates. At least one of these, however,requires Na+ for spore germination (61).The frequency of the various groups among isolates from

each plot (Fig. 3) showed that the composition of thebacterial sediment community forming colonies on unamend-ed ZoBell medium was markedly altered by sludge fertiliza-tion. Whereas the distribution of isolates among taxonomicgroups was not significantly different between UP and Cplots (P = 0.46, chi-square test) or between HF and XF plots(P = 0.35), the distribution in HF and XF plots was

significantly different from that in UP and C plots (P =

0.0003).

TAXONOMIC GROUPFIG. 3. Distribution among taxonomic groups of bacteria iso-

lated on unamended ZoBell medium from sediment of experimentalsalt marsh plots. The generic assignment of the taxonomic groups isas follows: groups 1 and 2, Cytophaga spp.; groups 3 to 6, Bacillusspp.; groups 7 to 13, Pseudomonas spp. (or Alteromonas sp. [6]);groups 14 to 20, Vibrio spp.; groups 21 to 25, Alcaligenes sp. (orDeleya sp. [5]); group 26, Spirillum sp.; groups 27 to 29, unidentifiednonmotile gram-negative rods; group 30, Moraxella sp.; group 31,Corynebacterium sp.; group 32, unidentified gram-positive rods insheaths.

TABLE 6. Prevalence of Cytophaga sp. group 1 CFUs

% of CFUs with group 1 morphology in:Type of ZoBell medium ZoBell medium + Hg

plot1981 1983 1981 1983

C 0.0 0.0 2.5 0.3UP 0.4 0.4 0.5 1.3HF 34.6 21.6 52.5 21.0XF 33.9 49.0 24.6 24.6

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TAXONOMIC GROUPFIG. 4. Distribution among taxonomic groups of bacteria from

experimental salt marsh plots isolated on mercury-amended ZoBellmedium. Genera of taxonomic groups are given in the legend to Fig.3. There were no mercury-resistant isolates in groups 23 to 32.

The most striking aspect of the difference in frequencydistributions was the predominance in XF and HF plots ofgroup 1, which consists of nonfruiting gliding bacteria thatform transparent yellow colonies with a distinctive brightgreen iridescence. Characterization of the 84 group 1 isolatesindicated that the members of this group constituted a single,previously undescribed, species of Cytophaga (N. Hamlett,A. Kropinski, C. Chan, and A. Fisher, manuscript in prep-

aration). Although we did not characterize isolated strainsfrom 1983 samples, enumeration of colonies with this uniquemorphology on all plates from the 1981 and 1983 samples(Table 6) showed a consistent enrichment of this species inboth years.The frequency distributions of the taxonomic groups

among isolates from mercury-amended ZoBell medium (Fig.4) showed an even more pronounced difference between theHF and XF plots and the UP and C plots. In all types ofplots, mercury resistance was largely confined to only a fewtaxonomic groups. Groups 7, 8 (both Pseudomonas sp.), and14 (Vibrio sp.) accounted for 90% of the mercury-resistantisolates from UP and C plots, whereas 90% of the mercury-

resistant isolates from HF and XF plots were accounted forby groups 1 (Cytophaga sp.), 3 (Bacillus sp.), and 7 (Pseu-domonas sp.).

Colony-blot DNA hybridization of DNA from representa-tives of groups 1, 3, 7, and 14 generally confirmed both thehomogeneity and unique identities of the four groups. DNAfrom a representative of each group hybridized strongly withall members of its own group, and DNA from groups 1 and

3 did not hybridize detectably with any other group. Group14 (Vibrio sp.) DNA also hybridized with group 16 (Vibriosp.), and group 7 (Pseudomonas sp.) hybridized with threeisolates assigned to Pseudomonas group 9 (data not shown).Community diversity. The numbers of isolates in each

taxonomic group were used to calculate Shannon's diversityindex and equitability (Table 7). Because Shannon's diver-sity index is sensitive to sample size, equitability is a fairerbasis for comparison. Comparison of the total heterotrophiccommunities (i.e., isolates obtained on unamended ZoBellmedium) showed that diversity was significantly decreasedin XF plots. As would be expected from the data shown inFig. 4, the mercury-resistant community was considerablyless diverse than the total community. Mercury-resistantcommunities from UP and XF (but not HF) plots weresignificantly less diverse than the C plot mercury-resistantcommunity.

DISCUSSION

Although previous work showed that sludge fertilization inthe Sippewissett Marsh has not impaired most measures ofmicrobial activity (including algal production [56], sulfatereduction [24], oxygen consumption [24], and litter decom-position [7, 31, 50]), my results indicate that fertilization hassignificantly altered the structure of the bacterial commu-nity. The observed increase in CFUs apparently reflects anincrease in bacteria able to form colonies aerobically onZoBell medium rather than an increase in total bacteria,since the numbers of bacteria enumerated by acridine-orange direct counts were not significantly different in XFand C plots (22). The increased CFUs appear to be causedeither directly or indirectly by nutrients in the sludge fertil-izer, since addition of equal amounts ofN and P as urea andphosphate produced an identical effect (Table 4, HF andUP). Two possible (nonexclusive) explanations are thathigher nutrient levels favor copiotrophic bacteria, which cangrow more readily on the rich peptone-yeast extract me-dium, or that the increased redox potential (Table 2) favorsaerobic bacteria. Increased microbial biomass, but not in-creased plate counts, was reported for Danish agriculturalsoils fertilized with sewage sludge (18). Although the totalyearly application of sludge in the Danish study was quitesimilar to that for XF plots, differences in the metal contentof the sludge, the frequency and length of application, andthe extreme differences in soil type make comparison diffi-cult.Other alterations were unique to sludge-fertilized plots.

The increased frequency of mercury- and cadmium-resistant

TABLE 7. Diversity indices for salt marsh sediment communities

Shannon diversity index (H )a Equitabilityb

plot Total Hg-resistant Total Hg-resistantcommunity community community community

C 3.62 2.10 0.732 0.427UP 3.98c 1.28e 0.801 0.260HF 3.30c 1.79c 0.671 0.365XF 2.69e 1.56d 0.547 0.318

a - X(nilN) log2(n,/N), where ni is the number of isolates in the ith speciesand N is the total number of isolates.

b H'/H'max, where H'm. is the maximum possible H' for that sample,calculated on the basis of an even distribution of the isolates among the 32taxonomic groups.

c Not significantly different from C plot (P > 0.05) (27).d Significantly different from C plot (P < 0.05) (27).e Significantly different from C plot (P < 0.01) (27).

ip-

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EFFECT OF SEWAGE SLUDGE ON MARSH BACTERIA 921

isolates supports the conclusion that a metal-resistant bac-terial community has developed. Mollura (Biol. Bull.[Woods Hole], 1980) also reported that bacteria capable ofgrowth on ZoBell medium plus 1 mM Cd were isolated fromXF but not from C plots in 1980. The percentage of mercury-resistant CFUs was highly correlated with sediment mercuryconcentration both in our study and in two other publishedstudies (39, 46) of estuarine and marine sediments (Fig. 5).Considering that these studies used different media, differentHg concentrations, and samples from very different types ofsediment, the relationships shown are remarkably consis-tent, although the actual percentages are lower in studiesusing higher mercury concentrations in the selective medium(I assume that 6 ppm of HgCl2 means 6 ppm of Hg in thestudy by Nelson and Colwell [39]).Olson and Thornton (41) proposed that tolerance of bac-

teria to metals may be used to determine the bioavailabilityand toxicity of metals. If this is the case, the data in Fig. 5suggest that even modest inputs of mercury are both avail-able and toxic in a variety of marine sediments, despite theknown mercury-binding ability of sediment components. Inthe Sippewissett, mercury appears to be tightly bound toparticulate organic matter (9), yet mercury-resistant bacteriaare selected for and mercury is adsorbed by Spartina litterduring decomposition (7).A particulary interesting consideration, discussed by

Giblin et al. (23), is that, in the chemical milieu of the saltmarsh sediment, sludge nutrients may have indirectly in-creased the availability of metals in sludge-fertilized plots.Fertilizer nitrogen stimulates the growth of S. alterniflora,resulting in increased water uptake by the plants, which inturn results in increased air entry into sediment, increasedEh, and decreased pore-water sulfide (Table 2; 25, 26; A. E.Giblin, Ph.D. dissertation, Boston University, Boston,Mass., 1982). The increased sediment oxidation greatlydecreases the importance of insoluble metal sulfides andincreases metal solubility in the pore water (23). The slightly

50.0

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0.0 0.5 1.0

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FIG. 5. Correlation of percentage of mercury-resistant bacteriawith mercury concentration in marine and estuarine sediments.Percentages were arcsine transformed before linear regression anal-ysis, and the scale of percent on the ordinate is proportional toarcsin(%/100)112. Symbols:[l, data from Great Sippewissett Marsh(6 mg of HgCl2 per liter [22,uM] in ZoBell Medium 2216E [r = 0.92;P = 0.005]);*, data from Great Sippewissett Marsh (6 mg of HgCl2per liter [22,uM] in MacConkey Agar [r = 0.99; P = 0.057]); 0, datafrom study of Chesapeake Bay by Nelson and Colwell (39) (6 ppm ofHgCl2 [30,uM] in a glucose-Casamino Acids-yeast extract-estuarinesalts agar [r = 0.97; P = 0.016]); 0, data from study of New YorkBight by Timoney et al. (46) (20,ug of Hg per ml [as HgCI2] [100FiM]in a tryptone-glucose-yeast extract agar plus 3.5% NaCl [r = 0.92; P= 0.005]).

lower pH in the fertilized plots would also favor moresoluble forms of some metals, including Zn, Cd, and Pb,although there would be little effect on Hg (23).The genetic basis of metal resistance in our isolates is at

this time unknown. Other workers (14, 46) interpreted asso-ciation of cadmium or mercury resistance with antibioticresistance as evidence for plasmid-mediated resistance. Theclose association of antibiotic and mercury resistance inisolates from C and UP plots is consistent with plasmid-mediated resistance, and the predominant mercury-resistantgenus is Pseudomonas, in which plasmid-mediated resist-ance has been widely observed. In HF and XF plots,however, the antibiotic resistance pattern largely resultedfrom the predominance of the group 1 Cytophaga sp., whichis uniformly resistant to ampicillin, tetracycline, kanamycin,and streptomycin, whether or not it is mercury resistant.This pattern suggests selection for a species that ischromosomally antibiotic resistant rather than selection forplasmids carrying metal and antibiotic resistance. Wattersonet al. (59) found that high levels of penicillin resistance inBacillus spp. from metalliferous soil resulted from selectionof Bacillus cereus, which has chromosomally encoded 1B-lactamase. This selection apparently results from an abun-dance in these soils of penicillin-producing molds (58), whichWatterson et al. (59) suggest may be due to the metal-bindingability of penicillamine, a main product of penicillin hydro-lysis. Further study is clearly required to understand thebasis for metal resistance and the significance of antibioticresistance in these and other environmental isolates.The observed decrease in community diversity agrees

with the observations of Barkay et al. (4) on the bacterialcommunities of sludge-amended agricultural land. However,in contrast to my results with the mercury-resistant commu-nities, they found the cadmium-resistant community to bemore diverse in sludge-amended soils than in control soils.These different results may simply reflect the nature of thetaxonomic groups used in the diversity calculations. If myindices are recalculated on the basis of genera as was doneby Barkay et al. (4), my results for both the total andmercury-resistant communities are very similar to theirs.Devanas et al. (14) found Shannon's index for cadmium-resistant communities from New York Bight to be lowest forisolates from the most heavily polluted sites.The most striking feature of the altered community struc-

ture is the predominance of the Cytophaga sp. constitutinggroup 1. Whereas this species is found rarely (<1% ofisolates) in C plots, it accounted for approximately one-thirdof the isolates from HF and XF plots (Fig. 3 and Table 6). Ifthe increase in CFUs (Table 4) is also considered, the actualenrichment is 10,000-fold. Such an enrichment for a partic-ular species has not previously been described.

I cannot say with certainty what aspects of sludge fertil-ization have produced the altered community structure.Although metals appear to be important, the selectionagainst the mercury-resistant bacteria found in control sed-iment and their replacement with different bacterial typessuggests that the situation is complex.The form of nutrients supplied by the sludge fertilizer may

be a contributing factor. An observation by Giblin et al. (22)suggests that the sludge-fertilized microbial community hasbecome adapted to new substrates. They showed that whensediment from C plots is used as a substrate, aerobicrespiration of microbes from XF plots is lower than that ofmicrobes from C plots, even though oxygen consumption insitu is greater in XF plots (24). The group 1 Cytophaga sp.cannot degrade cellulose but can utilize proteins, amino

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922 HAMLETT

acids, and lipids and is capable of using dead microbial cellsas a sole source of nutrients (Hamlett et al., in preparation).Other factors, such as novel organic compounds, may alsobe important, or the effects may be indirect, reflecting, forexample, changes in predators, competitors, or allelopathicorganisms. Additional investigation is clearly needed toelucidate the factors involved. Such investigations shouldimprove our understanding of the ramifications of sludgeaddition to coastal wetlands and our ability to assess itsenvironmental impact.

ACKNOWLEDGMENTS

I thank Jeanne Poindexter and Bill Reznikoff for suggesting thisstudy and providing helpful advice; Andrew Kropinski, MosheShilo, Ed Leadbetter, and Martin Dworkin for valuable suggestions;Kermit Hutcheson for advice on statistical analysis; William Har-rington for use of his facilities for percent G+C analysis; JosephTopping for atomic absorption spectroscopy; and Tamar Barkay forcritical reading of the manuscript. I also thank for technical assist-ance Tom Kieft (enumeration of cadmium-resistant bacteria), SeanThomas (statistical analysis), Debra Farina (stock culture mainte-nance), David Weiss (DNA hybridization), and Abby Fisher(Cytophaga taxonomy). I am especially grateful to the Arnold B.Gifford family for access to their salt marsh property, to the BostonUniversity Marine Program for permission to use their study sites,and to Anne Giblin for graciously sharing her unpublished results.

This work was supported by grants from the National Aeronauticsand Space Administration (NAGW-216), the Foundation for Micro-biology, the Division of Biological Research of the U.S. Departmentof Energy, the Faculty Development Program and the FacultyResearch Committee of Towson State University, and the ResearchSupport Committee of Swarthmore College.

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