Adaptacion de comunidades bacterianas a ambientes de trasicion.pdf

7/29/2019 Adaptacion de comunidades bacterianas a ambientes de trasicion.pdf

1/8

R E SE A R C H A R T IC L E

Subramanian Karthikeyan Darren R. Korber

Gideon M. Wolfaardt

Douglas E. Caldwell

Adaptation of bacterial communities to environmentaltransitions from labile to refractory substrates

Received: 1 September 2000 / Accepted: 30 January 2001 / Published online: 25 September 2001 Springer-Verlag and SEM 2001

Abstract The aim of this work was to assess theadaptation of bacterial communities to environmental

transitions from labile to refractory substrates. Thisinvolved testing the hypothesis that bacteria self-orga-nize and propagate not only as individual cellularsystems, but also as functional sets of interactingorganisms. A biolm community was cultivated in aow-cell irrigated with tryptic soy broth and subjected toa cyclic series of environmental transitions, from labileto refractory substrates, followed by a period of star-vation (30 days). The appearance and disappearance ofspecic colony morphotypes when the emigrants wereplated onto tryptic soy agar was used to monitor therestructuring of the community. Confocal laser micros-copy of ow cells showed that these transitions

decreased the biolm thickness and coverage. Substrateshifts also changed the architecture of the biolm com-munities. Repeated inoculation of ow-cell communitieswith a composite inoculum increased the number anddiversity of emigrants. Their biolms were thicker andcovered a wider area than those of communities that hadbeen inoculated only at the beginning of the experiment.With repeated inoculation, the time required for thecommunity to restructure and stabilize decreased duringmost transitions. This suggested that organismalrecombination acted as a mechanism of adaptation,enhancing the growth of microbial communities exposedto environmental stresses. Changes in the proles of

emigrants during the adaptation of biolm communitiesto environmental transitions showed the appearance and

disappearance of discrete sets of organisms. Thissuggested that the biolm communities responded to

environmental stresses as sets of interacting organisms.Enhanced growth of biolm communities due torepeated environmental cycling suggested that thefunctionality of cellular positioning accrued from onecycle to the next and was thus heritable, although it wasnot necessarily genetically encoded.

Keywords Biolms Restructuring Environmentaltransitions Biolm architecture Microbialcommunities

Introduction

The role of community-level bacterial strategies inbiodegradation has been well established [22, 27]. Indegradative microbial communities, the capability ofbreaking refractory organic compounds arises fromphysiological [20, 27] and genetic [12] interactionsamong community members. This capacity results fromthe metabolic diversity of its members, in terms of thenumber and variety of chemical bonds on which theycan act [20]. Horizontal transfer of degradative plasmidsamong genetically unrelated bacterial cell lines [12] alsoenhances reproduction in degradative communities,because more degradative phenotypes appear. These

adaptive strategies combine the adaptive potential ofcells (spatial recombination of molecules in cells) withthe adaptive potential of communities (spatial recom-bination of organisms in the communities). Thus, whenthese communities adapt to environmental perturba-tions, they undergo restructuring on multiple spatialscales [18, 23].

Synergisms among the members of communities arerequired for the optimal proliferation of the communi-ties themselves [5, 6, 7, 8], just as cellular and molecularsynergisms have been optimized for the proliferationof cells and molecules. In fact, the restructuring ofcommunities not only involves gene mutations and

Int Microbiol (2001) 4: 7380DOI 10.1007/s101230100016

S. Karthikeyan D.R. Korber D.E. Caldwell (&)Department of Applied Microbiology and Food Science,University of Saskatchewan, 51 Campus Drive,Saskatoon, Saskatchewan S7N 5A8, CanadaE-mail: [email protected].: +1-306-9665026Fax: +1-306-9668898

G.M. WolfaardtDepartment of Microbiology, University of Stellenbosch,7600 Stellenbosch, South Africa


2/8

recombination, but also recombination of cells andorganisms both within a single community and amongcommunities. However, the role of external organismalrecombination (i.e. the eects of immigration andemigration) in the process of adaptation of bacterialcommunities to environmental stresses has not beenstudied from this perspective. If adaptation to some kindof stress occurs at multiple spatial levels of organization,including the community, then an environmental shiftshould elicit co-ordinated responses among the com-munity members. Thus, the changes in communitystructure prole in response to substrate transitionsshould show sets of inections, as opposed to membersresponding individually.

This study tested the existence of communityadaptation strategies. We subjected a microbial com-munity to cyclic environmental stresses, from labile torefractory substrates [from triptic soy broth (TSB) tobenzoate, then to 2,4,6-trichlorobenzoic acid (2,4,6-T)]and to starvation. Then, we examined the role of

multiscalar recombination in the adaptation of biolmcommunities at various stages of environmentaltransition.

Materials and methods

Bacterial communities and culture conditions

A composite inoculum was prepared by mixing 0.5 g each of twooil-sludge samples (Kamsack and Baildon sites, Saskatchewan,Canada), a pristine soil sample (Matador grasslands, Saskatche-wan, Canada) and a pesticide disposal-pit soil sample (Iowa State

University, Iowa, USA), in 50 ml of mineral salts (M-salts) solution[27]. This was used to inoculate a ow cell [21] (channel dimensions:1340 mm) continuously irrigated with 0.3% (w/v) TSB broth ata laminar ow velocity of 0.1 mm/s. A quasi-steady state biolmcommunity developing on the solidliquid interface was deter-mined from the stability of its thickness, area coverage and overallarchitecture, as well as from the number and diversity of emigrants.The quasi-steady state community was then subjected to a sudden

environmental transition (to sodium benzoate as sole carbonsource). Subsequent transitions consisted of a shift to 2,4,6-T assole carbon substrate and then to a state of starvation (no carbon).The substrate shifts were accomplished by changing the ow-cellirrigation medium from 0.3% (w/v) TSB to M-salts supplementedwith 0.15% (w/v) sodium benzoate, 0.05% (w/v) 2,4,6-T, or nocarbon (when subjected to starvation), depending on the stage ofthe cycle. All chemicals were procured from Sigma Chemical Co.,St. Louis, Mo.

In the rst cycle (Cycle I-S), the communities were subjected tosudden environmental transitions that consisted of instantaneousshifts in the cultivation medium. The second set of cycles involvedeither sudden or gradual (stepwise) environmental transitions(Cycle II-S and Cycle II-G respectively; Fig. 1). During gradualtransitions, each shift extended over a period of 30 days andcomprised 10 steps (of 3 days each). Each step consisted of a

0.1-fold decrease in the concentration of the rst substrate,accompanied by a 0.1-fold supply of the second substrate. After30 days, the concentration of the rst substrate in the irrigationmedium was zero and that of the second was at its maximum.

During both cycles, biolm communities that were repeatedlyinoculated were compared with the communities inoculated only atthe beginning of the experiment. Repeated inoculation consisted ofinoculation at 12-h intervals, using 0.5 ml of a freshly preparedcomposite inoculum.

Monitoring biolm reorganization

During Cycle I-S and Cycle II-S (Fig. 1), the thickness, percentarea coverage and architecture of biolm communities were

Fig. 1 A ow-cell communitycultivated on tryptic soy broth(TSB) was subjected to threeenvironmental cycles as de-scribed in the text, BA Benzo-ate, 2,4,6-T 2,4,6-trichlorobenzoic acid

74


3/8

measured using scanning confocal laser microscopy (SCLM). InCycle I-S, the measurements were done at 12-h intervals, until thecommunity reached a quasi-steady state, and at longer intervals onthe stable biolm community. In Cycle II-S, SCLM measurementswere done less frequently (once before the transition and twice afterthe community stabilized in the new environment). Biolms nega-tively stained with 0.1% w/v uorescein (Sigma Chemical Co., St.Louis, Mo.) were scanned and digitized using a Bio-Rad MRC-600

Lasersharp uorescence scanning confocal laser system (Bio-RadMicroscience, Canada), mounted on a Nikon FA microscope,under a 60, 1.4 NA oil immersion objective (Nikon Corporation,Tokyo). An argon laser operated at 25 mW power and 1% beamtransmission (maximum excitation at 488 nm and 514 nm) wasused in the excitation of the uorophore. The images were collectedusing a Kalman lter. A Northgate 80846 host computer was usedto display the images. The focusing and stage control werecomputerized. The images were saved on an optical disc using aCorel-940 optical drive (WORM). The primary images wereanalyzed using Bio-Rad software in the Northgate host computer.The percent biolm area and the biolm thickness at eachobservation period were respectively the averages of 10 and20 replicate measurements. Communities subjected to gradualtransitions (Cycle II-G) were not examined by SCLM.

Monitoring the diversity of emigrant populations

The diversity of the emigrants (as measured by richness andShannon indices) was monitored at 12-h intervals until thecommunities reached a quasi-steady state and, less frequently, instable biolm communities. These determinations were done priorto the laser microscopy of biolm architecture in Cycle I-S andCycle II-S. In Cycle II-G, the emigrants were enumerated justbefore each stepwise transition. For this purpose, the ow-celleuent was collected for 1 h and plated onto 0.3% (w/v) TSB agarin triplicate. The plates were incubated at 22 C for 7 days and thenumber of various colony morphotypes that appeared on count-able plates of highest dilution, usually containing 30100 colonies,were counted. Colony morphotypes were determined, based on thedescriptions of Benson [4] and Eklund and Lankford [11]. Richnesswas based on the number of distinct colony morphotypes observed.The Shannon index of diversity H = S(pi log2pi) was thencalculated. The factor pi is the number of isolates classied as acertain morphotype divided by the total number of isolates in thesample being analyzed.

Results

Biolm thickness and percent area coverage

Shifts in carbon substrates, from TSB to benzoate to2,4,6-T, resulted in a general reduction in the thicknessand percent area coverage of biolm communities. In

Cycle I-S, when cultivated using TSB, the thickness andpercent area coverage of repeatedly inoculated andinitially inoculated biolm communities did not diersignicantly (P


4/8

substrate shift, the patterns of restructuring of emigrantpopulations showed that one morphotype (mpt-6)disappeared, whereas four new morphotypes (mpt-11,14, 15, 16) appeared in response to the transition. Moredrastic changes occurred in the repeatedly inoculated

biolm community, which lost three of its morphotypesand gained seven new members. The appearance,propagation and disappearance of some of the emi-grants occurred as sets. For example, morphotypes 7and 8 were present throughout the transition and

Fig. 2 Confocal scanning lasermicrographs of repeatedlyinoculated and initially inocu-lated biolm communitiessubjected to sudden environ-mental transitions from TSB tobenzoate to 2,4,6-T duringCycle I-S

Fig. 3 Relationship betweenthe refractory nature of thesubstrate and cell counts ofemigrant biolm communities.Starv No carbon source, 246T2,4,6-trichlorobenzoic acid,TSB tryptic soy broth, BAbenzoic acid

76


5/8

seemed to propagate as a set (Set 1). This set was present

in both kinds of communities. When those repeatedlyinoculated were subjected to the substrate shift,morphotypes 11, 14, and 16 and morphotypes 9, 12, 13and 15 appeared as two distinct sets (Set 2 and 3,respectively), whereas morphotypes 6, 9 and 10 disap-peared as a set (Set 4). Similarly, during adaptation ofthe initially inoculated biolm community, two distinctsets [morphotypes 11, 16 (Set 2) and morphotypes 14,15 (Set 3)] appeared. When communities were subjectedto stepwise environmental transitions, some morpho-types appeared, disappeared or propagated as sets.During most transitions, repeatedly inoculated commu-nities adapted to substrate shifts more rapidly than those

inoculated only initially. For example, when subjected to

a substrate shift from benzoate to 2,4,6-T, repeatedlyinoculated biolm communities required a shorterperiod (140 h) for restructuring and stabilization thaninitially inoculated communities (175 h) (Fig. 4).

Recoverable diversity of the emigrants measuredfrom colony morphotypes was generally higher inrepeatedly inoculated communities of both Cycle I-Sand Cycle II-S than in the initially inoculated commu-nities grown on the same substrate. However, Shannondiversity indices did not show any specic trend basedon the refractory nature of the substrate, the nature ofthe transition (sudden or gradual) or the cycle ofadaptation (I or II; data not shown).

Fig. 4 Eect of communityrestructuring on the emigrationpatterns detected when repeat-edly inoculated and initiallyinoculated biolm communitieswere subjected to a substratetransition from benzoate to2,4,6-T. Mpt Morphotype

77


6/8

Discussion

Earlier studies on the eects of environmental pertur-bations on microbial communities focused mainly on theimpacts of organic carbon amendments by pulse-feed,step-feed or periodic-feed patterns [16, 18, 23]. These

substrate perturbations led to changes in the structure ofmicrobial communities as a mechanism of adaptation.In nature, environmental perturbations are much morecomplex and comprise physicochemical (changes in theenvironmental concentrations and gradients of growthsubstrates, pH, redox potential, etc.) and biotic (eectsof micro- and macrobiota sharing the same ecosystem)factors. This is especially obvious in aquatic ecosystems,where ow inuences both the ux of nutrients and aninux and eux of other microorganisms. In such sys-tems, planktonic bacteria can be considered dynamic``gene pools'' that can colonize new environmentalniches. Their signicance in the new environment might

be reected by shifts in the structure and metaboliccapability of the native community. If proliferation oc-curs at the level of communities as well as at the level ofindividuals, then repeated inoculation with a diverseinoculum should optimize both community-leveladaptation in terms of the time required for thecommunity to restructure and stabilize and the diversityand productivity of the resulting assemblage oforganisms. Hence, we assessed the eect of repeatedinoculation as an external source of organismalrecombination (a process analogous to geneticrecombination at the cellular level), under conditions ofcyclic environmental shifts.

The results showed a decline in the thickness and areacoverage of biolm communities and a decline in thenumbers of emigrants upon successive substrate shifts.Repeated inoculation of biolm communities enhancedadaptation. This suggested that communities needsynergisms (resulting from both internal and externalrecombination processes) for their optimal growth, justas individuals need molecular and cellular synergismsfor their optimal growth. James et al. [19] indicated thatinterspecies interactions inuence the structure andphysiology of biolm communities during the course oftheir formation. Repeated inoculation may have alloweda larger genetic pool for those interactions. This is

consistent with the proliferation theory [5, 6, 7, 8], whichsuggests that bacterial self-organization not onlyinvolves mutation and recombination of genes, but alsothe restructuring of communities (immigration, emigra-tion, reshuing of members within a community) andthe spatial repositioning of organisms within geographicboundaries (the ow cell and its surface environments).

Techniques for the qualitative analysis of microbialcommunity structure include ribosomal RNA (rRNA)analysis using taxon-specic probes [13], rRNAsequence comparisons [10], whole community DNAhybridization [15], whole community phospholipidfatty-acid proles [26] and carbon source utilization

prole analysis using Biolog microtiter plates [14]. Onlysome of these methods can account for the overallchanges in community structure; and others are limitedin their application when the actual process of restruc-turing has to be described. In this work, we used colonymorphotypes that appeared when the euent obtainedfrom ow cells was plated on TSB agar to quantifyemigration (the number and diversity of emigrantpopulations) from the community. Haldeman and Amy[17], in their studies on the colony morphotypes ofbacterial isolates obtained from various rock samples,concluded that colony morphotypes can be consideredas biotypes and could provide an accurate basis to denerecoverable diversity. Colony development had beenpreviously used to characterize bacterial communities onwheat roots [9]. The selectivity of plating media andculture methods was unavoidable in this process. Wefollowed specic morphotypes through each transition,using countable plates of the lowest dilution containing30100 colonies, which we incubated for the same

duration. So, colony sizes and morphologies werecomparable between two sampling periods.

Atlas [2] suggested that diversity measurementswould be useful to monitor changes due to ecosystemperturbations and that the degree of diversity is lower incommunities under stress conditions. In the case ofco-metabolic interactions and sequential degradationof pesticides, species diversity is crucial to enable thebreakdown of complex bond structures. This seems toimply that stressed communities will be diverse. Duringthe present study, the diversity changes in response tosubstrate stress did not follow a specic pattern.However, emigrants of repeatedly inoculated biolm

communities tended to be richer and more diverse thanemigrants of initially inoculated communities. Thismight be due to the immigration of new members intothe community.

We used repeated environmental cycling to knowwhether pre-exposure of a microbial community to arefractory organic compound would enhance thecommunity's ability to utilize the same compound uponsubsequent exposures. Adaptation of microbialpopulations to use either refractory or toxic compoundsas carbon and energy sources has been well demon-strated [1, 3, 24]. Adaptation of microorganisms to hightoxicant concentrations would play a major role in

biodegradation [25]. Spain and van Veld [24] denedadaptation in the microbial community as any changethat increases the rate of transformation of a test com-pound as a result of a previous exposure to that com-pound. The higher cfu/ml of emigrants that we observedover Cycle II-S and Cycle II-G might have resulted frompre-exposure of the communities to these substratesduring Cycle I-S. Enhanced growth of biolm commu-nities due to repeated environmental cycling suggeststhat the cellular positioning information accrued fromone cycle to the next and may have been heritable.

A closer analysis of the emigration patterns thatresulted from sudden environmental transition (Fig. 4)

78


7/8

indicated that substrate perturbations led to the collapseof existing community structures. However, the contin-uous process of recombination probably led to thedevelopment of new, stable sets of emigrant populations.The emigration patterns of all communities showed theappearance or disappearance of sets of organisms duringbacterial adaptation to refractory substrates. During asudden environmental transition from benzoate to2,4,6-T, the appearance (Sets 2, 3), disappearance (Set 4)and propagation (Set 1) of stable sets of organisms sug-gests that the organisms in these sets might be related andthat bacterial adaptation to environmental transitionsmay occur as functional sets of interacting organisms.

Communities were subjected to gradual environ-mental transitions to better understand the process ofrestructuring in response to environmental transitions.Due to diculties in developing smooth temporalconcentration gradients of two substrates, we appliedstepwise concentration gradients. Although stepwisetransitions were not useful to dene the community

boundaries or to detect the presence of ecotones (whereone set of community-level relationships disappear andnew set of relationships appear), they were just as usefulas sudden environmental transitions to detect groups oforganisms that appeared or disappeared as sets. Thisagain suggested that the biolm communities adapted toenvironmental stresses as sets of interacting organisms,instead of functioning exclusively as unrelatedindividuals.

Each community is a network of organisms that haveadapted to propagate both as individuals and as part ofone or more associations. Networking of organisms in acommunity might allow ecient use of environmental

resources and production of favorable microenviron-ments within unfavorable macroenvironments [5, 6, 7, 8].Bacteria may respond to environmental stresses assets of interacting organisms, as revealed by theappearance and disappearance of sets of organismsduring adaptation of ow-cell communities to substrateshifts. Continual inoculation as a mechanism ofrecombination led to more rapid adaptation andenhanced growth of microbial communities subjected toenvironmental transitions, which suggested that prolif-eration might result from recombination events atmultiple levels of biological organization. Enhancedgrowth of biolm communities when they were

subjected to a second set of sudden environmentaltransitions suggests that the cellular positioning infor-mation accrued from one cycle to the next may havebeen heritable.

Acknowledgements The US Department of Energy, CanadianNSERC, GASReP and Saskatchewan ADF are acknowledged fornancial support.

References

1. Aelion CM, Swindoll CM, Pfaender FK (1987) Adaptation toand biodegradation of xenobiotic compounds by microbial

communities from a pristine aquifer. Appl Environ Microbiol53:22122227

2. Atlas RM (1984) Use of microbial diversity measurements toassess environmental stress. In: Klug MJ, Reddy CA (eds)Current perspectives in microbial ecology. American Societyfor Microbiology Press, Washington, D.C., pp 540545

3. Barkay T, Pritichard H (1988) Adaptation of aquatic microbialcommunities to pollutant stress. Microbiol Sci 5:165169

4. Benson HM (1990) Microbiological applications: a laboratorymanual in general microbiology. Brown, Dubuque, Iowa5. Caldwell DE, Costerton JW (1996) Are bacterial biolms

constrained to Darwin's concept of evolution through naturalselection? Microbiologia 12:347358

6. Caldwell DE, Atuku E, Wilkie DC, Wivcharuk KP, Kart-hikeyan S, Korber DR, Schmid DF, Wolfaardt GM (1997a)Germ theory versus community theory in understanding andcontrolling the proliferation of biolms. Adv Dent Res 11:413

7. Caldwell DE, Korber DR, Wolfaardt GM, Lawrence JR(1997b) Do bacterial communities transcend Darwinism? AdvMicrob Ecol 15:105191

8. Caldwell DE, Wolfaardt GM, Korber DR, Lawrence JR(1997c) Cultivation of microbial consortia and communities.In: Hurst CJ et al (eds) Manual of environmental microbiology.American Society for Microbiology Press, Washington, D.C.,

pp 79909. De Leij FAAM, Whipps JM, Lynch JM (1994) The use ofcolony development for the characterization of bacterial com-munities in soil and on roots. Microb Ecol 27:8197

10. Devereux R, Hines ME, Stahl DA (1996) S cycling: char-acterization of natural communities of sulfate-reducingbacteria by 16S rRNA sequence comparisons. Microb Ecol32:283292

11. Eklund C, Lankford CE (1967) Laboratory manual for generalmicrobiology. Prentice Hall, Englewood Clis, N.J.

12. Fulthorpe RR, Wyndham RC (1991) Transfer and expressionof the catabolic plasmid pBRC60 in wild bacterial recipients ina freshwater ecosystem. Appl Environ Microbiol 57:15461553

13. Fry NK, Fredrickson JK, Fishbain S, Wagner M, Stahl DA(1997) Population structure of microbial communities associ-ated with two deep, anaerobic, alkaline aquifers. Appl EnvironMicrobiol 63:14981504

14. Garland JL (1996) Analytical approaches to the characteriza-tion of samples of microbial communities using patterns ofpotential C source utilization. Soil Biol Biochem 28:213221

15. Griths BS, Ritz K, Glover LA (1996) Broad-scale approachesto the determination of soil microbial community structure:application of the community DNA hybridization technique.Microb Ecol 31:269280

16. Gupta A, Flora JRV, Gupta M, Sayles GD, Suidan MT (1994)Methanogenesis and sulfate reduction in chemostats. 1. Kineticstudies and experiments. Water Res 28:781793

17. Haldeman DL, Amy PS (1993) Diversity within a colonymorphotype: implications for ecological research. Appl Envi-ron Microbiol 59:933935

18. Hickey RF, Switzenbaum MS (1991) The response and utilityof hydrogen and carbon monoxide as process indicators ofanaerobic digestors subject to organic and hydraulic overloads.Res J Water Pollut Cont Fed 63:129140

19. James GA, Beaudette L, Costerton JW (1995) Interspeciesbacterial interactions in biolms. J Ind Microbiol 15:257262

20. Jime nez L, Breen A, Thomas N, Federle TW, Sayler GS (1991)Mineralization of linear alkylbenzene sulfonate by a four-member aerobic bacterial consortium. Appl Environ Microbiol57:15661569

21. Korber DR, Lawrence JR, Sutton B, Caldwell DE (1989)Eects of laminar ow velocity on the kinetics of surfacecolonization by mot+ and mot Pseudomonas uorescens.Microb Ecol 18:119

22. Rajagopal BD, Brahmaprakash GP, Sethunathan N (1984)Degradation of carbofuran by enrichment cultures and purecultures of bacteria from ooded soils. Environ Pollut SerA Ecol Biol 36:6174

79


8/8

23. Smith PD, McCarty PL (1989) Energetics and rate eects onmethanogenesis of ethanol and propionate in perturbedCSTRs. Biotechnol Bioeng 34:3954

24. Spain JC, Veld PA van (1983) Adaptation of naturalmicrobial communities to degradation of xenobioticcompounds: eects of concentration exposure time inoculumand chemical structure. Appl Environ Microbiol 45:428435

25. Spain JC, Pritchard PH, Bourquin AW (1980) Eects ofadaptation on biodegradation rates in sediment water cores

from estuarine and freshwater environments. Appl EnvironMicrobiol 40:726734

26. Sundh I, Nilsson M, Borga P (1997) Variation in microbialcommunity structure in two boreal peatlands as determined byanalysis of phospholipids fatty acid proles. Appl EnvironMicrobiol 63:14761482

27. Wolfaardt GM, Lawrence JR, Robarts RD, Caldwell DE(1994) The role of interactions sessile growth and nutrient

amendment on the degradative eciency of a bacterial con-sortium. Can J Microbiol 40:331340

80

Adaptacion de comunidades bacterianas a ambientes de trasicion.pdf

Documents

Transcript of Adaptacion de comunidades bacterianas a ambientes de trasicion.pdf