A soil microscale study to reveal the heterogeneity of Hg II impact

Lionel Ranjard

Description

Niveau: Supérieur, Doctorat, Bac+8
A soil microscale study to reveal the heterogeneity of Hg(II) impact on indigenous bacteria by quanti¢cation of adapted phenotypes and analysis of community DNA ¢ngerprints Lionel Ranjard a , Sylvie Nazaret a , Franc°ois Gourbie ' re a , Jean Thioulouse b , Philippe Linet c , Agne ' s Richaume a; * a Laboratoire d'Ecologie Microbienne, UMR CNRS 5557, Universite ? Claude Bernard, Lyon I, F-69622 Villeurbanne Cedex, France b Laboratoire de Biome ? trie, UMR CNRS 5558, Universite ? Claude Bernard, Lyon I, F-69622 Villeurbanne Cedex, France c Service Central d'Analyses du CNRS, Chemin du Canal, BP 22, 69390 Vernaison, France Received 19 April 1999; received in revised form 16 September 1999; accepted 4 October 1999 Abstract The short term impact of 50 WM Hg(II) on soil bacterial community structure was evaluated in different microenvironments of a silt loam soil in order to determine the contribution of bacteria located in these microenvironments to the overall bacterial response to mercury spiking. Microenvironments and associated bacteria, designated as bacterial pools, were obtained by successive soil washes to separate the outer fraction, containing loosely associated bacteria, and the inner fraction, containing bacteria retained into aggregates, followed by a physical fractionation of the inner fraction to separate aggregates according to their size (size fractions).

• unspiked control


• pools associated


• dna restriction


• nd nd


• control spiked


• soil


• bacterial community


• sand size


• bacteria

Sujets

• Yvelines
• Bernard
• Eberbach
• Nazaret

Informations

• Publié par: mijec
• Nombre de lectures: 17
• Langue: English

Extrait

FEMS Microbiology Ecology 31 (2000) 107^115
www.fems-microbiology.org
A soil microscale study to reveal the heterogeneity of Hg(II) impact
on indigenous bacteria by quanti¢cation of adapted phenotypes and
analysis of community DNA ¢ngerprints
a a a b'Lionel Ranjard , Sylvie Nazaret , Franc ?ois Gourbiere , Jean Thioulouse ,
c a;'Philippe Linet , Agnes Richaume *
a ¤Laboratoire d’Ecologie Microbienne, UMR CNRS 5557, Universite Claude Bernard, Lyon I, F-69622 Villeurbanne Cedex, France
b ¤ ¤Laboratoire de Biometrie, UMR CNRS 5558, Universite Claude Bernard, Lyon I, F-69622 Villeurbanne Cedex, France
c Service Central d’Analyses du CNRS, Chemin du Canal, BP 22, 69390 Vernaison, France
Received 19 April 1999; received in revised form 16 September 1999; accepted 4 October 1999
Abstract
The short term impact of 50WM Hg(II) on soil bacterial community structure was evaluated in different microenvironments of a silt loam
soil in order to determine the contribution of bacteria located in these microenvironments to the overall bacterial response to mercury
spiking. Microenvironments and associated bacteria, designated as bacterial pools, were obtained by successive soil washes to separate the
outer fraction, containing loosely and the inner fraction, containing bacteria retained into aggregates, followed by a
physical fractionation of the inner fraction to separate aggregates according to their size (size fractions). Indirect enumerations of viable
Rheterotrophic (VH) and resistant (Hg ) bacteria were performed before and 30 days after mercury spiking. A ribosomal intergenic spacer
analysis (RISA), combined with multivariate analysis, was used to compare modifications at the community level in the unfractionated soil
Rand in the microenvironments. The spatial heterogeneity of the mercury impact was revealed by a higher increase of Hg numbers in the
outer fraction and in the coarse size fractions. Furthermore, shifts in RISA patterns of total community DNA indicated changes in the
composition of the dominant bacterial populations in response to Hg(II) stress in the outer and in the clay size fractions. The heterogeneity
of metal impact on indigenous bacteria, observed at a microscale level, is related to both the physical and chemical characteristics of the soil
microenvironments governing mercury bioavailability and to the bacterial composition present before spiking. ? 2000 Federation of
European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords: Bacterial community; Mercury; Soil microenvironment; DNA ¢ngerprint; RISA; Indirect enumeration
1. Introduction versity within bacterial populations (for reviews see [1,2]).
Several studies reported an impact of heavy metals at the
Bacterial response to heavy metal contamination in soil community level using phenotypic or genetic ¢ngerprinting
provides a relevant model for ecological studies to assess techniques. Microbial community measurements based on
the in£uence of environmental characteristics on the quan- phospholipid fatty acid (PLFA) composition allowed the
titative and qualitative modi¢cations of soil bacterial com- detection of shifts in microbialsition in di¡erent
munities induced by hydrosoluble toxicants. Heavy metals soil types after short- and long-term metal exposures [3].
in soil are known to have a deleterious e¡ect on the num- Smit et al. [4] used ampli¢ed ribosomal DNA restriction
bers of bacteria, microbial biomass and activities, and di- analysis (ARDRA) as a genetic ¢ngerprinting tool to show
modi¢cations of the community structure in copper-con-
taminated soils. The shifts can re£ect an increase in bac-
terial community metal tolerance as demonstrated by
fi fiBaath [5] who used the thymidine-incorporation method.
* Corresponding author. Present address: UMR-CNRS 5557 - The increase in the relative abundance of adapted pheno-
¤ “Ecologie Microbienne, Universite Claude Bernard Lyon I, Bat 741,
types, generally evidenced in contaminated environments,
43 Bd du 11 Novembre 1918, F-69622 Villeurbanne cedex, France.
could be the mechanism responsible for such an increasedTel.: [phone]; Fax: [phone];
E-mail: [e-mail]. tolerance observed at the community level [6,7].
0168-6496 / 00 / $20.00 ? 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S0168-6496(99)00089-6
FEMSEC 1091 11-1-00108 L. Ranjard et al. / FEMS Microbiology Ecology 31 (2000) 107^115
When evaluating the bacterial response to heavy metal cosms were incubated for 30 days under the same condi-
contamination in soil, environmental parameters must also tions. Characteristics of unfractionated soil, outer and in-
be considered. Generally, more signi¢cant modi¢cations of ner soil fractions, and of various size fractions of the inner
bacterial activities, cell numbers and biomass occur in fraction are listed in Table 1.
light-textured soils than in soils rich in clay minerals and
organic matter [7^11]. Such observations are explained by 2.2. Soil fractionation
a reduced bioavailability of the metal bound by clay min-
erals and humic-like materials [12^14]. Metal accessibility The microenvironments were separated by using two
to bacterial cells is also dependent on circulation and dif- soil fractionation procedures: successive soil washes to
fusion processes in soil pores. Ranjard et al. [11] have separate the outer and the inner soil fractions followed
shown that the balance between macro- and microporosity by a physical fractionation of the inner fraction to sepa-
in di¡erent soil types controlled the magnitude of metal rate aggregates into size fractions. Theation was
impact. repeated twice using three microcosms for each incubation
Soil structural organization in aggregates of di¡erent time. The soil washing procedure was performed with 10 g
size and stability de¢nes a mosaic of microenvironments of soil as described by Ranjard et al. [11]. This procedure
di¡ering by their physical, chemical and structural proper- separated bacteria located in macropores, i.e., easily
ties [15^18]. Consequently, indigenous bacteria are sub- washed out from the surface of aggregates (outer fraction),
jected to heterogeneous conditions depending on their lo- from those located in micropores, i.e., retained in soil ag-
cation. Some microenvironments are more favorable gregates after washings (inner fraction). The supernatants
bacterial habitats due to a better nutrient and water status containing microorganisms released from the outer frac-
and to the absence of predation by protozoa [17,19^24]. In tions were pooled, centrifuged (9800Ug, 20 min) and re-
spite of the importance of cell location for the degree of suspended in 50 ml of sterile 0.8% NaCl solution. An
impact by a contamination, only a few studies have as- aliquot was dried (105‡C, 24 h) to determine the dry
sessed bacterial response at a microscale level [11,17,25]. weight of the soil.
The role of soil microenvironments in modulating quanti- The remaining washed soil (inner fraction), pooled from
tative (cell density) and qualitative (activity, diversity, three microcosms, was further fractionated to separate
community structure) bacterial responses and thus the microenvironments based on the size of stable aggregates
overall impact of metals on soil bacterial communities by the slightly modi¢ed procedure described by Kabir et
has never been investigated. al. [26]. Sand size fractions containing stable aggregates
In this study, the soil was fractionated to evaluate the above 50 Wm in size, including coarse and ¢ne sand par-
response of indigenous bacteria to heavy metal contami- ticles, fraction 250 to 2000 Wm and 50 to 250 Wm, respec-
nation in various microenvironments. Our objectives were tively, were obtained by wet sieving using sterile cool
(1) to evaluate the contribution of bacteria associated to water (6 10‡C) to reduce bacterial growth. The soil sus-
di¡erent microenvironments (designated hereafter as bac- pension, containing aggregates and particles below 50 Wm,
terial pools) to the overall response of the bacterial com- was aseptically transferred into a sedimentation £ask. The
munity and (2) to determine to what extent soil physical, silt size fraction containing aggregates and coarse silt par-
chemical and microbiological characteristics modulate the ticles (20^50 Wm) was obtained by gravity sedimentation.
impact of the metal on the soil micro£ora. These goals Aggregates and particles below 20 Wm were withdrawn.
were assessed with a model system consisting of a silt- The sedimentation step was repeated three times by resus-
loam soil arti¢cially polluted with 50 WM of Hg(II) as pending the sedimentated soil in cool sterile water. Fine
previously described [11]. silt particles and 2^20 Wm aggregates constituting the sec-
ond silt size fraction, were pelleted from the supernatant
by centrifugation at 90Ug at 10‡C in a swinging bucket
2. Materials and methods rotor in 250-ml centrifuge tubes. The dispersible clay frac-
tion (6 2 Wm) was obtained by an overnight £occulation
2.1. Soil and microcosm set up of the supernatant at 4‡C after addition of CaCl (50 mM2
¢nal). Moist size-fractions 250^2000, 50^250 and 20^50
The soil used was coll