Chromium biosorption with respect to growth phase for four bacterial isolates and two consortia following two hours of exposure to 100 mg/L trivalent or hexavalent chromium at pH 7.. sur
Introduction
Research hypothesis and significance of the work
Research was performed to elucidate previously-unexplored relationships between microbial habitat, cell physiology, and the biotransformation of metals and organics in the context of soil and sediment remediation The heavy metal used in this work was chromium The governing research hypothesis was that the degree of association of indigenous culturable, heterotrophic bacterial communities within vadose zone soil correlates with their propensity for chromium biosorption and reduction, chromium tolerance, cell surface properties, growth rate, and organic substrate affinity Communities more strongly associated with soil particles were expected to be more oligotrophic than those that were weakly-attached; that is, they should have a higher affinity for primary organic substrates and slower growth kinetics Their larger substrate affinity was expected to result from more efficient, tightly-controlled substrate uptake systems that would simultaneously provide microorganisms with greater resistance to chromium toxicity Strongly soil-associated bacterial communities were also expected to possess cell surface properties reflective of their attachment to soil, for example elevated hydrophobicity for enhanced interactions with hydrophobic soil organic matter Although these relationships were expected to hold at the community level, they were not expected to apply uniformly at the population level Instead, individual bacterial isolates cultured from larger communities were anticipated to exhibit unique cell surface properties and biotransformation behavior that would not necessarily reflect their original degree of soil association
Validation of the research hypothesis would have two main implications First, it would demonstrate potential experimental biases resulting from the use of “standard” microbiological elution methods that wash only easily-eluted organisms from soil Procedures for isolating soil bacteria for use in laboratory studies typically involve a relatively short, gentle soil washing step that recovers only a small fraction of easily- detached soil organisms (Dunbar et al., 1997) Microorganisms that remain trapped, and thus unstudied, in washed soil may possess useful traits, such as high substrate affinities and metal tolerance, that could be exploited for improved in situ or ex situ remediation performance Thus, the possible consequences of employing widely-accepted but flawed extraction measures are the underestimation of the rate and extent of comingled waste biotransformation and the incorrect extrapolation of laboratory data to the field
Second, a valid hypothesis would suggest that long-term immobilization of heavy metals such as chromium could be facilitated by manipulating nutrient loads to subsurface environments in order to shift microbial ecology for specific purposes Improved remediation could be achieved through a two-phased approach, for example During the first phase, copiotrophic microbial populations (those most often studied or cultivated in the laboratory, characterized by fast growth and a preference for high substrate and nutrient concentrations) could be stimulated in contaminated soil, perhaps through the addition of supplemental nutrients or electron donors With their relatively low substrate affinities, copiotrophs could transform or mineralize organics to moderate residual levels During the second phase, metal-reducing oligotrophic species could be stimulated, for example by decreasing or halting the provision of nutrients Oligotrophs would reduce metals to less toxic, less mobile species, and their greater substrate affinity would allow them to achieve lower residual organic pollutant concentrations than possible by copiotrophs alone, thereby achieving a “polishing step.”
Although previous studies have characterized subsurface bacteria based on their attachment to soil or other solid matrices (Campbell et al., 1999; DeFlaun et al., 1999; Holm et al., 1992; Johnson et al., 1996; Lehman et al., 2001a,b; Lehman and O’Connell, 2002; Van Schie and Fletcher, 1999), or according to their ability to degrade organics in the presence of heavy metals (Drzyzga et al., 2002; Kuo and Genther, 1996; Sandrin et al., 2000), this study examined relationships between bacterial association with soil, cell surface properties, metal transformation and resistance, and affinity for organic substrates in subsurface environments.
Experimental approach
The research utilized a combination of microbiological methods, chemical assays, and advanced nondisruptive spectroscopic analyses, including x-ray absorption near edge spectroscopy (XANES) and extended x-ray absorption fine structure spectroscopy (EXAFS), to examine the biotransformation of chromium, organics, and chromium- organic mixtures The bacteria used were aerobic, heterotrophic communities and isolates distinguished by their variable retention in vadose zone soil Specific goals included the following: refinement of a soil elution procedure for stepwise extraction of bacterial communities selection and preservation of eluted microbial consortia and isolates for detailed study establishment of the uniqueness of consortia and isolates through identification of distinguishing cell surface characteristics and other biochemical properties measurement of cultures’ growth kinetics and substrate affinity for organic electron donors determination of the tolerance of consortia and isolates to toxic hexavalent chromium in the presence of a labile substrate or a model organic pollutant establishment of whether metal tolerance was correlated to cell properties such as primary substrate affinity measurement of the rate and extent of chromium biosorption by the cultures assessment of whether microbial consortia and isolates were able to reduce hexavalent chromium to the trivalent form, and if so, whether this ability was related to substrate utilization characteristics.
Chromium pollution: sources and chemistry
This research focused on chromium The release of metals such as chromium into the environment is of great environmental concern because of their toxicity and persistence Although chromium is found naturally in soils and minerals, most chromium in the environment originates from industrial processes such as electroplating, steel and glass manufacturing, mining, dye and pigment synthesis, and leather tanning (Kimbrough et al., 1999; Mukherjee, 1998) The metal is also commonly found in industrial wastes such as sludges, fly ash, and slag Chromium has been found at concentrations as high as
2740 mg/L in groundwater (Office of Technology Assessment, 1984)
Chromium exists in multiple oxidation states, most of which are unstable in the environment Two major forms persist in environmental systems: trivalent chromium (Cr[III]) and hexavalent chromium (Cr[VI]) (Kroshwitz, 1993) Most chromium present in industrial wastes exists in the hexavalent form (Mukherjee, 1998) Hexavalent chromium is carcinogenic, mutagenic, and teratogenic to humans and other animals and is approximately one hundred times more toxic than the trivalent form (Langand, 1983; Petrilli and DeFlora, 1977) Persistent hexavalent chromium may prove toxic to subsurface microbial communities and negatively impact the biodegradation potential of organic substrates Because of its toxicological properties, legislative regulations for chromium are based on valence state, rather than total concentration
Hexavalent chromium compounds are generally more water soluble than trivalent chromium compounds, particularly in the biologically-relevant pH range of 5-7 For purposes of in situ metal immobilization, it is desirable to convert chromium (VI) contaminants to less soluble, less toxic chromium (III) compounds Aqueous solubilities of both trivalent and hexavalent chromium compounds vary over many orders of magnitude (Kimbrough et al., 1999) Chromium (III) precipitates as a hydroxide at a pH above 5.5 (Alloway, 1990), existing in natural aquatic environments primarily in the form of relatively insoluble metal hydroxides, including [Cr(OH) 2+ ], [Cr(OH)2 +], [Cr(OH)3], [Cr(OH)4 -], and [Cr3(OH)4 5+] (Faust and Aly, 1981) The solubilities of these compounds are particularly low at elevated pH Unlike trivalent chromium, which may be found as chromic ion (Cr 3+ ), hexavalent chromium does not exist as a free cation (Cr 6+ ) in waters
Instead, it is present as oxides that behave as divalent anions rather than hexavalent cations These oxides include chromate (CrO4 -2) and dichromate (Cr2O7 -2) ions, both soluble in water over a wide pH range Electron transfer between oxygen and various chromium compounds is key to the interconversion of trivalent and hexavalent chromium The most important natural oxygen source for subsurface redox transformations of chromium is water, but molecular oxygen from the atmosphere and from supplied sources such as ozone, hydrogen peroxide, and metal oxides must also be taken into account (i.e., during the use of aquifer oxygenation technologies) Even so, dissolved oxygen alone has not been found to induce measurable chromium (III) oxidation, even after more than four months (Saleh et al., 1989)
Chromium in soils and pore water exists in states similar to those found in aquatic systems: as trivalent chromium, trivalent chromium compounds, and hexavalent chromium oxides The chemical and sorptive properties of chromium in soil environments are complex and poorly understood Physisorbed chromium can be leached from soils, indicating that sorption of the metal is a reversible process Trivalent chromium compounds readily form stable complexes with negatively charged inorganic or organic particles in soils and sediments, including mineral solids with exposed surface hydroxy groups and silicates From a site management standpoint, chromium sorption to soil and sediment components is favorable since it leads to stabilization and immobilization of the metal Cr(III) that is not sorbed to the solid phase generally eventually precipitates as Cr(OH)3 (Barber and Stuckey, 2000)
Unlike Cr(III), hexavalent chromium is highly unstable and mobile in soils because it adsorbs poorly to soil components under natural conditions However, the interconversion between precipitated chromium, adsorbed chromium, and dissolved chromium in soil water is complicated It appears to be dependent on a variety of parameters including the species of chromium present; soil pH; biological activity in soil; oxygen concentration; oxidizing and/or reducing agents such as hydrogen sulfide, sulfur, iron sulfides, ammonium, nitrite, and ferrous iron (Bodek et al., 1988); and the presence of organic matter Organic matter has a greater influence on Cr(VI) reduction than most other chemical constituents, for it is able to promote accelerated reduction of Cr(VI) at almost any pH (Mukherjee, 1998) As long as anoxic sediments are themselves physically stable and are not depleted in reducing power, they can effectively promote chromium reduction, reduce chromium toxicity, and enhance chromium stability When reducing power becomes exhausted by high Cr(VI) concentrations, soil amendments may become necessary for continued long-term metal immobilization Aerobic environments often provide more of a challenge in controlling the speciation of chromium In well- aerated soils, the chemistry of chromium resembles that of iron, and in fact chromium speciation and mobility is strongly influenced by that of iron (Mukherjee, 1998).
Chromium uptake, transformation, and resistance by soil microorganisms
A number of studies have demonstrated the detoxification of chromium via microbial reduction of Cr(VI) to Cr(III) and subsequent precipitation of Cr(III) (Bopp and Ehrlich, 1988; Gvozdyak et al., 1985; Horitsu et al., 1987; Suzuki et al., 1992; Wang and Shen, 1995), which is less able to cross the cell membrane (Nieboer and Jusys, 1988) Microbial transformations of Cr(VI) to Cr(III) typically occur most quickly at neutral pH and at temperature, soil moisture, and nutrient conditions coinciding with the optimal growth of chromium-reducing microorganisms High cell densities are almost always necessary to obtain significant rates of Cr(VI) reduction (Wang and Shen, 1995) Known electron donors for Cr(VI) reduction under both aerobic and anaerobic conditions are generally limited to natural aliphatic compounds, amino acids, fatty acids, simple sugars and hydrogen, although one study demonstrated chromium reduction in a co-culture utilizing phenol (Shen and Wang, 1995) Under aerobic conditions, endogenous cell reserves and NADH (nicotinamide ademine dinucleotide, a carrier of electrons and protons), also may serve as the electron donor for Cr(VI) reduction (Wang and Shen, 1995) Both aerobic and anaerobic microorganisms from a wide range of subsurface environments and genera are able to reduce chromium
Reduction at cell surfaces may occur through a three-step process in which negatively charged dichromate (Cr2O7 2-) is bound to positively charged groups on cell surfaces; reduced by adjacent functional groups; and released as Cr(III) by electronic repulsion (Parka et al., 2005) Some bacteria are known to possess enzymes with large reduction potentials that are capable of transforming hexavalent chromium For example,
Pseudomonas ambigua G-1 has a NADPH-dependent reductase (Suzuki et al., 1992), Escherichia coli has a flavin reductase (Puzon et al., 2002), and Shewanella oneidensis
MR-1 has a nitrite reductase (Viamajala et al., 2002)
Microbial chromium resistance, a phenomenon not exclusive to the ability to reduce chromium, is often observed in microorganisms indigenous to chromium- impacted environments (Cervantes, 1991; Nieto et al., 1989; Ohtake et al., 1987; Wang et al., 1989) Resistance to the metal may be achieved through the adsorption of chromium(VI) to bacterial surfaces or by chromium accumulation inside cells
Chromium resistance appears to be more prevalent among prokaryotes than eukaryotes (Shakoori et al., 2000) and tends to diminish as chromium accumulates in the cell Microbial metal reduction and resistance have been shown to take place over concentrations ranging from trace levels up to 0.27 M total chromium or more (Badar et al., 2000; Fude et al., 1994; Shakoori et al., 2000)
Extracellular polymeric substances (EPS) are thought to play a major role in metal resistance and in the binding and accumulation of metals at cell surfaces EPS is often acidic (Czajka et al., 1997; Sutherland, 1982), and metals readily adsorb to the multiple high-affinity binding sites on polysaccharides and other EPS functional groups (Rudd et al., 1984; Kellems and Lion, 1989) Metals may also deposit on cell surfaces as oxides (Ghiorse, 1984; Lion et al., 1988; Nelson et al., 1994; Chen et al., 1994) EPS serves as a physical and chemical barrier that is thought to protect cells from compound toxicity (Brown and Gilbert, 1993; Chen, et al., 1993; Costerton et al., 1987; Evans et al., 1990; Gilbert et al., 1997; Gristina et al., 1987; Hoyle et al., 1990; Stewart, 1996; Xu et al., 2000) Much of what is known about toxicity to bacteria comes from studies of antibiotic resistance, which have shown that extracellular polymeric substances retard the diffusion of antibiotics (Ishida et al., 1998) and other solutes (Stewart, 1998), thus protecting biofilm organisms Bacteria living deep in a biofilm may exist in a slow-growing state (Brown et al., 1988), which further prevents the uptake of antibiotics and increases apparent resistance Comparable explanations have been offered for tolerance to heavy metals and toxic organics (Junter et al., 2002; Bessems and Terpstra, 2003)
Bacterial biomass is a very efficient metal biosorbent because of cells’ high surface area-to-volume ratio and the presence of functional groups that have high affinities for heavy metals (Beveridge, 1989a; Beveridge and Murray, 1980; Daughney and Fein, 1998; Daughney et al., 1998; Fein et al., 1997; Fowle and Fein, 1999) Two types of bacterial uptake systems for heavy metals have been postulated One is constitutively expressed and results in fast, nonspecific metal uptake that is dependent on chemiosmotic gradients across the cytoplasmic membrane The second system is inducible as needed and results in slower, metal-specific uptake that often requires ATP consumption in addition to a chemiosmotic gradient (Nies and Silver, 1995; Nies, 1999).
Microbial partitioning in soil and its implications for
The physical location or partitioning of microorganisms between soil surfaces, pore water, and groundwater is significant to in situ bioremediation applications For instance, control over cell attachment and detachment is necessary for bioaugmentation strategies that rely on the uniform delivery, without clogging near the injection point, of biodegradative organisms introduced into a contaminated zone Following introduction, bacteria should be adherent to an extent such that they are not displaced from the bioactive zone by groundwater flow or during delivery of supporting nutrients or substrates Cell adhesion also affects the types and quantities of substrates encountered by and available to cells, thus affecting remediation endpoints that can be expected for soluble and sorbed contaminants Bacteria adapted to living on contaminated surfaces and those that exist in the surrounding groundwater or pore water are expected to form communities with different capacities to degrade organics and to tolerate or transform heavy metals For example, adhesive bacteria may be expected to have greater exposure to strongly-sorbing organics, soil-sequestered heavy metals, and natural organic matter compared to planktonic organisms in a given contaminated microenvironment Cell attachment, particularly in biofilms, is also known to confer selective advantages to cells, including physical protection from predators, pH fluctuations, temperature gradients, and antimicrobial agents; the sharing of metabolites and extracellular enzymes; and horizontal gene transfer
Cell partitioning and physiological properties Microbial partitioning of bacteria between solid and aqueous phases has in fact been shown to be related to the physical distribution of physiological properties such as growth rates, exopolymer synthesis, and enzyme activity (Lehman and O’Connell, 2002; Van Loosdrecht et al., 1990), although it is often uncertain whether the differences are due to functional expression or community structure (Delong et al., 1993; Karner and Herndl, 1992; Murrell et al., 1999; Okabe et al., 1994) Individual bacterial species possess distinct capacities for degrading sorbed and dissolved organic contaminants such as naphthalene (Guerin and Boyd, 1992) and exhibit divergent metabolic traits depending upon the relative hydrophobicity of the microenvironment they occupy (Bastiaens et al., 2000) Lehman et al (2001b) found that attached heterotrophic bacteria isolated from aquifer core material were morphologically more diverse than free-living bacteria isolated from groundwater at the same depths Bacteria attached to particulates have been found to exhibit greater extracellular enzyme activity than free-living bacteria in aqueous habitats (Karner and Herndl, 1992; Middleboe et al., 1995)
Abundance and activity of planktonic versus attached bacteria The relative amount of biomass present as attached and free-living bacteria appears to be site- dependent A number of studies of aquifer material have identified the majority of total biomass as being attached to the solid phase (Holm et al., 1992; Kolbel-Boelke et al., 1988; Lehman et al., 2001a; Harvey et al., 1984; Alfreider et al., 1997; Escobar et al., 1996; Ghiorse and Wilson, 1988; Hazen et al., 1991; Pedersen and Ekendahl, 1990; Dispirito et al., 1983; Kirchman and Mitchell, 1982; Caron et al., 1982) One bench-scale column study demonstrated that about 99% of total biomass and 96% of phenol-oxidizing microorganisms were attached to the geologic medium (Lehman et al., 2001a) Other researchers have found greater quantities of free-living bacterial biomass (Lehman et al., 2001b; Bidle and Fletcher, 1995; Griffith et al., 1994; Turley and Mackie, 1994; Kirchman and Mitchell, 1982; Middleboe et al., 1995; Simon et al, 1990; Unanue et al., 1992; Kogure, 1989, Smith et al., 1995; Bekins et al., 1999; Thomas et al., 1987) Based on this information, it is difficult to state presumptively whether sessile or planktonic organisms will prevail in a given environment
Mechanisms of attachment Microbial attachment is a complicated process involving physicochemical and biological reactions between solid surfaces, bacteria, and the ambient environment The first step in attachment is a weak physicochemical interaction between bacterium and surface that may be reversed by gentle shearing forces (Marshall et al., 1971; Marshall, 1992) Stronger, potentially irreversible adhesion may subsequently develop (Dankert et al., 1986; Marshall, 1985; Marshall et al., 1971), for example through specific or nonspecific bridging (Marshall, 1992) of extracellular polymeric substances including polysaccharides, proteins, glycoproteins, or lipopolysaccharides (Allison et al., 1987; Costerton et al., 1985; Fletcher and Floodgate, 1973; Marshall, 1992; Marshall, 1985, Underwood et al., 1995; Heissenberger et al.,
1996; Bennett et al., 1999; Ransom et al., 1999; Makin and Beveridge, 1996) In fact, a lack of bacterial EPS has been linked to less adherence (Christensen et al., 1982, Davey and O’Toole, 2000)
Both long-range and short-range forces are involved in attachment (An and Friedman, 1997; Dankert et al., 1986; Krekeler et al., 1989; Marshall, 1992; Marshall, 1985) Attractive forces between cells and surfaces include van der Waals forces, gravitational forces, and electrostatic and hydrophobic interations (Dankert et al., 1986; Krekeler et al., 1989) Chemical fluctuations can cause changes in cell surface properties, attractive forces, and attachment tendences; for example, lactic acid was shown to alter the hydrophobicity of Listeria monocytogenes, affecting the microorganism’s adherence (Briandet et al., 1999)
Attachment is both environment- and species-specific One study that used Tween 20 to treat attached cells found that hydrophobic interactions were not involved in maintaining the structure of marine biofilms, but were important in certain freshwater organisms (Fletcher, 1989) Another study determined that attached Cyanobacteria had hydrophobic surfaces, whereas planktonic cells were hydrophilic (Fattom and Shilo, 1984) Such phenomena may be explained by the activation of specific genes following attachment; these genes may trigger production of extracellular materials (e.g., EPS) that stabilize attachment and alter cell surface properties (McCarter and Silverman, 1990) A number of studies have demonstrated the genetic control of biofilm development (e.g., Costerton et al., 1999; Davies et al., 1993; Watnick et al., 2001; Fan and Macnab, 1996; Stephens et al., 1997; Davies and Geesey, 1995; Prigent-Combaret et al., 1999)
Solid surfaces and microbial attachment The chemical composition of surfaces is among the most important factors influencing microbial attachment (Harvey et al., 1989) Biomaterials have been found to permit easier cell attachment (Sugarman and Musher, 1981) than abiotic surfaces (e.g., steel, glass, plastic, and soil), which are mostly non-nutritive and may require bacteria to produce exudates to facilitate adhesion Specific components of abiotic surfaces have varying effects on cell attachment Iron and aluminum hydroxides in sediments have been found to enhance bacterial retention in aquifers (Knapp et al., 1998; Ghiorse, 1984; Lion et al., 1988; Dong et al., 2002), possibly by altering surface charge or nutrient conditions (Nelson et al., 1994) However, organic matter adsorbed to the surfaces of metal oxide coatings can preclude adhesion (Dong et al., 2002) Hydrophobic surfaces, such as those bearing silicon, can also decrease cell attachment (van Schie and Fletcher, 1999) Granular activated carbon is relatively favorable for attachment, comparable to sand and anthracite (Camper et al., 1987; Malony et al., 1984)
The physical nature of surfaces is also important to attachment Surface roughness, topography, and grain size are all significant Deep narrow pits provide a safe environment for bacteria to avoid predators (Murray and Jumers, 2002; DeFlaun and Mayer, 1983) Boyd et al., (2002) used atomic force microscopy to detect the attachment of individual bacteria on surfaces of different topographies and discovered that bacteria became more strongly attached to abraded surfaces than to smooth layers Knoell et al (1999) found that irregular or elliptical pores discouraged bacterial retention
Cell surface properties and microbial attachment Many studies have focused on hydrophobic and electrostatic interactions between cells and surfaces and their role in attachment (Fattom et al., 1984; Fletcher and Marshall, 1982; Kjelleberg and Hermansson, 1984; Gannon et al., 1991b; McCalou et al., 1995; Absolom et al., 1983; Busscher et al., 1984; Hermansson et al., 1982; Hermesse et al., 1988; Morisaki, 1986; Marshall et al., 1971; van Loosdrecht et al., 1987a; Briandet et al., 1999; Dickson and Koohmaraie, 1989; Gordon and Millero, 1984; Fletcher, 1996; Taylor et al., 1997; van Loosdrecht et al., 1990; Daniels, 1980; Rijnaarts et al., 1999; Bendinger et al., 1993; Dickson, 1989; Marshall, 1985; Rutter and Vincent, 1984; van Oss, 1993) Makin and Beveridge (1996) showed that hydrophobic cells such as those of Pseudomonas aeruginosa easily attached to hydrophobic surfaces, whereas hydrophilic cells’ attachment was controlled more by surface charge (Makin and Beveridge, 1996) Hydrophobic bacteria were shown not to attach effectively to hydrophilic materials (Yousefi, et al., 1998; Knoell et al., 1999; Gómez-Suarez et al., 2001; Cunliffe et al., 1999) Nevertheless, bacteria are normally negatively charged because of the presence of carboxyl and phosphate groups, among others, and these contribute a measure of hydrophilicity In fact, electronegatively charged bacteria can bind to either positively- or negatively-charged surfaces, the latter as a result of counterions involved in cation bridging mechanisms (Urrutia and Beveridge, 1993; Ueshima et al., 2002)
Cell surface properties can change with growth phase Although growth phase affects bacterial attachment (Weiss et al., 1995), it appears to be species-dependent, with some strains showing greater adhesion during exponential phase and others during stationary phase (van Schie and Fletcher, 1999; Grasso et al., 1996; Stewart et al., 1997)
Cell structures and microbial attachment Cell structures that facilitate attachment include capsules, pili, and fimbriae (rigid, straight protein filaments, 0.2-20 nm in length and numbering 10-1000 per cell) (Jones and Isaacson, 1983; Michiels et al., 1991) Fimbriated bacteria tend to be more hydrophobic than nonfimbriated cells and are often gram negative Flagella and the microbial motility they impart may also facilitate attachment by increasing the likelihood and force of cell-surface contact (Fletcher, 1996; Morisaki et al., 1999), but motility is not a prerequisite (Dan, 2003)
Biochemical stimulation of attachment Certain bacteria are known to have the ability to sense surfaces and respond phenotypically or behaviorally For example, biofilms grown on submerged stones were found to produce the quorum-sensing signal molecules acylated homoserine lactones (acyl-HSLs or AHLs) which can stimulate attachment (McLean et al., 1997) Studies have also shown a tendency for antagonistic behavior by particle-associated bacteria, particularly at high cell densities (Long and Azam, 2001; Grossart et al., 2003) Interactions between species could be especially prevalent and important in controlling microbial community composition at surfaces.
Oligotrophy, copiotrophy and bacterial attachment
Microbial communities in soil evolve under a variety of environmental pressures, including fluctuations in nutrient and carbonaceous substrate availability Populations comprising soil communities have been classified into two main groups based upon their niche, or function, their general response to environmental stresses, and their kinetic behavior (MacArthur and Wilson, 1967; Panikov, 1995) Oligotrophic microorganisms are characterized by broad substrate specificity, high substrate affinity, low cell maintenance requirements, slow growth, and resistance to starvation conditions Hu et al (1999) demonstrated that soil oligotrophs were in fact inhibited by highly-available mineralizable carbon Copiotrophic microorganisms, on the other hand, display narrower substrate specificity, lower substrate affinity, fast or explosive growth, and less tolerance to starvation Given the low-energy maintenance needs and high-affinity substrate transport systems of oligotrophs, these organisms may be expected to thrive in mass transfer-limited, substrate-poor environments, achieving threshold concentrations, or residual levels, of contaminants that are orders of magnitude lower than those achieved by copiotrophic organisms (Bosma et al., 1997) In carbon-poor environments contaminated by weathered organic compounds that are released slowly over long periods of time, oligotrophs could outcompete copiotrophs (Button, 1991) and contribute more significantly to pollutant biodegradation It should be noted that oligotrophy and copiotrophy are not strictly defined but can be considered extremes on a wide spectrum of microbial carbon requirements (Semenov, 1991) Furthermore, oligotrophs and copiotrophs can coexist, for instance in the presence of multiple limiting substrates or nutrients, or under conditions of dormancy (Hu et al., 1999)
Oligotrophic or copiotrophic behavior is not necessarily an inherent trait of a microbial species Cells are known to adjust their metabolism during periods of stress in order to compete for nutrients, adopting a feast-or-famine type of strategy (Egli, 1995; Kovárová-Kovar and Egli, 1998) Nutrient limitations or starvation conditions can cause cells to change in size, rates of endogenous respiration, protein synthesis, or cell surface hydrophobicity, for instance The effect of starvation on cell attachment appears to be species- and environment-specific Starved marine bacteria were found to become more adhesive in oligotrophic habitats (Marshall, 1989), and certain microorganisms have been found to synthesize unique membrane and periplasmic proteins during starvation, which could contribute to observed increases in their attachment (Nystrửm et al., 1989) In other species, starvation results in decreased attachment (van Schie and Fletcher, 1999)
Other environmental stresses that are known to impact bacterial metabolism and attachment include fluctuations in pH (Nyvad and Kilian, 1990; ỉrstavik, 1977; Gordon et al., 1981; Harber et al., 1983; Scholl and Harvey, 1992; Kinoshita et al., 1993; Jewett et al., 1994); salinity (Zita and Hermansson, 1994; Gordon and Millero, 1984); ionic strength (Fontes et al., 1991; Gannon et al., 1991a; Gannon et al., 1991b; Jewett et al., 1994; van Loosdrecht et al., 1990; ỉrstavik, 1977; Abbot et al., 1983; Sampson and Blake, 1999); specific ions such as Fe 3+ (van Schie and Fletcher, 1999; O’Toole and Kolter, 1998a), nitrate, and sulfate (van Loosdrecht et al., 1990); fluid dynamics (van Loosdrecht et al., 1990; Gómez-Suarez et al., 2000; Gómez-Suarez al 1999a; Gómez- Suarez et al., 1999b, Gómez-Suarez et al., 2001; Harvey et al., 2002; McClaine and Ford, 2002; Sjollema et al., 1989; Boyd et al., 2002; Klausen, 2003); carbon dioxide concentration (Denyer, 1990); osmolarity (Fletcher, 1996); and organic pollutants and surfactants (Bekins et al., 1999; Harvey et al., 1984; Harvey and George, 1987; Hazen et al., 1991; Niels et al., 1995; van Schie and Fletcher, 1999)
The community structure, kinetic characteristics, and adaptability of microorganisms also appear to be related to their degree of attachment to soil Rihana-Abdallah (2000) found ecological variation (measured using phospholipid fatty acid profiles) between loosely-attached and tightly-attached microbial fractions eluted from soil Differences between fractions diminished with increasing soil organic carbon content Divergence in kinetic parameters was also observed between microbial fractions, most notably in low-carbon soils Tightly-attached microorganisms adapted more readily to changes in substrate type and concomitantly exhibited decreased membrane fluidity, a phenomenon that has been linked to resistance to environmental stresses, for example changes in temperature (Annous et al., 1999) This type of membrane response by tightly-attached, oligotroph-like populations could lead to increased metal resistance and enhanced organic pollutant biodegradation compared to the behavior of loosely-attached, copiotroph-like organisms Attached bacteria have also been found to show higher uptake rates for certain substrates (Palumbo et al., 1984; Unanue et al., 1992); greater biodegradative capability for organics (Jeffrey and Paul, 1986; van Loosdrecht et al., 1987a), and faster decomposition of labile matter associated with particles (Cho et al., 1988) Furthermore, bench-scale column studies revealed that attached bacteria had a preference for more complex organics than unattached bacteria (Lehman et al., 2001a).
Isolation and Physiological Characterization of Microbial
Communities and Isolates from Unsaturated Soil
This research study commenced with the selection of a field site from which soil samples and indigenous soil bacteria could be obtained An undisturbed pasture at a research farm managed by Case Western Reserve University was chosen because it was accessible and was not impacted by tilling, agriculture, pesticide application, or historic contamination Thus, it could reasonably represent conditions in a subsurface environment before the onset of a contamination event Soil cores were collected from the shallow vadose zone The vadose zone is defined as the unsaturated soil region above the groundwater table, extending from the bottom of the capillary fringe to the soil surface It includes surface soil, which is the uppermost soil layer normally disturbed during tilling Surface soil in noncultivated environments typically ranges in depth from
7 to 20 centimeters (Sylvia et al., 1999) The vadose zone was expected to be aerobic and to support microbial populations accustomed to living in close physical association with soil material and utilizing natural organic matter as a primary substrate Many of the functional groups present in natural organic compounds are analogous to chemical structures found in xenobiotic chemicals
Soil samples were used to test and refine a serial elution procedure that employed increasingly stringent physical and chemical methods to remove microorganisms Microbiology studies that seek to detach cells with improved efficiency can employ chemical procedures in which samples are suspended in buffer with sodium chloride, sodium pyrophosphate, or surfactants (Ogram and Feng, 1997; Rihana-Abdallah, 2000); mechanical processes that use shaking, vortexing, centrifugation and ultrasonication; or enzymatic treatments that use lipases, α-glucosidase, or β-galactosidase to degrade the extracellular polymeric substances binding cells to soil “Standard” microbiology practices such as shaking or vortexing are considered insufficient for removing tightly- attached bacteria (McLean et al., 2001)
In this study, cells that were collected during the first, gentlest elution step and during the final, harshest step comprised two consortia or “microbial fractions” that were the focus of much of the research These consortia were generically categorized as
“loosely-attached” and “tightly-attached,” respectively, although the specific chemistry of their interactions with the soil matrix was outside the scope of this work Individual bacterial isolates were also cultured from the two fractions with the goal of comparing their physiological traits to those of the parent consortia
Microbial consortia and isolates were characterized using Gram stains, catalase tests, and other common microbiology assays, along with partitioning tests to determine cell surface properties The microbial adhesion to hexadecane (MATH) test was used to measure cell surface hydrophobicity by comparing the partitioning of a cell suspension in organic solvent and water phases (Rosenberg et al., 1980) Hydrophobic interaction chromatography was used to differentiate the elution of cell suspensions through small columns packed with hydrophobic and control resins Electrostatic interaction chromatography was employed to compare the elution of cells through anion-exchange and control resins These tests were performed to establish the uniqueness of the cultures and to examine whether surface properties correlated to biotransformation behavior examined in subsequent chapters
Soil sampling and characterization Soil samples were collected in April, 2003 and January, 2004 at Case Western Reserve University’s Squire Valleevue Farm, a 400- acre research and teaching facility located in Hunting Valley, Ohio Soil cores were removed from the vadose zone of untilled grassland at depths of approximately 12 to 18 inches using a hand auger Cores were sealed in plastic and stored at 4°C prior to subdivision and use in laboratory studies Soil pH was measured in a suspension of 10 g air-dried soil and 25 mL distilled, deionized water after shaking at 175 rpm for 15 minutes at 19°C (Rowell, 1994) Soil moisture content was determined gravimetrically following sample drying for 24 hours at 105°C Total organic carbon content was measured as weight loss following combustion of oven-dried samples at 550°C for 30 minutes
Adhesion-based extraction of soil microorganisms Soil core material was processed by hand to remove plant roots, leaves, and other detritus before being subjected to a serial elution procedure based on that of Rihana-Abdallah (2000), which fractionates microbial communities according to the strength of their adhesion to soil particles During each extraction test a suspension of 100 g soil and 350 mL of autoclaved mineral medium was shaken for four hours at 225 rpm and room temperature Mineral medium consisted of nitrogen-augmented BOD dilution water (American Public Health Association et al., 1998) with a pH of 7.0 and the following composition: 8.5 mg/L
KH2PO4; 21.75 mg/L K2HPO4; 33.4 mg/L Na2HPO4•7H2O; 22.5 mg/L MgSO4•7H2O; 27.5 mg/L CaCl2; 0.25 mg/L FeCl3•6H2O; and 1.0 g/L NH4Cl
The soil suspension was centrifuged at 4,500×g for 10 minutes and the supernatant was subsequently filtered with Whatman 50 filter paper (>2.7 μm particle retention) to collect eluent containing relatively easily-detached microorganisms The filtrate from this stage was termed “loosely-attached fraction F1,” representative of easily-eluted microbial communities that would be obtained by traditional microbiological extraction methods A second elution was performed by resuspending the drained soil with 300 mL of fresh, sterile high-nitrogen BOD water containing 0.2% (v/v) Tween 80 surfactant, agitating the mixture for 2 hours at 100 rpm, and centrifuging as before The collected filtrate, termed the “F2” fraction, represented microbial communities of intermediate attachment strength It was not used in experiments A third elution of the same soil sample was performed to extract microbial communities of greater attachment strength This final step entailed resuspending the twice-eluted soil in
300 mL of fresh medium, agitating as before, and then mildly sonicating the slurry for 15 minutes prior to centrifugation The filtrate, termed fraction “F3,” contained cells that were not detached during the previous two elutions and was assumed to include the most strongly adherent cells recovered by the method
Isolation of dominant microbial populations “Whole” microbial communities were harvested from filtrate fractions F1 and F3 by centrifuging at 10,000×g for 15 minutes, washing cell pellets twice with sterile 10 mM phosphate buffer (pH 7.4; composed of 1.08 g/L Na2HPO4•7H2O; 0.135 g/L NaH2PO4•H2O), and resuspending pellets in fresh phosphate buffer Aliquots of the concentrated community cultures were used immediately or amended with 20% (v/v) glycerol and stored cryogenically at -80°C to provide an inoculum source of consistent composition for subsequent experiments
Predominant culturable microbial populations were isolated from fractions F1 and F3 using serial dilution and plating techniques First, concentrated aliquots of F1 and F3 were transferred to high-nitrogen BOD water containing 100 ppm yeast extract (Difco) and 200 ppm cycloheximide (Aldrich), a fungal inhibitor Cultures were shake-incubated at 100 rpm and room temperature for 24 hours Next, aliquots were removed from the shake flasks and spread onto agar plates prepared with granulated agar and one-tenth strength nutrient broth (Difco; final concentration 0.3 g/L beef extract and 0.5 g/L peptone) Colony counting was used to enumerate culturable aerobic, heterotrophic bacterial cells extracted from the field soil samples in elution fractions F1 and F3 In addition, colonies of distinct morphology and appearance were selected from the plates and further isolated by culturing in liquid medium and streak-plating three times in sequence The resulting five distinct isolates were used as representatives of predominant culturable populations present in fractions F1 and F3 The “loosely-attached” isolates from fraction F1 were designated F11, F12, and F13, while the “tightly-attached” isolates from fraction F3 were designated F31 and F32 These five isolates were cultured, harvested, and cryogenically stored for future use as described previously It should be noted that the isolation methods used to obtain microbial isolates may have introduced a culture bias Even at one-tenth strength, nutrient broth may be considered a rich substrate medium, and its use may have inhibited the growth of highly sensitive oligotrophic microorganisms
Characterization of variably-attached microbial communities and isolates
Assays were performed to characterize the structural and biochemical properties of variably-attached bacterial communities and populations isolated by the soil elution protocol Standard biochemical assays included Gram staining, the bicinchoninic acid (BCA) method of protein analysis (Sigma-Aldrich, Inc., 2004), and catalase tests Cell surface properties, including charge and hydrophobicity, were characterized using four techniques: the microbial adhesion to hydrocarbons (MATH) assay, hydrophobic interaction chromatography (HIC), electrostatic interaction chromatography (EIC), and colloid titrations
The Gram stain reaction forms an insoluble, intracellular complex of crystal violet and iodine inside cells This complex is alcohol-extractable in gram-negative bacteria but not in gram-positive organisms, which have thicker cell walls and peptidoglycan layers The BCA determination of protein is a colorimetric method that first complexes protein to Cu +2 under alkaline conditions, then uses bicinchoninic acid to reduce the copper to a purple-blue Cu + complex The absorbance is measured at 562 nm and is compared to that of known protein standards (Sigma-Aldrich, Inc., 2004) The catalase test determines the presence of catalase, an enzyme typically produced by aerobic and facultatively aerobic bacteria to destroy hydrogen peroxide, a toxic byproduct of respiration Catalase positive cells are indicated by the formation of bubbles within thirty seconds of placing a drop of hydrogen peroxide atop a bacterial colony (Colome et al., 1986)
MATH assays MATH assays are used to measure the relative surface hydrophobicity of cells Hexadecane was used as the hydrocarbon phase according to the method of Sanin et al., (2003) Cell cultures to be tested were grown in high-nitrogen