34 Enrichment and Isolation of Hydrocarbon Degraders

34 Enrichment and Isolation of Hydrocarbon Degraders Z. Shao Key Laboratory of Marine Biogenetic Resources, The Third Institute of Oceanography, State Oceanic Administration, Xiamen, Fujian, Republic of China [email protected] 1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3778

2 2.1 2.1.1 2.1.2 2.1.3 2.2 2.2.1 2.2.2 2.3 2.4

Experimental Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3779 Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3779 Enrichment of Marine HDB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3779 Isolation of HDB by Plate Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3781 Isolate Characterization and Preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3782 Solutions and Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3782 Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3782 Stock Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3783 Time Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3783 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3784

K. N. Timmis (ed.), Handbook of Hydrocarbon and Lipid Microbiology, DOI 10.1007/978-3-540-77587-4_297, # Springer-Verlag Berlin Heidelberg, 2010

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Enrichment and Isolation of Hydrocarbon Degraders

Introduction

Hydrocarbons are organic compounds that contain only carbon and hydrogen. They constitute the main component of crude oil, and can be divided into two classes, the saturates and the aromatics. Saturated hydrocarbons have two forms: the cyclic alkanes and straight chain n-alkanes. The aromatic hydrocarbons have two groups varying in benzene ring number: the single-ring and more than one ring. The first group is represented by benzene, toluene, ethylbenzene and xylene (BTEX); the latter is designated as polycyclic aromatic hydrocarbon (PAH), and is represented by, e.g., naphthalene, phenanthrene, pyrene and benzo[a]pyrene, with ring numbers increasing from 2 to 5. Hydrocarbons can serve as carbon and energy sources for bacterial growth (Head et al., 2006). Bacterial degradation of various hydrocarbons has been intensively studied, and previous reviews describe this in great detail (Atlas, 1981; Leahy and Colwell, 1990; Van Hamme et al., 2003). As hydrocarbons in the environment have a wide range of distributions both spatially and temporally, it is not surprising to find hydrocarbon-degrading bacteria (HDB) to be ubiquitous over the globe. Even in the deep sea or pelagic environments, various HDBs were found (Cui et al., 2008; Wang et al., 2008a, b; Yuan et al., 2009). Many different types of HDB have been isolated and many of those stem from marine environments (Head et al., 2006; Leahy and Colwell, 1990). In the past decade several obligate marine HDBs have been isolated [as reviewed by (Head et al., 2006)], including Alcanivorax spp. (Liu and Shao, 2005; Yakimov et al., 1998), Cycloclasticus spp. (Chung and King, 2001; Dyksterhouse et al., 1995), Oleiphilus spp. (Golyshin et al., 2002), Oleispira spp. (Yakimov et al., 2003), and Thalassolituus spp. (Yakimov et al., 2004). Cycloclasticus spp. have evolved to use a range of PAHs (Churchill et al., 1999; Dyksterhouse et al., 1995; Wang et al., 2008a), while others can use a variety of branched- and/ or straight-chain saturated hydrocarbons. To isolate a HDB, an enrichment in liquid medium prior to plate cultivation with hydrocarbons is usually necessary. The technique of coating plates with a PAH film was first used to screen phenanthrene-assimilating bacteria (Kiyohara et al., 1982), and later shown to be a rapid method to isolate PAH-degrading bacteria (Heitkamp et al., 1988). Although in most cases hydrocarbons can be used as the sole carbon and energy source, some bacteria need the presence of other organic compounds for efficient hydrocarbon metabolism. This is often the case for bacteria mineralizing high-molecular-mass (HMM) aromatics. For instance, Mycobacterium sp. required the presence of common organic carbon sources such as peptone or yeast extract to completely degrade pyrene (Heitkamp and Cerniglia, 1988; Heitkamp et al., 1988). Later, another isolate of Mycobacterium sp., strain CH1 was discovered that utilized phenanthrene or pyrene as sole carbon and energy source (Churchill et al., 1999). Alkane-assimilating bacteria are not easily isolated by plate cultivation with alkanes as a sole carbon source. This is due to the fact that in contrast to PAHs, alkanes can not be used to ‘coat’ agar surfaces, and, consequently, clear zones as an indication for metabolism can not be observed. Moreover, residual growth may occur on trace carbon sources in agar plates. Silica can be used as solidifying agent instead of agar, which reduces the amount of residual carbon in the medium other than alkanes (Atlas, 1981; Seki, 1973, 1976). In addition, Tween 80 has been used to emulsify the alkanes, although in this method it has to be excluded that Tween itself does not serve as a carbon source. In the case of short-chain alkanes and other volatile hydrocarbons like BEXT and naphthalene, they can be provided through the vapor phase by placing a droplet of pure substance on the lid of a Petri dish (Liu and Shao, 2005; Wang et al., 2008b). Any colonies growing from enrichment cultures on plates with hydrocarbons should be further confirmed to be true HDBs.


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Considering the variety of environmental parameters and ecosystems across the globe, it is likely that many more HDBs are to be discovered. Here I focus on describing a number of enrichment and isolation protocols for marine HDBs under aerobic conditions and atmospheric pressure. These protocols can certainly be modified and applied to HDB from other environments.

2

Experimental Approach

2.1

Procedures

Hydrocarbons can be used as carbon and energy sources for bacteria. Thus, a supplement of hydrocarbons to the medium as the main sources of carbon and energy can select for the growth of HDB. Samples used for enrichment are usually water, soil or sediment aseptically taken from marine and land environments. Selected growth of HDB occurs under the given conditions, including temperature, pH, salinity, and the presence of other nutrients besides hydrocarbons (Leahy and Colwell, 1990). Purified isolates are usually obtained by plate cultivation, and further subjected to degradation tests and taxonomic analyses.

2.1.1

Enrichment of Marine HDB

The density of HDBs in the environment is usually not high enough for direct isolation on selective plates. Therefore, an enrichment prior to plate isolation is necessary. To increase the population size of alkane-degrading bacteria under laboratory conditions, the enrichment can be conducted by supplying one kind or a mixture of hydrocarbons as the carbon sources in liquid medium, as described in the following. 2.1.1.1

Preparing Medium for HDB Enrichment

Artificial seawater media with hydrocarbons as the sole carbon sources can be used for HDB enrichment, like ONR7a medium (Dyksterhouse et al., 1995), MMC medium (Liu and Shao, 2005), or nutrient-amended natural seawater (Yakimov et al., 2003). For convenience and to avoid precipitation, MMC medium is prepared from different components, which are sterilized separately. MMC can be separated in a basal part, a solution of MgSO4, a solution of a phosphorus source, a trace element solution, and carbon sources. It is important not to sterilize MgSO4 and phosphate together. Here, I will describe preparation of MMC with pyrene as an example: Add 60 ml of pyrene stock solution (see section medium and solutions) to an empty flask (250 ml), and blow with nitrogen gas in a fume hood to form a thin film of pyrene on the bottom of the flask. Add 100 ml of basal part of MMC medium and sterilize at 121 C for 20 minutes. Let the medium cool down to room temperature and add one by one the other stock solutions (sterilized MgSO4 and trace elements). Note: 1. Volatile hydrocarbons such as BTEX and naphthalenes should be added to the medium separately after autoclaving the basal part and cooling down; they can be sterilized by filtration through a 0.22 mm membrane, if necessary. Naphthalene can be predissolved in an organic solvent. 2. Alkanes longer than hexadecane can be added to medium directly and sterilized together.

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3. Solvents to dissolve PAHs must be allowed to evaporate after addition to the medium, especially those that might be used as carbon sources, like acetone, or methanol. Potentially harmful solvents, such as chloroform or dichloromethane should also be evaporated and removed from the medium flask. 4. BTEX and short-chain alkanes are often toxic in direct contact to the cells. Toxicity can be diminished by lowering the medium concentration to below aqueous phase solubility. More substrate can be delivered by sequential amendment to the culture. Alternative is to provide those substances via the vapor phase, by using a separate central reservoir in the flask. 5. Iron is often crucial to hydrocarbon degradation; the concentration in MMC medium is ten times that in ONR7a. 6. Aged seawater can be used to prepare the enrichment medium. In this case it is necessary to add sources of N, P, and Fe, in addition to hydrocarbons, but trace elements are provided naturally by the seawater. 7. A PAH mixture is sometime necessary for enrichment of HMM-PAH degraders. For example, phenanthrene was necessary for Cycloclasticus sp. N3 to degrade pyrene (Dyksterhouse et al., 1995). Thus, co-metabolism or co-induction of different substrates should be taken into consideration in medium design. 8. Phosphate tends to precipitate magnesium ions during autoclaving. Both compounds are preferably sterilized separately. 2.1.1.2 Inoculation with Environment Samples Sampling and Subsampling In order to avoid contamination during sampling, samplers

and containers are sterilized prior to use if possible. Sometimes sterility is difficult to control, especially during deep sea sampling or deep sea sediment sampling with single or multi-core devices. In this case, subsampling from the inner part of the core should be done aseptically in the laboratory afterwards. A similar situation occurs with seawater sampling at varying depths. This is typically carried out by using Niskin bottles that are attached to a CTD (conductivity, temperature and depth) circular rosette platform, which, obviously, is very hard to sterilize. Enrichment Initiation Add 2.0 g sediment or 2 ml seawater to 100 ml of MMC medium in a 250 ml flask and set up a control without sample addition for each hydrocarbon treatment, meanwhile parallel another control without carbon source addition for each sample treatment. Endogenous organic compounds contained within the samples might bring about a weak growth of oligotrophic bacteria. Moreover, undefined micronutrients in the sample may be beneficial and even essential for growth of PAH-degrading bacteria. Thus, the added sample might influence the final enrichment result. The amount of inoculated sample used for enrichment depends on your experimental design, such as the size of the enrichment system. On board of a research vessel, HDB enrichment can be rapidly initiated by adding N, P, and Fe to the seawater or sediment samples. For sediment samples, a slurry with sterilized seawater can be prepared on board. 2.1.1.3

Cultivation and Subculturing

1. Cultivation: Agitate the inoculated flasks on an orbital shaker at a speed of 280 rpm or as convenient. Temperature is usually set to suit the sampling environment.


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2. At a certain interval transfer a sample from the enrichment to fresh medium. Inoculum volume is usually taken as 1%. Two or more successive enrichments should be carried out. In the case of PAH enrichment, the PAH dosage can be increased step by step for each subsequent enrichment. 3. Bacterial growth in the enrichment should be monitored and is typically accompanied by changes in culture turbidity and/or color. Note: to avoid the growth of photoautotrophic bacteria, it is better to keep the enrichments in the dark during incubation.

2.1.2

Isolation of HDB by Plate Purification

Agar or agarose plates are usually used to isolate HDB from the enriched cultures. PAH film coatings on plates are frequently used to visualize growth of potential PAH degraders via formation of a clear zone in which the PAH is solubilized and removed. Also, one could chose to make plates using simple carbon sources other than hydrocarbons. In that case it is necessary to confirm the hydrocarbon degradation ability on the purified isolate. 2.1.2.1

Culture Dilution

Homogenize the culture by vigorous shaking, then remove 1 ml of culture to make tenfold serial dilutions down to 10–6 in MMC medium (basal part) or sterilized seawater. 2.1.2.2 Plate Spreading Protocol 1: Screen HDBs on PAH Coated Plates

1. Pipette 100 ml of diluted enrichment culture, spread on the surface of an ONR7a or MMC medium agar plate without any carbon source. 2. Wait for the liquid on the surface to be absorbed, this takes about 15 min. 3. Then spray the PAH solution on the plate surface with the help of a parfum dispenser or similar. PAHs can be prepared in ethereal solution at concentrations between 50 and 100 mg per ml. 4. Wait for the ether to vaporize from the surface at ambient temperature, leaving a thin layer of PAH on the entire surface. 5. Then incubate the plate until colonies are appearing. Look for colonies around which a clear zone has produced. 6. Pick those colonies showing a cleared zone with sterile toothpicks and streak them for purification on marine agar 2216 (Difco™). Notes: 1. PAH stock solutions can be prepared in other solvents than petroleum ether, but be careful for solvent toxicity to the cells. 2. Similar procedures to culture bacteria on plates can be used for all solid hydrocarbons other than naphthalene, which easily disappears due to sublimation. 3. The PAH-ether solution can be sterilized by passing through a 0.22 mm membrane filter. 4. Control for hydrocarbon growth should consist of an inoculated plate but without hydrocarbon addition.

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Protocol 2 Screen HDBs on Plates by Supplying Hydrocarbon Vapor

1. Plate the enriched and diluted culture as in Protocol 1 above. 2. Supply short-chain alkanes or BTEX in a 0.2 ml slow-release tube. The hydrocarbon can be drained out from the capped tube with a slip of Whatman 3MM filter paper. Alkanes from n-decane (C10) to n-tetradecane (C14) can be supplied by placing a Whatman paper disk with 1 ml of n-alkane in the lid of the petri dishes. Naphthalene can be supplied as fine crystals or sprayed on the inverted upper lid. 3. Seal bottom and lid of the Petri dish with Parafilm. 4. Incubate and purify any grown colonies as described above. Protocol 3 Isolation on Solid Medium with Other Carbon Sources

1. Dilute and spread the enriched culture as above, but on a plate containing a nonhydrocarbon C-source such as citrate or acetate, or even use a complex medium such as marine agar 2216 (Difco™), HLB medium or M2 medium (Cui et al., 2008; Wang et al., 2008a). 2. Incubate as above. 3. Pick all colonies of unique appearance and purify further on the same medium. Proceed with extensive testing for hydrocarbon degradation on purified cultures.

2.1.3

Isolate Characterization and Preservation

The capability of purified isolates to degrade hydrocarbons needs further confirmation in strict mineral medium by measuring growth at the expense of hydrocarbon disappearance, or by using 14C-labeled PAHs or alkanes (Churchill et al., 1999; Hilyard et al., 2008). 1. Inoculate the purified isolate into liquid medium with hydrocarbon(s) as the sole carbon and energy source(s), in a flask containing 100 ml of ONR7a medium or similar. 2. Incubate under the same conditions as during the enrichment procedure. 3. Examine occurrence of degradation by monitoring bacterial growth, the decrease of hydrocarbon concentration, or the production of an intermediate. In case of using a 14C labeled substrate, measure [14C]-CO2 evolution and prepare a complete mass balance for 14 C in the system at the end of the experiment. 4. Identify the taxonomy of the bacterial isolate by e.g., 16S rRNA sequence analysis (See > Chapter 58, Vol. 5, Part 3). 5. Preserve purified strains at 80 C or 196 C in 15% glycerol, 5% dimethylsulfoxide or similar.

2.2

Solutions and Materials

2.2.1

Media

ONR7a medium, an artificial seawater mineral salts medium used for enrichment cultures and isolation of HDB (Dyksterhouse et al., 1995). It was used successfully to isolate the key marine PAH-degrading bacterium Cycloclasticus pugetii. ONR7a contains (per liter of distilled or


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deionized water) 22.79 g of NaCl, 11.18 g of MgCl26H2O, 3.98 g of Na2SO4, 1.46 g of CaC122H2O, 1.3 g of TAPSO {3-[N-tris(hydroxymethyl) methylamino]-2-hydroxypropanesulfonic acid}, 0.72 g of KCl, 0.27 g of NH4Cl, 89 mg of Na2HPO47H2O, 83 mg of NaBr, 31 mg of NaHCO3, 27 mg of H3BO3, 24 mg of SrCl26H2O, 2.6 mg of NaF, and 2.0 mg of FeCl24H2O. The detailed preparation of this medium was described elsewhere (Dyksterhouse et al., 1995). MMC medium, an artificial seawater mineral salts medium, also used for enrichment cultures and isolation. MMC contains (per liter distilled water): 24 g NaCl, 9.0 g MgSO47H2O, 1 g NH4NO3, 0.7 g KCl, 0.2 g K2HPO4, 10 ml trace element stock solution (100), and 10 ml Ferrous stock (100). MgSO4 is added separately to avoid phosphate precipitation. When agar plates are prepared, 1.5% (w/v) Bacto agar (Difco) is melted in MMC by autoclaving and solidified. M2 medium, a complex medium prepared with seawater, containing (per liter) 5.0 g CH3COONa, 0.5 g tryptone, 0.5 g yeast extract, 0.5 g glucose, 0.5 g sucrose, 0.05 g sodium citrate, 0.05 g malic acid, 1.0 g NH4NO3, 0.2 g NH4Cl, 0.5 g KH2PO4, (pH 7.6). 1.5% (w/v) agar is added to make plates. Phosphate is dissolved and autoclaved separately. HLB medium: modified from Luria-Bertani (LB) medium (Sambrook et al., 1989). The concentration of NaCl is increased to 30 g per L.

2.2.2

Stock Solutions

PAH stock solutions: PAHs such as phenanthrene, fluoranthene, pyrene, chrysene, or benzo [a]pyrene are dissolved to 50 mg per ml in an organic solvent (acetone, ether or chloroform). Naphthalene can be dissolved in methanol at a final concentration of 100 mg per ml. The solutions can be sterilized by filtration through a 0.22 mm pore solvent-resistant membrane. Solutions should be stored in a tight-capped glass bottles kept away from light and heat. Trace element stock solution (100): used for MMC medium, containing (per liter distilled water) 15 g of CaCl22H2O, 8 g of KBr, 2.7 g of H3BO3, 2.4 g of SrCl26H2O, 10 mg of CuSO45H2O, 50 mg of MnCl24H2O, 10 mg of ZnSO47H2O, autoclaved for 30 min at 121 C. FeCl2 stock (100): 0.20 g FeCl24H2O, dissolved in 100 ml distilled water, and filtered through a 0.22 mm pore size filter, is used for MMC medium. N/P stock solution (100): 10 g NH4NO3 and 2 g K2HPO4, dissolved in 100 ml distilled water, natural pH (7.6), autoclaved for 30 min at 121 C. Used in marine medium prepared with natural seawater.

2.3

Time Considerations

1. Initiate the enrichment as soon as possible after sampling. 2. For an enrichment with PAH-degrading bacteria: The first incubation time with environment samples to detect growth usually takes 2–4 weeks; Every further subculture usually takes between 1 and 2 weeks. An enrichment for alkane-degrading bacteria usually takes half the time of that for PAH-degrading bacteria at each step. 3. Bacterial isolation: marine obligate HDBs usually appear as oligotrophic bacteria, taking longer time to form visible colonies on plates. This may take at least 2 weeks.

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Troubleshooting

No bacterial growth during enrichment. Check the toxicity of substrates caused by high concentration, especially in the case of BTEX or short chain alkanes; inoculate a higher quantity or volume of the original sample to the enrichment; match the medium and cultivation conditions as closely as possible to in situ conditions. Bacteria in the enrichment culture cannot be isolated on plate. Examine the community structure by 16S rDNA analysis based on denaturing gradient gel electrophoresis (DGGE) or library construction to observe their relative abundance (See > Chapter 58, Vol. 5, Part 3). Use a dilution-to-extinction method to obtain pure HDB isolates or bacteria associated closely. Investigate any possible micronutrients that may be required for pure culture growth; start by amending tiny amounts of yeast extract or vitamines.

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