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Interaction between cyanobacteria and aerobic heterotrophic bacteria in the degradation of hydrocarbons
International Biodeterioration & Biodegradation 64 (2010) 58e64
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Interaction between cyanobacteria and aerobic heterotrophic bacteria in the degradation of hydrocarbons Raeid M.M. Abed* Sultan Qaboos University, College of Science, Biology Department, P.O. Box: 36, Al Khoud, Postal Code 123, Muscat, Sultanate of Oman
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 August 2009 Received in revised form 14 October 2009 Accepted 14 October 2009 Available online 10 November 2009
Four strains of aerobic heterotrophic bacteria were isolated on crude oil with the aim to test whether their presence and activity might support the growth of cyanobacteria in oil-polluted microbial mats and whether the cyanobacterial exudates might play a role in stimulating their degradative activities. The strains were phylogenetically related to known oil-degrading species from the genera Marinobacter, Pseudomonas and Sphingomonas. Three strains (GM41, GM61 and GM63) grew well on C5eC18 alkanes but not on 7 tested aromatics, whereas one strain (i.e. GM42) grew best on phenanthrene and pentane. All strains showed ability to metabolize a range of cyanobacterial photosynthetic and fermentative exudates. In coculture experiments, the addition of the Pseudomonas-related GM41 strain to the cyanobacterium Synechocystis PCC6803, found in the same mat, resulted in 8-fold increase in the cyanobacterial biomass. This growth was more pronounced when hexadecane was added to the culture medium. The addition of representative substrates of cyanobacterial exudates to the phenanthrene-degrading strain GM42 resulted in variable effects. While acetate, pyruvate and glucose enhanced phenanthrene degradation, alanine and butanol showed no effect. We conclude that aerobic heterotrophic bacteriaecyanobacteria consortia can be very useful for bioremediating oil-polluted sites, circumventing the costly use of organic and inorganic fertilizers. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Cyanobacteria Aerobic heterotrophic bacteria Cyanobacterial mats Cyanobacterial exudates Hydrocarbons
1. Introduction The uppermost layer of cyanobacterial mats, dominated by cyanobacteria and aerobic heterotrophic bacteria, was shown to degrade € tzschel et al., 2002; Benhydrocarbons (Abed et al., 2002, 2006; Gro nchez et al., 2006). Heterotrophic bacteria but thien et al., 2004; Sa not the cyanobacteria, were chiefly responsible for the observed €ster, 2005). This raised the questions why degradation (Abed and Ko cyanobacteria dominate polluted sites and whether their presence may contribute to the degradation of petroleum compounds. It has been postulated that cyanobacteria require aerobic heterotrophic bacteria for their growth, which is evident from the difficulty to obtain them in axenic cultures (Rippka, 1988). Cyanobacteria play an indirect role in oil biodegradation by providing the associated aerobic heterotrophs with: oxygen (by-product of photosynthesis) needed for the breakdown of aliphatic and aromatic compounds (Abed et al., € ster, 2005); fixed nitrogen (through nitrogen 2002; Abed and Ko fixing strains) that is often limited in marine environments (Musat et al., 2006); and simple organics produced by photosynthesis and
* Tel.: þ968 24142406. E-mail address: [email protected] 0964-8305/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2009.10.008
fermentation. So far, the effect of cyanobacterial photosynthetic and fermentative organic exudates on the activity of oil-degrading microorganisms has not been investigated. The organics produced by cyanobacteria in microbial mats nourish other functional bacterial groups including aerobic heterotrophic bacteria (Bateson and Ward, 1988). These bacteria oxidize organic compounds directly to CO2. Cyanobacterial exudates include low molecular weight compounds and extrapolymeric substances (EPS) composed primarily of proteins, lipids and nucleic acids (Decho, 1990; Flemming and Wingender, 2001). Mannitol and arabinose were identified as excretion products (Hellebust,1965). Glycolate was found to be the main compound among dissolved photosynthates under hyperoxic and alkaline conditions (Bateson and Ward,1988). In the dark and under anoxic conditions, cyanobacteria ferment and produce organics different from those produced during the day (Nold and Ward, 1996; Jonkers and Abed, 2003). Fermentation products like acetate, propionate, lactate and ethanol were identified (Anderson et al., 1987; Jrgensen et al., 1992; Stal, 1995; Stal and Moezelaar, 1997). Studies on the diversity and degradative capabilities of aerobic heterotrophic bacteria in microbial mats demonstrated the presence of different populations that are specialized in the consumption of specific cyanobacterial exudates (Jonkers and Abed, 2003; Abed et al., 2007).
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In this study, a culture-based approach was used to study the interaction between cyanobacteria and associated aerobic heterotrophic bacteria in the degradation of hydrocarbons. Enrichment cultivation, although known to underestimate bacterial diversity (Amann et al., 1995), is still an important technique to study the physiological characteristics of individual strains and to test and expand hypotheses in microbial ecology. Four aerobic heterotrophic bacteria were isolated on crude oil as the sole source of carbon and were phylogenetically and physiologically characterized. The effect of the presence of an aerobic heterotroph (i.e. GM41 strain) on the growth of Synechocystis PCC6803 axenic culture was tested. Several cyanobacterial exudates were checked for their effects on the degradation rate of phenanthrene by the strain GM42. 2. Materials and methods 2.1. Isolation of aerobic heterotrophic bacteria Aerobic heterotrophic bacteria were isolated from a marine polluted cyanobacterial mat (Abed et al., 2002) on a defined artificial seawater medium. The medium contained MgCl2$6H2O (5.6 g l1), MgSO4$7H2O (6.8 g l1), CaCl2$2H2O (1.47 g l1), KCl (0.66 g l1) and KBr (0.09 g l1). After autoclaving, 0.15 and 0.2 g l1, respectively, of KH2PO4 and NH4Cl solutions were added. Autoclaved solutions of trace elements (Widdel and Bak, 1992), selenite and tungstate, and vitamins (Heijthuijsen and Hansen, 1986) were then added (1 ml l1). Solid media were prepared using 1% (w/v) Bactoagar. The salinity of the medium was adjusted to 2%, similar to the salinity of the field site where the strains originated from. Cultivation enrichments were done in 20 ml screw-cap culture tubes containing 10 ml culture medium under aerobic conditions. Serial dilution was used as an isolation technique. The enrichment was performed on Maya crude oil (de Oteyza and Grimalt, 2004) at a concentration of 1% (w/v) and all enrichments were incubated at 28 C. Axenic strains of aerobic heterotrophic bacteria were obtained from high dilutions (104 and 106) by plating on agar acetatecontaining medium. The obtained strains were then tested for growth on crude oil. 2.2. Characterization of aerobic heterotrophic bacterial isolates Polymerase chain reaction (PCR) was performed on the DNA extracts of the isolates using GM3 and GM4 primers (Muyzer et al., 1995). The PCR products were purified using the QIAquick PCR € sseldorf, Germany) and then sequenced. purification kit (Diagen, Du The 16S rRNA sequences of the strains (ca. 1400 bp) were analyzed using the ARB software (Pruesse et al., 2007). Phylogenetic trees were constructed based on almost complete 16S rRNA gene sequences (>1300 bp) by applying different methods integrated in the ARB software such as maximum likelihood, maximum parsimony and neighbor joining. Partial sequences were not included in the calculation of the trees. The final maximum likelihood tree was minimized for simplicity in presentation. The sequences of the four strains were submitted to the GenBank (Accession numbers GQ273962eGQ273965). The growth of the obtained strains on 26 different chemical compounds known to be cyanobacterial exudates as sole carbon source was tested. The tested substrates, listed in Table 1, included fatty acids, alcohols, amino acids and sugars. Each individual substrate was added to 10 ml of the medium to give the final concentration of 10 mM. Each tube was inoculated with 100 ml of the treated strain pre-grown on acetate. Growth on individual substrate was measured by following the changes of the optical densities of the cultures at 660 nm against biotic (without a substrate) and sterile (without bacteria) controls.
59
Table 1 Growth of the four aerobic heterotrophic bacterial strains on various aliphatic and aromatic compounds. (þþ): maximum growth reached in <3 days; (þ): maximum growth reached in 3e10 days; (): no growth. Growth was monitored by following daily changes in optical density at 660 nm against biotic (without a substrate) and sterile (without bacteria) controls. Hydrocarbons
Strains GM41
GM42
GM61
GM63
Alkanes n-Pentane n-Hexane n-Octane n-Decane n-Dodecane n-Tetradecane n-Hexadecane n-Octadecane
þ þþ þþ þþ þþ þþ þþ þþ
þþ þ þ þ
þ þ þþ þ þþ þþ þþ
þþ þþ þþ þ þ þþ þþ
Mono-aromatics Benzene Toulene
Di-aromatics Naphthalene
Tri-aromatics Phenanthrene Anthracene
þþ
Cycloalkanes Cyclohexane
Sulfur aromatics Dibenzothiophene
Table 2 Substrate spectra of the four isolates on previously identified cyanobacterial exudates including compatible solutes, photosynthetic and fermentative products (see text for references). The substrates were added at a final concentration of 10 mM. (þþ): maximum growth reached in <3 days; (þ): maximum growth reached in 3e10 days; (): no growth. Growth was monitored by following daily changes in optical density at 660 nm against biotic (without a substrate) and sterile (without bacteria) controls. Substrate
Strains GM41
GM42
GM61
GM63
Fatty acids Acetic acid Glycolic acid Citric acid Malic acid Succinic acid Butyric acid Methyl succinic acid Fumaric acid Pyruvic acid Hydroxybutyric acid Iso-valeric acid Propionic acid Formic acid
þþ þ þþ þþ þþ þþ þþ þþ
þþ þ þ þþ þ þþ
þþ þ þþ þþ þþ þþ þþ þþ
þþ þ þþ þþ þþ þþ þþ þþ
Alcohols Methanol Ethanol Propanol Butanol Glycerol
þþ þþ þþ þþ
þþ þ
þ þ þþ þþ
þþ þþ þþ þþ
Amino acids Glycine Betain Alanine
þþ
þþ
þþ
þþ
Sugars Glucose Fructose Galactose Sucrose Ribose
þþ þþ þþ þþ þþ
þ þ þ þ
þþ þþ þ þ
þþ þþ þþ þþ þþ
Pseudomonas denitrificans (AB021419) Pseudomonas pertucinogena (AB021380) Strain GM41 (GQ273962) Strain GM61 (GQ273964) Pseudomonas sp. (AB030085) Pseudomonas oleovorans (D84018) Pseudomonas alcaligenes (Z76653) Pseudomonas mendocina (AF232713) Pseudomonas alcalophila (AB030583) Marinobacter sp. (U61848) Marinobacter hydrocarbonoclasticus (AB019148) Marinobacter aquaeolei (AJ000726) Strain GM63 (GQ273965) Pseudomonas nautica (D11189) Sphingomonas adhaesiva (D13722) Sphingomonas macrogoltabidus (D84530) Strain GM42 (GQ273963) Sphingomonas sp.(AB025720) Sphingomonas stygia (AB025013) Sphingomonas aromaticivorans (AB025012) "Microcystis aeruginosa“ (U53589) Cyanothece PCC7424 (AJ000715) "Gloeothece membranacea" (X78680) Synechocystis PCC6803 (D90916) Synechocystis UNIWG Synechococcus PCC7002 (AJ000716) Oscillatoria rosea (AB003164) Cyanothece sp. (AF296873)
Alpha-
10
Gamma-proteobacteria
R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e64
Cyanobacteria
60
Fig. 1. 16S rRNA-based phylogenetic reconstruction of the four aerobic heterotrophic bacterial strains and the cyanobacterium Synechocystis PCC6803 used in this study. The strains were isolated from the uppermost layer of a heavily polluted mat at high dilution levels (104 and 106) after enrichments on Maya crude oil. Synechocystis UNIWG denotes the sequence obtained directly from the original mat.
The growth of the obtained strains in the presence of 16 different hydrocarbons, listed in Table 2, as sole carbon source was measured. Individual hydrocarbon was added to 10 ml of the medium in a final concentration of 0.5e2% depending on the toxicity and the solubility of the compound. Each tube was inoculated with 100 ml of bacterial cultures pre-grown on acetate. Growth was monitored by following the changes of the optical densities of the cultures at 660 nm over a period of 2 weeks. 2.3. Coculture of Synechocystis PCC6803 and the Pseudomonas-related GM41 strain Each 25 ml sterile test tube received 10 ml of the autoclaved medium described above. The selected strains for this experiment were the axenic Synechocystis strain PCC6803 (obtained from the PCC Pasteur collection, Paris, France) and the heterotrophic bacterial strain GM41. This cyanobacterial strain was selected because of its dominance in the original field mat (Abed et al., 2002, Fig. 1). Four experiments were prepared each in replicates: 1) Synechocystis PCC6803 alone; 2) Synechocystis PCC6803 þ the GM41 strain þ hexadecane (1% v/v); 3) Synechocystis PCC6803 þ hexadecane (1% v/v) and 4) Synechocystis PCC6803 þ the GM41 strain without hexadecane. This experiment was designed in order to test 1) whether Synechocystis PCC6803 could grow on hexadecane, 2) whether the presence of associated aerobic heterotrophs could stimulate/inhibit the growth of the cyanobacterium and 3) whether the growth of the aerobic heterotrophs on hydrocarbons might stimulate/inhibit the growth of the cyanobacterium. The tubes were inoculated with 5% (v/v) pre-grown cultures of Synechocystis PCC6803 and the GM41 strain. Incubation was at 28 C and under 12 light/12 darkness for 42 days. Samples were collected, centrifuged and the pellet was stored at 20 C for chlorophyll a (Chl a) determination. Chl a was extracted by addition of 3 ml methanol to the microbial pellets after centrifugation (Stal et al., 1984). The tubes were shaken for 3 h at 100 rpm/min, and then centrifuged at 4500 rpm/m for 10 min. The supernatant was transferred to a fresh tube and the pellet was re-extracted with methanol. The supernatants from both steps were pooled together. The absorbance of the supernatant
was measured at 662 nm using a spectrophotometer (Shimadzu, Duisburg, Germany). All steps were performed in a dim light 2.4. Effects of organics known to be cyanobacterial exudates on degradation rates of phenanthrene by the aerobic heterotrophic strain GM42 The used medium was prepared by mixing equal volumes of seawater with distilled water to obtain a salinity of about 2%. Nitrogen and phosphate sources were added to the medium as 1 mM ammonium chloride and 8 mM sodium dihydrophosphate, respectively. The inoculum was 100 ml of the GM42 strain, after 24 h pre-growth on acetate, per 100 ml medium. The GM42 strain was incubated in the presence of 0.15 mM phenanthrene with and without 0.15 mM of an individual cyanobacterial exudate. The tested exudate substrates were acetate, pyruvate, glucose, alanine and butanol. Phenanthrene was added after adsorbing it on hydrophobic clay (montmorillonite KSF, Aldrich) as a carrier to ensure its accessibility. The phenanthreneeclay complex was prepared as described earlier (Abed et al., 2002). Two control flasks were prepared in which phenanthrene was incubated with and without the added cyanobacterial substrate but in the absence of the GM42 strain. All treatments were run in triplicates. All flasks were incubated at 28 C with constant shaking at 100 rpm for 12 days. Samples for chemical analysis (2 ml each) were taken every 2 days. The residual phenanthrene was extracted with dichloromethane (DCM) and analyzed by gas chromatography. The growth of the GM42 strain on phenanthrene with and without the cyanobacterial exudate was determined in terms of protein concentrations according to Lowry et al. (1951). The bacterial cells were collected by centrifugation and 1 ml of 1 M sodium hydroxide was added to the pellet. The suspension was heated for 10 min at 100 C and after cooling to room temperature, 5 ml of a mixture of 2% Na2CO3, 1% CuSO4$5H2O and 2% sodium potassium tartarate (100:1:1 vol/vol) was added. The suspension was vortexed and incubated for 10 min. At the end of incubation, 0.5 ml of Folin reagent was added and the tubes were left for 30 min. Absorption was measured at 663 nm using a double beam spectrophotometer. 2.5. Statistical analysis Chl a (Section 2.3), biodegration of phenanthrene and protein (Section 2.4) data were analyzed by one-way ANOVA using the SPSS software (10th edition, Chicago, USA). P-values were adjusted using the sequential Bonferroni (Quinn and Keough, 2002) and Tukey's test was used to determine differences between individual means. P-values were used to directly compare data among different treatments at each time point. 3. Results 3.1. Characterization of aerobic heterotrophic bacterial strains Four pure bacterial strains were isolated at the 104 (GM41 and GM42) and 106 (GM61 and GM63) dilution levels on crude oil. The strains belonged to the Alphaproteobacteria and Gammaproteobacteria based on the phylogenetic reconstruction of their 16S rRNA gene sequences (Fig. 1). The GM63 strain was closely affiliated to the known oil-degrading bacteria Marinobacter aquaeolei and Marinobacter hydrocarbonclasticus (Gauthier et al., 1992, Huu et al., 1999) with 98.2 and 98.0% sequence similarity, respectively. The strains GM41 and GM61 shared 97.0% 16S rRNA gene sequence similarity and fell phylogenetically within the Pseudomonas group, which contains strains with the ability to grow on both alkanes and aromatic compounds (Eggink et al., 1988; Yen et al., 1991; Tian et al.,
R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e64
2002). The GM42 strain belonged to the Alphaproteobacteria within the Sphingomonas group. Growth spectra on hydrocarbons showed that the strains GM41, GM61 and GM63 grew on alkanes but not aromatics (Table 1). Growth was observed in the presence of all tested alkanes except in the case of the GM61 strain on n-octadecane and GM63 on n-pentane. The strain GM42 grew poorly on alkanes compared to other strains. The most pronounced growth of this strain was detected in the presence of n-pentane and phenanthrene. The tested strains showed growth on most cyanobacterial exudates with slight differences (Table 2). All strains grew on most tested carbohydrates. While all tested strains readily grew on acetate, succinate, butyrate, propionate and pyruvate, none grew on citrate, malate and formate. Other fatty acids like glycolate and fumarate supported the growth of the strains GM41, GM61 and GM63 but not GM42. All strains grew on glycerol and butanol but not on methanol. The four strains grew on alanine but not glycine and the compatible solute betain.
3.2. Growth of Synechocystis PCC6803 in the presence of hexadecane and/or the GM41 strain The effect of the presence of hexadecane and/or the strain GM41, which is capable of growing on hexadecane, on the growth of the axenic Synechocystis PCC6803 culture was monitored over a period of 42 days (Fig. 2). Growth of Synechocystis PCC6803 in the presence and absence of hexadecane, but without the GM41 strain, was comparable, with a typical bacterial growth curve. There was no statistical difference between these two treatments at all time points (Fig. 2). Addition of cells of the GM41 strain apparently stimulated the growth of the cyanobacterium and maximum growth was recorded after 20 days. The growth after 20 days was 8 times higher, based on the optical density values of extracted Chl a, with the GM41 strain than without it. This difference was statistically significant (P ¼ 0.013). The best growth of Synechocystis PCC6803 strain was observed in the presence of hexadecane and the GM41 strain. The cyanobacterium exhibited a lag phase of about 10 days, and growth reached the maximum after 35 days, after which it was significantly higher than other treatments (P < 0.05).
Chlorophyll a (Chl a) at 662 nm
3.5 PCC6803 PCC6803+hexadecane PCC6803+GM41 PCC6803+GM41+hexadecane
3.0
*a a* a*
2.5
a*
2.0 1.5 1.0
*a
0.5
0
b c
b 10
b
c
b
* *
0.0
b
20
30
b 40
50
Days Fig. 2. Growth of Synechocystis PCC6803 with and without hexadecande and in the presence and absence of the hexadecane-degrading strain GM41 (n ¼ 3). The growth of the cyanobacterium was monitored by measuring Chlorophyll a (Chl a) at 662 nm. Stars indicate the time points at which different incubations exhibit statistically significant difference (P < 0.05). Common alphabetic superscripts indicate no significant difference using Tukey's test within each time point.
61
The growth of the cyanobacterium in the presence of both hexadecane and the GM41 strain was 2 times higher than with only the GM41 strain and 12 times higher than without both. 3.3. Effect of organics known to be cyanobacterial exudates on phenanthrene consumption by the GM42 strain The effects of acetate, pyruvate, glucose, alanine and butanol addition on the phenanthrene consumption by the GM42 strain was tested (Fig. 3). Phenanthrene completely disappeared after 7 days at a rate of 0.34 mg d1 in the absence of any added organic substrate. The addition of alanine and butanol did not show any significant effect on phenanthrene degradation and the consumption rate was comparable to the control (p > 0.05). In contrast, glucose, pyruvate and acetate significantly stimulated the degradation process (p < 0.02). Phenanthrene disappeared after 6 days at a rate of 0.48 and 0.54 mg d1 in the presence of glucose and pyruvic acid, respectively. The highest degradation rate was recorded in the presence of acetate at a rate of 0.8 mg d1 within about 5 days. The growth of the GM42 strain in the presence of phenanthrene with and without the tested cyanobacterial exudates (acetate, pyruvate and glucose) was followed using protein estimation in order to check whether the observed increase in phenanthrene degradation rate was due to increased biomass or increased activity of the GM42 strain (Fig. 4). The GM42 strain accumulated more biomass in the presence of phenanthrene and one cyanobacterial exudate than in the presence of phenanthrene alone. This difference in growth was statistically significant with P-values < 0.05. Maximum growth was reached already after 4 days in the presence of acetate but took 6e7 days in the presence of glucose and pyruvate, after which it remained more or less constant. In spite of that, more bacterial biomass was detected in the presence of glucose and pyruvate than acetate. 4. Discussion This study supports earlier reports that cyanobacteria and associated aerobic heterotrophs constitute an efficient consortium for the degradation of hydrocarbons (Radwan et al., 2002; Abed and €ster, 2005; Sa nchez et al., 2005). The richness of polluted cyanoKo bacterial mats in aerobic heterotrophic bacteria with the ability to grow on aliphatic and aromatic hydrocarbons as well as on cyanobacterial exudates is congruent with our previous findings (Abed et al., 2007). Our isolates belonged to the genera Marinobacter, Pseudomonas and Sphingomonas, known to include hydrocarbondegrading species in other ecosystems (Sagardıa et al., 1975; Barathi and Vasudevan, 2001; Cohen, 2002; Ying et al., 2004; Leys et al., 2004; Yakimov et al., 2004, 2007). Marinobacter-related species were always encountered in cultures from oil-contaminated cyanobacterial mats (Cohen, 2002; McGowan et al., 2004; Abed et al., 2007). The isolation of these strains at higher dilution levels suggests their numerical abundance in the studied mat. Our strains showed also growth on most of the tested cyanobacterial organic exudates. This suggests that these strains could play an essential role in carbon cycling within cyanobacterial mats. They probably compete with other coexisting non-oil-degrading aerobic heterotrophs for these cyanobacterial metabolites. It has been demonstrated that aerobic heterotrophs with the ability to grow on oil prefer these organics over oil components because of their simple structure and no toxicity (Kirkwood et al., 2006). A previous study demonstrated the occurrence of two functional guilds of aerobic heterotrophs in polluted microbial mats, one degrading the cyanobacterial exudates but not hydrocarbons and the other degrading both (Abed et al., 2007). The former group included strains belonging to the genera Marinobacter, Halomonas, Roseobacter and Rhodobacter whereas the strains from the later group belonged to Marinobacter
62
R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e64
of the initially added amount of phenanthrene
120
T = 0 days
T = 1 day b b
a b
100
T = 3 days
T = 2 days
b a,b
a,b
a
b,c
b,c
80
b,c
c
b,c
a,b
d,e
e
c,d,e
a
b
b,c
c,d
60 40 a
20 0
T = 4 days
T = 5 days
T = 6 days
T = 7 days
d
a
100 80
d
e c,d
60
c,d
d
c, d b,c
40 20
c
c
b
a,b
a
c
b
a
b
a,b
Con No
Acet Pyru Gluc Ala
0 Con No
Acet Pyru Gluc Ala
But
Con No
Acet Pyru Gluc Ala
But
But
Con No
Acet Pyru Gluc Ala
But
Fig. 3. Degradation of phenanthrene by the GM42 isolate in the presence of different organic compounds previously identified as cyanobacterial excretion products as followed by gas chromatography (GC) analysis (n ¼ 3). The data are expressed as percentage relative to the amount of compound initially added (i.e. 3.33 mg/100 mg organo-clay). Different letters indicate a significant difference (P < 0.05) among the different treatments. Con: control; No: no additional substrate was added; Acet: acetate; Pyru: pyruvate; Gluc: glucose; Ala: alanine; But: butanol.
and Alcanivorax (Jonkers and Abed, 2003; Abed et al., 2007). Such heterotrophs apparently reside in close proximity to cyanobacteria in the photic layer of microbial mats in order to ensure a continuous access to cyanobacterial exudates as well as to the photosynthetically-produced oxygen. 4.1. Aerobic heterotrophic bacteria and cyanobacterial growth Synechocystis PCC6803 exhibited similar growth in the presence and absence of hexadecane, indicating its tolerance but inability to grow on hexadecane, although this strain became dominant in the original mats after degradation of four petroleum compounds including phenanthrene (Abed et al., 2002). On the contrary, the growth of Synechocystis was clearly stimulated in the presence of the GM41 strain and more evidently when hexadecane was added. The addition of the aerobic heterotroph might have supported the growth of the cyanobacterium by utilizing the photosyntheticallyproduced carbon compounds, whose accumulation is known to inhibit photosynthesis (Bateson and Ward, 1988). The oil-degrading
Protein concentration (µg/ml)
180
*a
Phenanthrene+acetate Phenanthrene+glucose Phenanthrene+pyruvic acid Phenanthrene
160
*a
140
a*
* a a,b
120
b b
100 80
b
60
*
a* a,b
c
c
b
types of aerobic heterotrophs reduce the concentrations of toxic aromatics and alkanes around cyanobacteria, which were shown to inhibit photosynthesis, growth and enzyme activity (Megharaj et al., 2000). Safonova et al. (1999) demonstrated that the presence of alkane utilizing bacteria in association with algae and cyanobacteria restored the growth of sensitive strains exposed to black oil and stimulated the growth of the tolerant species. The utilization of cyanobacterial exudates and petroleum compounds by aerobic heterotrophs in microbial mats results in increased rates of oxygen consumption, thus reducing the high concentrations of photosynthetic O2 in mats, which can be toxic to a variety of metabolic and biosynthetic pathways (Garcia-Pichel et al., 1999). Using microsensors, the addition of crude oil and its components to microbial mats was shown to induce prominent changes in rates of light respiration, gross and net photosynthesis and pH gradients €tzschel et al., 2002; Benthien et al., 2004). The respiring (Gro bacteria counteract the chemical changes in O2, CO2 and pH € hl, 2006). induced by photosynthesis (Wieland and Ku Cyanobacteria could feed directly on metabolites (e.g. organic acids) produced upon the degradation of oil components by aerobic heterotrophic bacteria. Alkanes are known to be utilized via their oxidation to fatty acids that are further oxidized to acetate (Radwan and Al-Hasan, 2000). There are several reports that demonstrated the heterotrophic growth of several strains of cyanobacteria on organic compounds like sugars, acetate and glycerol (Rippka, 1972; More et al., 1979; Radwan and Al-Hasan, 2000). Cyanobacteria could also use the carbon dioxide, formed as the final product of hexadecane degradation, directly for their photosynthesis. 4.2. The role cyanobacterial exudates in hydrocarbons degradation by aerobic heterotrophs
c
b
40 20
d
0 0
2
4
6
8
10
Days Fig. 4. The growth of the GM42 strain on phenanthrene with and without acetate, glucose and pyruvate, as determined by protein estimation over a period of 8 days. Stars indicate the time points where statistically significant differences among different incubations were detected. Different alphabetic superscripts indicate a significant difference between incubations at P < 0.05 within each time point using Tukey's test.
The results of this study showed that cyanobacterial organic exudates play a role in supporting the growth of aerobic heterotrophic bacteria and consequently the degradation of hydrocarbons. Alanine and butanol did not affect the degradation rate of phenanthrene by the GM42 strain, while acetate, pyruvate and glucose exhibited a stimulatory effect. When phenanthrene and an exudate were provided together, it should be expected that that aerobic heterotrophic bacteria would prefer to utilize easily assimilated, low molecular weight, cyanobacterial exudates rather than phenanthrene. A number of studies have shown that cyanobacterial exudates can be easily
R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e64
assimilated and recycled by the associated aerobic heterotrophs (Bauld and Brock, 1974; Bateson and Ward, 1988; Epping et al., 1999). Oil-degrading bacteria will grow initially on these organics, leading to a significant increase in their numbers, after which they degrade hydrocarbons. This explanation is evident from the protein analysis which showed around 3e4-fold increase in the bacterial biomass when the exudate was added. Previous studies have demonstrated the significance of cyanobacterial exudates in accelerating rates of hydrocarbon degradation. Glucose and lipids were shown to enhance degradation of alkanes in polluted soils from Kuwait (Radwan et al., 2000, 2001). Kirkwood and his coworkers demonstrated a species-specific effect of cyanobacterial exudates from four strains on the biodegradation rates of phenol and dichloroacetate by Pseudomonas and Ancylobacter, respectively (Kirkwood et al., 2006). Exudates from Aphanocapsa and Pseudoanabaena spp. repressed the degradation of phenol but enhanced the degradation of dichloroacetate although all exudates supported the growth of the bacterial strains. The authors attributed the inhibition of phenol degradation to a toxic rather than a competitive effect of the exudates. In rhizospheres of higher plants, hydrocarbon-degrading bacteria were found in high densities benefiting from the root exudates which include easily assimilated compounds like sugars and amino acids (Lee et al., 1995; Radwan et al., 1998). Indeed, the addition of organic fertilizers that contain fatty acids, alcohols and sugars was successfully used in the bioremediation of several polluted sites worldwide including the well known Exxon Valdez oil spill in 1989 (Rivet et al., 1993; Bragg et al., 1994; Al-Hadhrami et al., 1996). The use of cyanobacteria in the cleanup of pollution may circumvent the costly use of these fertilizers by directly supplying the degrading bacteria with necessary organics to increase their biomass and with fixed nitrogen that can be limited in marine environments (Ward and Brock, 1976). In conclusion, the aerobic heterotrophs-cyanobacterial mat consortia offer a cost-effective and promising system for bioremediating oil-polluted coastal sites. Our study demonstrated that cyanobacteria grew better in the presence of the associated aerobic heterotrophic bacteria and could provide them with necessary organics for efficient degradation activities. Further investigations should extrapolate our observations in culture experiments to study this relationship in intact mats and under field conditions. The use of simple organics compounds like acetate in accelerating biodegradation rates in oilpolluted sediments should be tested in the field. However, it should be noted that these compounds have to be added as a starter at concentrations enough to induce growth of oil-degrading bacteria without enriching specific bacterial populations.
Acknowledgment I would like to thank Henk Jonkers and Dirk de Beer from the Max-Planck Institute, Bremen, Germany for fruitful discussions and suggestions. I would also like to thank Florin Musat for his intro€ rgen Ko €ster for preparing duction to gas chromatography (GC) and Ju the organo-clay complexes. Special thanks go to Michael Barry for his help in the statistical analysis. Natuschka Lee and Derek Roberts are thanked for reviewing the paper. This research was financially supported by the Deutsche Forschungsgemeinschaft (grant BE 2167/ 4) and by the Max-Planck Society.
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Interaction between cyanobacteria and aerobic heterotrophic bacteria in the degradation of hydrocarbons Raeid M.M. Abed* Sultan Qaboos University, College of Science, Biology Department, P.O. Box: 36, Al Khoud, Postal Code 123, Muscat, Sultanate of Oman
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 August 2009 Received in revised form 14 October 2009 Accepted 14 October 2009 Available online 10 November 2009
Four strains of aerobic heterotrophic bacteria were isolated on crude oil with the aim to test whether their presence and activity might support the growth of cyanobacteria in oil-polluted microbial mats and whether the cyanobacterial exudates might play a role in stimulating their degradative activities. The strains were phylogenetically related to known oil-degrading species from the genera Marinobacter, Pseudomonas and Sphingomonas. Three strains (GM41, GM61 and GM63) grew well on C5eC18 alkanes but not on 7 tested aromatics, whereas one strain (i.e. GM42) grew best on phenanthrene and pentane. All strains showed ability to metabolize a range of cyanobacterial photosynthetic and fermentative exudates. In coculture experiments, the addition of the Pseudomonas-related GM41 strain to the cyanobacterium Synechocystis PCC6803, found in the same mat, resulted in 8-fold increase in the cyanobacterial biomass. This growth was more pronounced when hexadecane was added to the culture medium. The addition of representative substrates of cyanobacterial exudates to the phenanthrene-degrading strain GM42 resulted in variable effects. While acetate, pyruvate and glucose enhanced phenanthrene degradation, alanine and butanol showed no effect. We conclude that aerobic heterotrophic bacteriaecyanobacteria consortia can be very useful for bioremediating oil-polluted sites, circumventing the costly use of organic and inorganic fertilizers. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Cyanobacteria Aerobic heterotrophic bacteria Cyanobacterial mats Cyanobacterial exudates Hydrocarbons
1. Introduction The uppermost layer of cyanobacterial mats, dominated by cyanobacteria and aerobic heterotrophic bacteria, was shown to degrade € tzschel et al., 2002; Benhydrocarbons (Abed et al., 2002, 2006; Gro nchez et al., 2006). Heterotrophic bacteria but thien et al., 2004; Sa not the cyanobacteria, were chiefly responsible for the observed €ster, 2005). This raised the questions why degradation (Abed and Ko cyanobacteria dominate polluted sites and whether their presence may contribute to the degradation of petroleum compounds. It has been postulated that cyanobacteria require aerobic heterotrophic bacteria for their growth, which is evident from the difficulty to obtain them in axenic cultures (Rippka, 1988). Cyanobacteria play an indirect role in oil biodegradation by providing the associated aerobic heterotrophs with: oxygen (by-product of photosynthesis) needed for the breakdown of aliphatic and aromatic compounds (Abed et al., € ster, 2005); fixed nitrogen (through nitrogen 2002; Abed and Ko fixing strains) that is often limited in marine environments (Musat et al., 2006); and simple organics produced by photosynthesis and
* Tel.: þ968 24142406. E-mail address: [email protected] 0964-8305/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2009.10.008
fermentation. So far, the effect of cyanobacterial photosynthetic and fermentative organic exudates on the activity of oil-degrading microorganisms has not been investigated. The organics produced by cyanobacteria in microbial mats nourish other functional bacterial groups including aerobic heterotrophic bacteria (Bateson and Ward, 1988). These bacteria oxidize organic compounds directly to CO2. Cyanobacterial exudates include low molecular weight compounds and extrapolymeric substances (EPS) composed primarily of proteins, lipids and nucleic acids (Decho, 1990; Flemming and Wingender, 2001). Mannitol and arabinose were identified as excretion products (Hellebust,1965). Glycolate was found to be the main compound among dissolved photosynthates under hyperoxic and alkaline conditions (Bateson and Ward,1988). In the dark and under anoxic conditions, cyanobacteria ferment and produce organics different from those produced during the day (Nold and Ward, 1996; Jonkers and Abed, 2003). Fermentation products like acetate, propionate, lactate and ethanol were identified (Anderson et al., 1987; Jrgensen et al., 1992; Stal, 1995; Stal and Moezelaar, 1997). Studies on the diversity and degradative capabilities of aerobic heterotrophic bacteria in microbial mats demonstrated the presence of different populations that are specialized in the consumption of specific cyanobacterial exudates (Jonkers and Abed, 2003; Abed et al., 2007).
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In this study, a culture-based approach was used to study the interaction between cyanobacteria and associated aerobic heterotrophic bacteria in the degradation of hydrocarbons. Enrichment cultivation, although known to underestimate bacterial diversity (Amann et al., 1995), is still an important technique to study the physiological characteristics of individual strains and to test and expand hypotheses in microbial ecology. Four aerobic heterotrophic bacteria were isolated on crude oil as the sole source of carbon and were phylogenetically and physiologically characterized. The effect of the presence of an aerobic heterotroph (i.e. GM41 strain) on the growth of Synechocystis PCC6803 axenic culture was tested. Several cyanobacterial exudates were checked for their effects on the degradation rate of phenanthrene by the strain GM42. 2. Materials and methods 2.1. Isolation of aerobic heterotrophic bacteria Aerobic heterotrophic bacteria were isolated from a marine polluted cyanobacterial mat (Abed et al., 2002) on a defined artificial seawater medium. The medium contained MgCl2$6H2O (5.6 g l1), MgSO4$7H2O (6.8 g l1), CaCl2$2H2O (1.47 g l1), KCl (0.66 g l1) and KBr (0.09 g l1). After autoclaving, 0.15 and 0.2 g l1, respectively, of KH2PO4 and NH4Cl solutions were added. Autoclaved solutions of trace elements (Widdel and Bak, 1992), selenite and tungstate, and vitamins (Heijthuijsen and Hansen, 1986) were then added (1 ml l1). Solid media were prepared using 1% (w/v) Bactoagar. The salinity of the medium was adjusted to 2%, similar to the salinity of the field site where the strains originated from. Cultivation enrichments were done in 20 ml screw-cap culture tubes containing 10 ml culture medium under aerobic conditions. Serial dilution was used as an isolation technique. The enrichment was performed on Maya crude oil (de Oteyza and Grimalt, 2004) at a concentration of 1% (w/v) and all enrichments were incubated at 28 C. Axenic strains of aerobic heterotrophic bacteria were obtained from high dilutions (104 and 106) by plating on agar acetatecontaining medium. The obtained strains were then tested for growth on crude oil. 2.2. Characterization of aerobic heterotrophic bacterial isolates Polymerase chain reaction (PCR) was performed on the DNA extracts of the isolates using GM3 and GM4 primers (Muyzer et al., 1995). The PCR products were purified using the QIAquick PCR € sseldorf, Germany) and then sequenced. purification kit (Diagen, Du The 16S rRNA sequences of the strains (ca. 1400 bp) were analyzed using the ARB software (Pruesse et al., 2007). Phylogenetic trees were constructed based on almost complete 16S rRNA gene sequences (>1300 bp) by applying different methods integrated in the ARB software such as maximum likelihood, maximum parsimony and neighbor joining. Partial sequences were not included in the calculation of the trees. The final maximum likelihood tree was minimized for simplicity in presentation. The sequences of the four strains were submitted to the GenBank (Accession numbers GQ273962eGQ273965). The growth of the obtained strains on 26 different chemical compounds known to be cyanobacterial exudates as sole carbon source was tested. The tested substrates, listed in Table 1, included fatty acids, alcohols, amino acids and sugars. Each individual substrate was added to 10 ml of the medium to give the final concentration of 10 mM. Each tube was inoculated with 100 ml of the treated strain pre-grown on acetate. Growth on individual substrate was measured by following the changes of the optical densities of the cultures at 660 nm against biotic (without a substrate) and sterile (without bacteria) controls.
59
Table 1 Growth of the four aerobic heterotrophic bacterial strains on various aliphatic and aromatic compounds. (þþ): maximum growth reached in <3 days; (þ): maximum growth reached in 3e10 days; (): no growth. Growth was monitored by following daily changes in optical density at 660 nm against biotic (without a substrate) and sterile (without bacteria) controls. Hydrocarbons
Strains GM41
GM42
GM61
GM63
Alkanes n-Pentane n-Hexane n-Octane n-Decane n-Dodecane n-Tetradecane n-Hexadecane n-Octadecane
þ þþ þþ þþ þþ þþ þþ þþ
þþ þ þ þ
þ þ þþ þ þþ þþ þþ
þþ þþ þþ þ þ þþ þþ
Mono-aromatics Benzene Toulene
Di-aromatics Naphthalene
Tri-aromatics Phenanthrene Anthracene
þþ
Cycloalkanes Cyclohexane
Sulfur aromatics Dibenzothiophene
Table 2 Substrate spectra of the four isolates on previously identified cyanobacterial exudates including compatible solutes, photosynthetic and fermentative products (see text for references). The substrates were added at a final concentration of 10 mM. (þþ): maximum growth reached in <3 days; (þ): maximum growth reached in 3e10 days; (): no growth. Growth was monitored by following daily changes in optical density at 660 nm against biotic (without a substrate) and sterile (without bacteria) controls. Substrate
Strains GM41
GM42
GM61
GM63
Fatty acids Acetic acid Glycolic acid Citric acid Malic acid Succinic acid Butyric acid Methyl succinic acid Fumaric acid Pyruvic acid Hydroxybutyric acid Iso-valeric acid Propionic acid Formic acid
þþ þ þþ þþ þþ þþ þþ þþ
þþ þ þ þþ þ þþ
þþ þ þþ þþ þþ þþ þþ þþ
þþ þ þþ þþ þþ þþ þþ þþ
Alcohols Methanol Ethanol Propanol Butanol Glycerol
þþ þþ þþ þþ
þþ þ
þ þ þþ þþ
þþ þþ þþ þþ
Amino acids Glycine Betain Alanine
þþ
þþ
þþ
þþ
Sugars Glucose Fructose Galactose Sucrose Ribose
þþ þþ þþ þþ þþ
þ þ þ þ
þþ þþ þ þ
þþ þþ þþ þþ þþ
Pseudomonas denitrificans (AB021419) Pseudomonas pertucinogena (AB021380) Strain GM41 (GQ273962) Strain GM61 (GQ273964) Pseudomonas sp. (AB030085) Pseudomonas oleovorans (D84018) Pseudomonas alcaligenes (Z76653) Pseudomonas mendocina (AF232713) Pseudomonas alcalophila (AB030583) Marinobacter sp. (U61848) Marinobacter hydrocarbonoclasticus (AB019148) Marinobacter aquaeolei (AJ000726) Strain GM63 (GQ273965) Pseudomonas nautica (D11189) Sphingomonas adhaesiva (D13722) Sphingomonas macrogoltabidus (D84530) Strain GM42 (GQ273963) Sphingomonas sp.(AB025720) Sphingomonas stygia (AB025013) Sphingomonas aromaticivorans (AB025012) "Microcystis aeruginosa“ (U53589) Cyanothece PCC7424 (AJ000715) "Gloeothece membranacea" (X78680) Synechocystis PCC6803 (D90916) Synechocystis UNIWG Synechococcus PCC7002 (AJ000716) Oscillatoria rosea (AB003164) Cyanothece sp. (AF296873)
Alpha-
10
Gamma-proteobacteria
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Cyanobacteria
60
Fig. 1. 16S rRNA-based phylogenetic reconstruction of the four aerobic heterotrophic bacterial strains and the cyanobacterium Synechocystis PCC6803 used in this study. The strains were isolated from the uppermost layer of a heavily polluted mat at high dilution levels (104 and 106) after enrichments on Maya crude oil. Synechocystis UNIWG denotes the sequence obtained directly from the original mat.
The growth of the obtained strains in the presence of 16 different hydrocarbons, listed in Table 2, as sole carbon source was measured. Individual hydrocarbon was added to 10 ml of the medium in a final concentration of 0.5e2% depending on the toxicity and the solubility of the compound. Each tube was inoculated with 100 ml of bacterial cultures pre-grown on acetate. Growth was monitored by following the changes of the optical densities of the cultures at 660 nm over a period of 2 weeks. 2.3. Coculture of Synechocystis PCC6803 and the Pseudomonas-related GM41 strain Each 25 ml sterile test tube received 10 ml of the autoclaved medium described above. The selected strains for this experiment were the axenic Synechocystis strain PCC6803 (obtained from the PCC Pasteur collection, Paris, France) and the heterotrophic bacterial strain GM41. This cyanobacterial strain was selected because of its dominance in the original field mat (Abed et al., 2002, Fig. 1). Four experiments were prepared each in replicates: 1) Synechocystis PCC6803 alone; 2) Synechocystis PCC6803 þ the GM41 strain þ hexadecane (1% v/v); 3) Synechocystis PCC6803 þ hexadecane (1% v/v) and 4) Synechocystis PCC6803 þ the GM41 strain without hexadecane. This experiment was designed in order to test 1) whether Synechocystis PCC6803 could grow on hexadecane, 2) whether the presence of associated aerobic heterotrophs could stimulate/inhibit the growth of the cyanobacterium and 3) whether the growth of the aerobic heterotrophs on hydrocarbons might stimulate/inhibit the growth of the cyanobacterium. The tubes were inoculated with 5% (v/v) pre-grown cultures of Synechocystis PCC6803 and the GM41 strain. Incubation was at 28 C and under 12 light/12 darkness for 42 days. Samples were collected, centrifuged and the pellet was stored at 20 C for chlorophyll a (Chl a) determination. Chl a was extracted by addition of 3 ml methanol to the microbial pellets after centrifugation (Stal et al., 1984). The tubes were shaken for 3 h at 100 rpm/min, and then centrifuged at 4500 rpm/m for 10 min. The supernatant was transferred to a fresh tube and the pellet was re-extracted with methanol. The supernatants from both steps were pooled together. The absorbance of the supernatant
was measured at 662 nm using a spectrophotometer (Shimadzu, Duisburg, Germany). All steps were performed in a dim light 2.4. Effects of organics known to be cyanobacterial exudates on degradation rates of phenanthrene by the aerobic heterotrophic strain GM42 The used medium was prepared by mixing equal volumes of seawater with distilled water to obtain a salinity of about 2%. Nitrogen and phosphate sources were added to the medium as 1 mM ammonium chloride and 8 mM sodium dihydrophosphate, respectively. The inoculum was 100 ml of the GM42 strain, after 24 h pre-growth on acetate, per 100 ml medium. The GM42 strain was incubated in the presence of 0.15 mM phenanthrene with and without 0.15 mM of an individual cyanobacterial exudate. The tested exudate substrates were acetate, pyruvate, glucose, alanine and butanol. Phenanthrene was added after adsorbing it on hydrophobic clay (montmorillonite KSF, Aldrich) as a carrier to ensure its accessibility. The phenanthreneeclay complex was prepared as described earlier (Abed et al., 2002). Two control flasks were prepared in which phenanthrene was incubated with and without the added cyanobacterial substrate but in the absence of the GM42 strain. All treatments were run in triplicates. All flasks were incubated at 28 C with constant shaking at 100 rpm for 12 days. Samples for chemical analysis (2 ml each) were taken every 2 days. The residual phenanthrene was extracted with dichloromethane (DCM) and analyzed by gas chromatography. The growth of the GM42 strain on phenanthrene with and without the cyanobacterial exudate was determined in terms of protein concentrations according to Lowry et al. (1951). The bacterial cells were collected by centrifugation and 1 ml of 1 M sodium hydroxide was added to the pellet. The suspension was heated for 10 min at 100 C and after cooling to room temperature, 5 ml of a mixture of 2% Na2CO3, 1% CuSO4$5H2O and 2% sodium potassium tartarate (100:1:1 vol/vol) was added. The suspension was vortexed and incubated for 10 min. At the end of incubation, 0.5 ml of Folin reagent was added and the tubes were left for 30 min. Absorption was measured at 663 nm using a double beam spectrophotometer. 2.5. Statistical analysis Chl a (Section 2.3), biodegration of phenanthrene and protein (Section 2.4) data were analyzed by one-way ANOVA using the SPSS software (10th edition, Chicago, USA). P-values were adjusted using the sequential Bonferroni (Quinn and Keough, 2002) and Tukey's test was used to determine differences between individual means. P-values were used to directly compare data among different treatments at each time point. 3. Results 3.1. Characterization of aerobic heterotrophic bacterial strains Four pure bacterial strains were isolated at the 104 (GM41 and GM42) and 106 (GM61 and GM63) dilution levels on crude oil. The strains belonged to the Alphaproteobacteria and Gammaproteobacteria based on the phylogenetic reconstruction of their 16S rRNA gene sequences (Fig. 1). The GM63 strain was closely affiliated to the known oil-degrading bacteria Marinobacter aquaeolei and Marinobacter hydrocarbonclasticus (Gauthier et al., 1992, Huu et al., 1999) with 98.2 and 98.0% sequence similarity, respectively. The strains GM41 and GM61 shared 97.0% 16S rRNA gene sequence similarity and fell phylogenetically within the Pseudomonas group, which contains strains with the ability to grow on both alkanes and aromatic compounds (Eggink et al., 1988; Yen et al., 1991; Tian et al.,
R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e64
2002). The GM42 strain belonged to the Alphaproteobacteria within the Sphingomonas group. Growth spectra on hydrocarbons showed that the strains GM41, GM61 and GM63 grew on alkanes but not aromatics (Table 1). Growth was observed in the presence of all tested alkanes except in the case of the GM61 strain on n-octadecane and GM63 on n-pentane. The strain GM42 grew poorly on alkanes compared to other strains. The most pronounced growth of this strain was detected in the presence of n-pentane and phenanthrene. The tested strains showed growth on most cyanobacterial exudates with slight differences (Table 2). All strains grew on most tested carbohydrates. While all tested strains readily grew on acetate, succinate, butyrate, propionate and pyruvate, none grew on citrate, malate and formate. Other fatty acids like glycolate and fumarate supported the growth of the strains GM41, GM61 and GM63 but not GM42. All strains grew on glycerol and butanol but not on methanol. The four strains grew on alanine but not glycine and the compatible solute betain.
3.2. Growth of Synechocystis PCC6803 in the presence of hexadecane and/or the GM41 strain The effect of the presence of hexadecane and/or the strain GM41, which is capable of growing on hexadecane, on the growth of the axenic Synechocystis PCC6803 culture was monitored over a period of 42 days (Fig. 2). Growth of Synechocystis PCC6803 in the presence and absence of hexadecane, but without the GM41 strain, was comparable, with a typical bacterial growth curve. There was no statistical difference between these two treatments at all time points (Fig. 2). Addition of cells of the GM41 strain apparently stimulated the growth of the cyanobacterium and maximum growth was recorded after 20 days. The growth after 20 days was 8 times higher, based on the optical density values of extracted Chl a, with the GM41 strain than without it. This difference was statistically significant (P ¼ 0.013). The best growth of Synechocystis PCC6803 strain was observed in the presence of hexadecane and the GM41 strain. The cyanobacterium exhibited a lag phase of about 10 days, and growth reached the maximum after 35 days, after which it was significantly higher than other treatments (P < 0.05).
Chlorophyll a (Chl a) at 662 nm
3.5 PCC6803 PCC6803+hexadecane PCC6803+GM41 PCC6803+GM41+hexadecane
3.0
*a a* a*
2.5
a*
2.0 1.5 1.0
*a
0.5
0
b c
b 10
b
c
b
* *
0.0
b
20
30
b 40
50
Days Fig. 2. Growth of Synechocystis PCC6803 with and without hexadecande and in the presence and absence of the hexadecane-degrading strain GM41 (n ¼ 3). The growth of the cyanobacterium was monitored by measuring Chlorophyll a (Chl a) at 662 nm. Stars indicate the time points at which different incubations exhibit statistically significant difference (P < 0.05). Common alphabetic superscripts indicate no significant difference using Tukey's test within each time point.
61
The growth of the cyanobacterium in the presence of both hexadecane and the GM41 strain was 2 times higher than with only the GM41 strain and 12 times higher than without both. 3.3. Effect of organics known to be cyanobacterial exudates on phenanthrene consumption by the GM42 strain The effects of acetate, pyruvate, glucose, alanine and butanol addition on the phenanthrene consumption by the GM42 strain was tested (Fig. 3). Phenanthrene completely disappeared after 7 days at a rate of 0.34 mg d1 in the absence of any added organic substrate. The addition of alanine and butanol did not show any significant effect on phenanthrene degradation and the consumption rate was comparable to the control (p > 0.05). In contrast, glucose, pyruvate and acetate significantly stimulated the degradation process (p < 0.02). Phenanthrene disappeared after 6 days at a rate of 0.48 and 0.54 mg d1 in the presence of glucose and pyruvic acid, respectively. The highest degradation rate was recorded in the presence of acetate at a rate of 0.8 mg d1 within about 5 days. The growth of the GM42 strain in the presence of phenanthrene with and without the tested cyanobacterial exudates (acetate, pyruvate and glucose) was followed using protein estimation in order to check whether the observed increase in phenanthrene degradation rate was due to increased biomass or increased activity of the GM42 strain (Fig. 4). The GM42 strain accumulated more biomass in the presence of phenanthrene and one cyanobacterial exudate than in the presence of phenanthrene alone. This difference in growth was statistically significant with P-values < 0.05. Maximum growth was reached already after 4 days in the presence of acetate but took 6e7 days in the presence of glucose and pyruvate, after which it remained more or less constant. In spite of that, more bacterial biomass was detected in the presence of glucose and pyruvate than acetate. 4. Discussion This study supports earlier reports that cyanobacteria and associated aerobic heterotrophs constitute an efficient consortium for the degradation of hydrocarbons (Radwan et al., 2002; Abed and €ster, 2005; Sa nchez et al., 2005). The richness of polluted cyanoKo bacterial mats in aerobic heterotrophic bacteria with the ability to grow on aliphatic and aromatic hydrocarbons as well as on cyanobacterial exudates is congruent with our previous findings (Abed et al., 2007). Our isolates belonged to the genera Marinobacter, Pseudomonas and Sphingomonas, known to include hydrocarbondegrading species in other ecosystems (Sagardıa et al., 1975; Barathi and Vasudevan, 2001; Cohen, 2002; Ying et al., 2004; Leys et al., 2004; Yakimov et al., 2004, 2007). Marinobacter-related species were always encountered in cultures from oil-contaminated cyanobacterial mats (Cohen, 2002; McGowan et al., 2004; Abed et al., 2007). The isolation of these strains at higher dilution levels suggests their numerical abundance in the studied mat. Our strains showed also growth on most of the tested cyanobacterial organic exudates. This suggests that these strains could play an essential role in carbon cycling within cyanobacterial mats. They probably compete with other coexisting non-oil-degrading aerobic heterotrophs for these cyanobacterial metabolites. It has been demonstrated that aerobic heterotrophs with the ability to grow on oil prefer these organics over oil components because of their simple structure and no toxicity (Kirkwood et al., 2006). A previous study demonstrated the occurrence of two functional guilds of aerobic heterotrophs in polluted microbial mats, one degrading the cyanobacterial exudates but not hydrocarbons and the other degrading both (Abed et al., 2007). The former group included strains belonging to the genera Marinobacter, Halomonas, Roseobacter and Rhodobacter whereas the strains from the later group belonged to Marinobacter
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of the initially added amount of phenanthrene
120
T = 0 days
T = 1 day b b
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T = 3 days
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b a,b
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T = 5 days
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T = 7 days
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Acet Pyru Gluc Ala
0 Con No
Acet Pyru Gluc Ala
But
Con No
Acet Pyru Gluc Ala
But
But
Con No
Acet Pyru Gluc Ala
But
Fig. 3. Degradation of phenanthrene by the GM42 isolate in the presence of different organic compounds previously identified as cyanobacterial excretion products as followed by gas chromatography (GC) analysis (n ¼ 3). The data are expressed as percentage relative to the amount of compound initially added (i.e. 3.33 mg/100 mg organo-clay). Different letters indicate a significant difference (P < 0.05) among the different treatments. Con: control; No: no additional substrate was added; Acet: acetate; Pyru: pyruvate; Gluc: glucose; Ala: alanine; But: butanol.
and Alcanivorax (Jonkers and Abed, 2003; Abed et al., 2007). Such heterotrophs apparently reside in close proximity to cyanobacteria in the photic layer of microbial mats in order to ensure a continuous access to cyanobacterial exudates as well as to the photosynthetically-produced oxygen. 4.1. Aerobic heterotrophic bacteria and cyanobacterial growth Synechocystis PCC6803 exhibited similar growth in the presence and absence of hexadecane, indicating its tolerance but inability to grow on hexadecane, although this strain became dominant in the original mats after degradation of four petroleum compounds including phenanthrene (Abed et al., 2002). On the contrary, the growth of Synechocystis was clearly stimulated in the presence of the GM41 strain and more evidently when hexadecane was added. The addition of the aerobic heterotroph might have supported the growth of the cyanobacterium by utilizing the photosyntheticallyproduced carbon compounds, whose accumulation is known to inhibit photosynthesis (Bateson and Ward, 1988). The oil-degrading
Protein concentration (µg/ml)
180
*a
Phenanthrene+acetate Phenanthrene+glucose Phenanthrene+pyruvic acid Phenanthrene
160
*a
140
a*
* a a,b
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*
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types of aerobic heterotrophs reduce the concentrations of toxic aromatics and alkanes around cyanobacteria, which were shown to inhibit photosynthesis, growth and enzyme activity (Megharaj et al., 2000). Safonova et al. (1999) demonstrated that the presence of alkane utilizing bacteria in association with algae and cyanobacteria restored the growth of sensitive strains exposed to black oil and stimulated the growth of the tolerant species. The utilization of cyanobacterial exudates and petroleum compounds by aerobic heterotrophs in microbial mats results in increased rates of oxygen consumption, thus reducing the high concentrations of photosynthetic O2 in mats, which can be toxic to a variety of metabolic and biosynthetic pathways (Garcia-Pichel et al., 1999). Using microsensors, the addition of crude oil and its components to microbial mats was shown to induce prominent changes in rates of light respiration, gross and net photosynthesis and pH gradients €tzschel et al., 2002; Benthien et al., 2004). The respiring (Gro bacteria counteract the chemical changes in O2, CO2 and pH € hl, 2006). induced by photosynthesis (Wieland and Ku Cyanobacteria could feed directly on metabolites (e.g. organic acids) produced upon the degradation of oil components by aerobic heterotrophic bacteria. Alkanes are known to be utilized via their oxidation to fatty acids that are further oxidized to acetate (Radwan and Al-Hasan, 2000). There are several reports that demonstrated the heterotrophic growth of several strains of cyanobacteria on organic compounds like sugars, acetate and glycerol (Rippka, 1972; More et al., 1979; Radwan and Al-Hasan, 2000). Cyanobacteria could also use the carbon dioxide, formed as the final product of hexadecane degradation, directly for their photosynthesis. 4.2. The role cyanobacterial exudates in hydrocarbons degradation by aerobic heterotrophs
c
b
40 20
d
0 0
2
4
6
8
10
Days Fig. 4. The growth of the GM42 strain on phenanthrene with and without acetate, glucose and pyruvate, as determined by protein estimation over a period of 8 days. Stars indicate the time points where statistically significant differences among different incubations were detected. Different alphabetic superscripts indicate a significant difference between incubations at P < 0.05 within each time point using Tukey's test.
The results of this study showed that cyanobacterial organic exudates play a role in supporting the growth of aerobic heterotrophic bacteria and consequently the degradation of hydrocarbons. Alanine and butanol did not affect the degradation rate of phenanthrene by the GM42 strain, while acetate, pyruvate and glucose exhibited a stimulatory effect. When phenanthrene and an exudate were provided together, it should be expected that that aerobic heterotrophic bacteria would prefer to utilize easily assimilated, low molecular weight, cyanobacterial exudates rather than phenanthrene. A number of studies have shown that cyanobacterial exudates can be easily
R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e64
assimilated and recycled by the associated aerobic heterotrophs (Bauld and Brock, 1974; Bateson and Ward, 1988; Epping et al., 1999). Oil-degrading bacteria will grow initially on these organics, leading to a significant increase in their numbers, after which they degrade hydrocarbons. This explanation is evident from the protein analysis which showed around 3e4-fold increase in the bacterial biomass when the exudate was added. Previous studies have demonstrated the significance of cyanobacterial exudates in accelerating rates of hydrocarbon degradation. Glucose and lipids were shown to enhance degradation of alkanes in polluted soils from Kuwait (Radwan et al., 2000, 2001). Kirkwood and his coworkers demonstrated a species-specific effect of cyanobacterial exudates from four strains on the biodegradation rates of phenol and dichloroacetate by Pseudomonas and Ancylobacter, respectively (Kirkwood et al., 2006). Exudates from Aphanocapsa and Pseudoanabaena spp. repressed the degradation of phenol but enhanced the degradation of dichloroacetate although all exudates supported the growth of the bacterial strains. The authors attributed the inhibition of phenol degradation to a toxic rather than a competitive effect of the exudates. In rhizospheres of higher plants, hydrocarbon-degrading bacteria were found in high densities benefiting from the root exudates which include easily assimilated compounds like sugars and amino acids (Lee et al., 1995; Radwan et al., 1998). Indeed, the addition of organic fertilizers that contain fatty acids, alcohols and sugars was successfully used in the bioremediation of several polluted sites worldwide including the well known Exxon Valdez oil spill in 1989 (Rivet et al., 1993; Bragg et al., 1994; Al-Hadhrami et al., 1996). The use of cyanobacteria in the cleanup of pollution may circumvent the costly use of these fertilizers by directly supplying the degrading bacteria with necessary organics to increase their biomass and with fixed nitrogen that can be limited in marine environments (Ward and Brock, 1976). In conclusion, the aerobic heterotrophs-cyanobacterial mat consortia offer a cost-effective and promising system for bioremediating oil-polluted coastal sites. Our study demonstrated that cyanobacteria grew better in the presence of the associated aerobic heterotrophic bacteria and could provide them with necessary organics for efficient degradation activities. Further investigations should extrapolate our observations in culture experiments to study this relationship in intact mats and under field conditions. The use of simple organics compounds like acetate in accelerating biodegradation rates in oilpolluted sediments should be tested in the field. However, it should be noted that these compounds have to be added as a starter at concentrations enough to induce growth of oil-degrading bacteria without enriching specific bacterial populations.
Acknowledgment I would like to thank Henk Jonkers and Dirk de Beer from the Max-Planck Institute, Bremen, Germany for fruitful discussions and suggestions. I would also like to thank Florin Musat for his intro€ rgen Ko €ster for preparing duction to gas chromatography (GC) and Ju the organo-clay complexes. Special thanks go to Michael Barry for his help in the statistical analysis. Natuschka Lee and Derek Roberts are thanked for reviewing the paper. This research was financially supported by the Deutsche Forschungsgemeinschaft (grant BE 2167/ 4) and by the Max-Planck Society.
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