lable at ScienceDirect

International Biodeterioration & Biodegradation 64 (2010) 58e64

Contents lists avai

International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ib iod

Interaction between cyanobacteria and aerobic heterotrophic bacteriain 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

Article history:Received 29 August 2009Received in revised form14 October 2009Accepted 14 October 2009Available online 10 November 2009

Keywords:CyanobacteriaAerobic heterotrophic bacteriaCyanobacterial matsCyanobacterial exudatesHydrocarbons

* Tel.: þ968 24142406.E-mail address: rabed@mpi-bremen.de

0964-8305/$ e see front matter � 2009 Elsevier Ltd.doi:10.1016/j.ibiod.2009.10.008

a b s t r a c t

Four strains of aerobic heterotrophic bacteria were isolated on crude oil with the aim to test whethertheir presence and activity might support the growth of cyanobacteria in oil-polluted microbial matsand whether the cyanobacterial exudates might play a role in stimulating their degradative activities. Thestrains 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 butnot on 7 tested aromatics, whereas one strain (i.e. GM42) grew best on phenanthrene and pentane. Allstrains 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 cyanobacteriumSynechocystis 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 ofrepresentative substrates of cyanobacterial exudates to the phenanthrene-degrading strain GM42 resultedin variable effects. While acetate, pyruvate and glucose enhanced phenanthrene degradation, alanine andbutanol showed no effect. We conclude that aerobic heterotrophic bacteriaecyanobacteria consortia canbe very useful for bioremediating oil-polluted sites, circumventing the costly use of organic and inorganicfertilizers.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The uppermost layer of cyanobacterial mats, dominated by cya-nobacteria and aerobic heterotrophic bacteria,was shown to degradehydrocarbons (Abed et al., 2002, 2006; Gr€otzschel et al., 2002; Ben-thien et al., 2004; S�anchez et al., 2006). Heterotrophic bacteria butnot the cyanobacteria, were chiefly responsible for the observeddegradation (Abed and K€oster, 2005). This raised the questions whycyanobacteria dominate polluted sites and whether their presencemay contribute to the degradation of petroleum compounds. It hasbeen postulated that cyanobacteria require aerobic heterotrophicbacteria for their growth, which is evident from the difficulty toobtain them in axenic cultures (Rippka, 1988). Cyanobacteria play anindirect role in oil biodegradation by providing the associated aerobicheterotrophs with: oxygen (by-product of photosynthesis) neededfor the breakdown of aliphatic and aromatic compounds (Abed et al.,2002; Abed and K€oster, 2005); fixed nitrogen (through nitrogenfixing strains) that is often limited in marine environments (Musatet al., 2006); and simple organics produced by photosynthesis and

All rights reserved.

fermentation. So far, the effect of cyanobacterial photosyntheticand fermentative organic exudates on the activity of oil-degradingmicroorganisms has not been investigated.

The organics produced by cyanobacteria in microbial matsnourish other functional bacterial groups including aerobic hetero-trophic bacteria (Bateson and Ward, 1988). These bacteria oxidizeorganic compounds directly to CO2. Cyanobacterial exudates includelow molecular weight compounds and extrapolymeric substances(EPS) composed primarily of proteins, lipids andnucleic acids (Decho,1990; Flemming and Wingender, 2001). Mannitol and arabinosewere identified as excretionproducts (Hellebust,1965). Glycolatewasfound to be the main compound among dissolved photosynthatesunderhyperoxic and alkaline conditions (Bateson andWard,1988). Inthe dark and under anoxic conditions, cyanobacteria ferment andproduce organics different from those produced during the day (Noldand Ward, 1996; Jonkers and Abed, 2003). Fermentation productslike acetate, propionate, lactate and ethanol were identified (Ander-son et al., 1987; J�rgensen et al., 1992; Stal, 1995; Stal and Moezelaar,1997). Studies on the diversity and degradative capabilities ofaerobic heterotrophic bacteria in microbial mats demonstratedthe presence of different populations that are specialized in theconsumption of specific cyanobacterial exudates (Jonkers and Abed,2003; Abed et al., 2007).

Table 1Growth of the four aerobic heterotrophic bacterial strains on various aliphatic andaromatic compounds. (þþ): maximum growth reached in <3 days; (þ): maximumgrowth reached in 3e10 days; (�): no growth. Growth was monitored by followingdaily changes in optical density at 660 nm against biotic (without a substrate) andsterile (without bacteria) controls.

Hydrocarbons Strains

GM41 GM42 GM61 GM63

Alkanesn-Pentane þ þþ þ �n-Hexane þþ � þ þþn-Octane þþ þ þþ þþn-Decane þþ � þ þþn-Dodecane þþ þ þþ þn-Tetradecane þþ þ þþ þn-Hexadecane þþ � þþ þþn-Octadecane þþ � � þþMono-aromaticsBenzene � � � �Toulene � � � �Di-aromaticsNaphthalene � � � �Tri-aromaticsPhenanthrene � þþ � �Anthracene � � � �CycloalkanesCyclohexane � � � �Sulfur aromaticsDibenzothiophene � � � �

Table 2Substrate spectra of the four isolates on previously identified cyanobacterialexudates including compatible solutes, photosynthetic and fermentative products(see text for references). The substrates were added at a final concentration of10 mM. (þþ): maximum growth reached in <3 days; (þ): maximum growthreached in 3e10 days; (�): no growth. Growth was monitored by following dailychanges in optical density at 660 nm against biotic (without a substrate) and sterile(without bacteria) controls.

Substrate Strains

GM41 GM42 GM61 GM63

Fatty acidsAcetic acid þþ þþ þþ þþGlycolic acid þ � þ þCitric acid � � � �Malic acid � � � �Succinic acid þþ þ þþ þþButyric acid þþ þ þþ þþMethyl succinic acid � � � �Fumaric acid þþ � þþ þþPyruvic acid þþ þþ þþ þþHydroxybutyric acid þþ þ þþ þþIso-valeric acid � � � �Propionic acid þþ þþ þþ þþFormic acid � � � �AlcoholsMethanol � � � �Ethanol þþ � þ þþPropanol þþ � þ þþButanol þþ þþ þþ þþGlycerol þþ þ þþ þþAmino acidsGlycine � � � �Betain � � � �Alanine þþ þþ þþ þþSugarsGlucose þþ þ þþ þþFructose þþ þ þþ þþGalactose þþ � þ þþSucrose þþ þ � þþRibose þþ þ þ þþ

R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e64 59

In this study, a culture-based approach was used to study theinteraction between cyanobacteria and associated aerobic hetero-trophic bacteria in the degradation of hydrocarbons. Enrichmentcultivation, although known to underestimate bacterial diversity(Amann et al., 1995), is still an important technique to study thephysiological characteristics of individual strains and to test andexpand hypotheses in microbial ecology. Four aerobic heterotro-phic bacteria were isolated on crude oil as the sole source of carbonand were phylogenetically and physiologically characterized. Theeffect 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 onthe 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 marinepolluted cyanobacterial mat (Abed et al., 2002) on a defined artificialseawater medium. The medium contained MgCl2$6H2O (5.6 g l�1),MgSO4$7H2O (6.8 g l�1), CaCl2$2H2O (1.47 g l�1), KCl (0.66 g l�1) andKBr (0.09 g l�1). After autoclaving, 0.15 and 0.2 g l�1, respectively,of KH2PO4 and NH4Cl solutions were added. Autoclaved solutions oftrace elements (Widdel and Bak, 1992), selenite and tungstate,and vitamins (Heijthuijsen and Hansen, 1986) were then added(1 ml l�1). Solid media were prepared using 1% (w/v) Bactoagar. Thesalinity of the medium was adjusted to 2%, similar to the salinity ofthe field site where the strains originated from.

Cultivation enrichments were done in 20 ml screw-cap culturetubes containing 10 ml culture medium under aerobic conditions.Serial dilution was used as an isolation technique. The enrichmentwas performed on Maya crude oil (de Oteyza and Grimalt, 2004)at a concentration of 1% (w/v) and all enrichments were incubated at28 �C. Axenic strains of aerobic heterotrophic bacteriawere obtainedfrom high dilutions (10�4 and 10�6) by plating on agar acetate-containing medium. The obtained strains were then tested forgrowth on crude oil.

2.2. Characterization of aerobic heterotrophic bacterial isolates

Polymerase chain reaction (PCR) was performed on the DNAextracts of the isolates using GM3 and GM4 primers (Muyzer et al.,1995). The PCR products were purified using the QIAquick PCRpurification kit (Diagen, D€usseldorf, Germany) and then sequenced.The 16S rRNA sequences of the strains (ca. 1400 bp) were analyzedusing the ARB software (Pruesse et al., 2007). Phylogenetic treeswere constructed based on almost complete 16S rRNA genesequences (>1300 bp) by applying different methods integrated inthe ARB software such as maximum likelihood, maximum parsi-mony and neighbor joining. Partial sequences were not included inthe calculation of the trees. The final maximum likelihood treewas minimized for simplicity in presentation. The sequences of thefour strains were submitted to the GenBank (Accession numbersGQ273962eGQ273965).

The growth of the obtained strains on 26 different chemicalcompounds known to be cyanobacterial exudates as sole carbonsource was tested. The tested substrates, listed in Table 1, includedfatty acids, alcohols, amino acids and sugars. Each individual substratewas added to 10 ml of the medium to give the final concentration of10 mM. Each tube was inoculated with 100 ml of the treated strainpre-grown on acetate. Growth on individual substrate was measuredby following the changes of the optical densities of the cultures at660 nm against biotic (without a substrate) and sterile (withoutbacteria) controls.

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 hydrocarbonoclasticus (AB019148)Marinobacter sp. (U61848)

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 UNIWGSynechococcus PCC7002 (AJ000716)Oscillatoria rosea (AB003164)

Cyanothece sp. (AF296873)

10

Cya

noba

cter

iaG

amm

a-pr

oteo

bact

eria

Alp

ha-

Fig. 1. 16S rRNA-based phylogenetic reconstruction of the four aerobic heterotrophicbacterial strains and the cyanobacterium Synechocystis PCC6803 used in this study.The strains were isolated from the uppermost layer of a heavily polluted mat at highdilution levels (10�4 and 10�6) after enrichments on Maya crude oil. SynechocystisUNIWG denotes the sequence obtained directly from the original mat.

R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e6460

The growth of the obtained strains in the presence of 16 differenthydrocarbons, listed in Table 2, as sole carbon source was measured.Individual hydrocarbonwas added to 10 ml of the medium in a finalconcentration of 0.5e2% depending on the toxicity and the solubilityof the compound. Each tube was inoculated with 100 ml of bacterialcultures pre-grown on acetate. Growth was monitored by followingthe changes of the optical densities of the cultures at 660 nm overa period of 2 weeks.

2.3. Coculture of Synechocystis PCC6803 and thePseudomonas-related GM41 strain

Each 25 ml sterile test tube received 10 ml of the autoclavedmedium described above. The selected strains for this experimentwere the axenic Synechocystis strain PCC6803 (obtained from the PCCPasteur collection, Paris, France) and the heterotrophic bacterial strainGM41. This cyanobacterial strain was selected because of its domi-nance in the original field mat (Abed et al., 2002, Fig. 1). Four exper-iments were prepared each in replicates: 1) Synechocystis PCC6803alone; 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. Thisexperiment was designed in order to test 1) whether SynechocystisPCC6803 could grow on hexadecane, 2) whether the presence ofassociated aerobic heterotrophs could stimulate/inhibit the growthof the cyanobacterium and 3) whether the growth of the aerobicheterotrophs on hydrocarbons might stimulate/inhibit the growthof 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 awas extracted by addition of 3mlmethanol to themicrobialpellets after centrifugation (Stal et al., 1984). The tubes were shakenfor 3 h at 100 rpm/min, and then centrifuged at 4500 rpm/m for10 min. The supernatant was transferred to a fresh tube and thepellet was re-extracted with methanol. The supernatants from bothsteps 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 exudateson degradation rates of phenanthrene by the aerobicheterotrophic strain GM42

The used medium was prepared by mixing equal volumes ofseawater with distilled water to obtain a salinity of about 2%.Nitrogen andphosphate sourceswere added to themediumas 1mMammonium chloride and 8 mM sodium dihydrophosphate, respec-tively. The inoculum was 100 ml of the GM42 strain, after 24 hpre-growth on acetate, per 100 ml medium. The GM42 strain wasincubated in the presence of 0.15 mM phenanthrene with andwithout 0.15mMof an individual cyanobacterial exudate. The testedexudate substrates were acetate, pyruvate, glucose, alanine andbutanol. Phenanthrenewas added after adsorbing it on hydrophobicclay (montmorillonite KSF, Aldrich) as a carrier to ensure its acces-sibility. The phenanthreneeclay complex was prepared as describedearlier (Abed et al., 2002). Two controlflaskswere prepared inwhichphenanthrene was incubated with and without the added cyano-bacterial substrate but in the absence of the GM42 strain. All treat-ments were run in triplicates. All flaskswere incubated at 28 �Cwithconstant shaking at 100 rpm for 12 days. Samples for chemicalanalysis (2 ml each) were taken every 2 days. The residual phen-anthrene was extracted with dichloromethane (DCM) and analyzedby gas chromatography.

The growth of the GM42 strain onphenanthrenewith andwithoutthe cyanobacterial exudate was determined in terms of proteinconcentrations according to Lowry et al. (1951). The bacterial cellswere collected by centrifugation and 1 ml of 1 M sodium hydroxidewas added to the pellet. The suspension was heated for 10 min at100 �C and after cooling to room temperature, 5 ml of a mixture of2% Na2CO3, 1% CuSO4$5H2O and 2% sodium potassium tartarate(100:1:1 vol/vol) was added. The suspension was vortexed and incu-bated for 10min. At the end of incubation, 0.5 ml of Folin reagent wasadded and the tubeswere left for 30min. Absorptionwasmeasured at663 nm using a double beam spectrophotometer.

2.5. Statistical analysis

Chl a (Section 2.3), biodegration of phenanthrene and protein(Section 2.4) datawere analyzed by one-way ANOVAusing the SPSSsoftware (10th edition, Chicago, USA). P-values were adjusted usingthe sequential Bonferroni (Quinn and Keough, 2002) and Tukey'stest was used to determine differences between individual means.P-values were used to directly compare data among differenttreatments at each time point.

3. Results

3.1. Characterization of aerobic heterotrophic bacterial strains

Four pure bacterial strains were isolated at the 10�4 (GM41 andGM42) and 10�6 (GM61 and GM63) dilution levels on crude oil. Thestrains belonged to the Alphaproteobacteria and Gammaproteobac-teria based on the phylogenetic reconstruction of their 16S rRNAgene sequences (Fig. 1). The GM63 strainwas closely affiliated to theknown oil-degrading bacteria Marinobacter aquaeolei and Mar-inobacter hydrocarbonclasticus (Gauthier et al.,1992, Huu et al.,1999)with 98.2 and 98.0% sequence similarity, respectively. The strainsGM41 and GM61 shared 97.0% 16S rRNA gene sequence similarityand fell phylogenetically within the Pseudomonas group, whichcontains strains with the ability to grow on both alkanes andaromatic compounds (Eggink et al., 1988; Yen et al., 1991; Tian et al.,

R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e64 61

2002). The GM42 strain belonged to the Alphaproteobacteriawithinthe Sphingomonas group.

Growth spectra on hydrocarbons showed that the strains GM41,GM61 andGM63 grewon alkanes but not aromatics (Table 1). Growthwas observed in the presence of all tested alkanes except in the case ofthe GM61 strain on n-octadecane and GM63 on n-pentane. The strainGM42 grew poorly on alkanes compared to other strains. The mostpronounced growth of this strain was detected in the presence ofn-pentane and phenanthrene.

The tested strains showed growth on most cyanobacterialexudates with slight differences (Table 2). All strains grew onmost tested carbohydrates. While all tested strains readily grew onacetate, succinate, butyrate, propionate and pyruvate, none grewon citrate, malate and formate. Other fatty acids like glycolate andfumarate supported the growth of the strains GM41, GM61 andGM63 but not GM42. All strains grew on glycerol and butanol butnot on methanol. The four strains grew on alanine but not glycineand the compatible solute betain.

3.2. Growth of Synechocystis PCC6803 in the presenceof hexadecane and/or the GM41 strain

The effect of the presence of hexadecane and/or the strainGM41, which is capable of growing on hexadecane, on the growthof the axenic Synechocystis PCC6803 culture was monitored overa period of 42 days (Fig. 2). Growth of Synechocystis PCC6803 in thepresence and absence of hexadecane, but without the GM41 strain,was comparable, with a typical bacterial growth curve. There wasno statistical difference between these two treatments at alltime points (Fig. 2). Addition of cells of the GM41 strain apparentlystimulated the growth of the cyanobacterium and maximumgrowth was recorded after 20 days. The growth after 20 days was 8times higher, based on the optical density values of extracted Chl a,with the GM41 strain than without it. This difference was statisti-cally significant (P ¼ 0.013). The best growth of SynechocystisPCC6803 strain was observed in the presence of hexadecane andthe GM41 strain. The cyanobacterium exhibited a lag phase of about10 days, and growth reached the maximum after 35 days, afterwhich it was significantly higher than other treatments (P < 0.05).

Days0 10 20 30 40 50

Chl

orop

hyll

a (C

hla)

at 6

62 n

m

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

PCC6803PCC6803+hexadecanePCC6803+GM41PCC6803+GM41+hexadecane

*

*

**

*

a

b

a

b

a

b

c

a

b

c

a

b

b*

*

Fig. 2. Growth of Synechocystis PCC6803 with and without hexadecande and in thepresence and absence of the hexadecane-degrading strain GM41 (n ¼ 3). The growth ofthe cyanobacterium was monitored by measuring Chlorophyll a (Chl a) at 662 nm.Stars indicate the time points at which different incubations exhibit statisticallysignificant difference (P < 0.05). Common alphabetic superscripts indicate no signifi-cant difference using Tukey's test within each time point.

The growth of the cyanobacterium in the presence of both hex-adecane and the GM41 strainwas 2 times higher thanwith only theGM41 strain and 12 times higher than without both.

3.3. Effect of organics known to be cyanobacterial exudateson phenanthrene consumption by the GM42 strain

The effects of acetate, pyruvate, glucose, alanine and butanoladdition on the phenanthrene consumption by the GM42 strainwas tested (Fig. 3). Phenanthrene completely disappeared after 7days at a rate of 0.34 mg d�1 in the absence of any added organicsubstrate. The addition of alanine and butanol did not show anysignificant effect on phenanthrene degradation and the consump-tion rate was comparable to the control (p > 0.05). In contrast,glucose, pyruvate and acetate significantly stimulated the degra-dation process (p< 0.02). Phenanthrene disappeared after 6 days ata rate of 0.48 and 0.54 mg d�1 in the presence of glucose and pyruvicacid, respectively. The highest degradation rate was recorded in thepresence of acetate at a rate of 0.8 mg d�1 within about 5 days.

The growth of the GM42 strain in the presence of phenanthrenewith and without the tested cyanobacterial exudates (acetate,pyruvate and glucose)was followedusing protein estimation inorderto check whether the observed increase in phenanthrene degrada-tion rate was due to increased biomass or increased activity of theGM42 strain (Fig. 4). The GM42 strain accumulated more biomass inthe presence of phenanthrene and one cyanobacterial exudate thanin the presence of phenanthrene alone. This difference in growthwasstatistically significant with P-values < 0.05. Maximum growth wasreached already after 4 days in the presence of acetate but took 6e7days in the presence of glucose and pyruvate, after which it remainedmore or less constant. In spite of that, more bacterial biomass wasdetected in the presence of glucose and pyruvate than acetate.

4. Discussion

This study supports earlier reports that cyanobacteria and asso-ciated aerobic heterotrophs constitute an efficient consortium forthe degradation of hydrocarbons (Radwan et al., 2002; Abed andK€oster, 2005; S�anchez et al., 2005). The richness of polluted cyano-bacterial mats in aerobic heterotrophic bacteria with the abilityto grow on aliphatic and aromatic hydrocarbons as well as on cya-nobacterial exudates is congruent with our previous findings (Abedet al., 2007). Our isolates belonged to the genera Marinobacter,Pseudomonas and Sphingomonas, known to include hydrocarbon-degrading species in other ecosystems (Sagard�ıa et al., 1975; Barathiand Vasudevan, 2001; Cohen, 2002; Ying et al., 2004; Leys et al.,2004; Yakimovet al., 2004, 2007).Marinobacter-related specieswerealways encountered in cultures from oil-contaminated cyanobacte-rial mats (Cohen, 2002; McGowan et al., 2004; Abed et al., 2007).The isolation of these strains at higher dilution levels suggests theirnumerical abundance in the studied mat.

Our strains showed also growth on most of the tested cyano-bacterial organic exudates. This suggests that these strains could playan essential role in carbon cycling within cyanobacterial mats. Theyprobably compete with other coexisting non-oil-degrading aerobicheterotrophs for these cyanobacterial metabolites. It has beendemonstrated that aerobic heterotrophs with the ability to grow onoil prefer these organics over oil components because of their simplestructure and no toxicity (Kirkwood et al., 2006). A previous studydemonstrated the occurrence of two functional guilds of aerobicheterotrophs in polluted microbial mats, one degrading the cyano-bacterial exudates but not hydrocarbons and the other degradingboth (Abed et al., 2007). The former group included strains belongingto the generaMarinobacter, Halomonas, Roseobacter and Rhodobacterwhereas the strains from the later group belonged to Marinobacter

T = 0 days

of

the

init

iall

y ad

ded

amou

nt o

f ph

enan

thre

ne

0

20

40

60

80

100

120T = 1 day T = 2 days T = 3 days

T = 4 days

Con No Acet Pyru Gluc Ala But0

20

40

60

80

100

T = 5 days

Con No Acet Pyru Gluc Ala But

T = 6 days

Con No Acet Pyru Gluc Ala But

T = 7 days

Con No Acet Pyru Gluc Ala But

ab

b baa,b

ba,b

b,c

a

ca,bb,c

a

c,d,eb,c

d,e

bc,d

a

b

c, dc,d

b,c

d

c,d

a,b ba

d

cc

e d

b a,b

c

b,cb,c e

a

Fig. 3. Degradation of phenanthrene by the GM42 isolate in the presence of different organic compounds previously identified as cyanobacterial excretion products as followed bygas 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). Differentletters 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.

R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e6462

and Alcanivorax (Jonkers and Abed, 2003; Abed et al., 2007). Suchheterotrophs apparently reside in close proximity to cyanobacteria inthe photic layer of microbial mats in order to ensure a continuousaccess to cyanobacterial exudates as well as to the photosyntheti-cally-produced oxygen.

4.1. Aerobic heterotrophic bacteria and cyanobacterial growth

Synechocystis PCC6803 exhibited similar growth in the presenceand absence of hexadecane, indicating its tolerance but inability togrow on hexadecane, although this strain became dominant inthe original mats after degradation of four petroleum compoundsincluding phenanthrene (Abed et al., 2002). On the contrary, thegrowth of Synechocystis was clearly stimulated in the presence ofthe GM41 strain and more evidently when hexadecane was added.The addition of the aerobic heterotroph might have supported thegrowth of the cyanobacterium by utilizing the photosynthetically-produced carbon compounds, whose accumulation is known toinhibit photosynthesis (Bateson andWard,1988). The oil-degrading

Days0 2 4 6 8 10

0

20

40

60

80

100

120

140

160

180Phenanthrene+acetatePhenanthrene+glucosePhenanthrene+pyruvic acidPhenanthrene

Prot

ein

conc

entr

atio

n (µ

g/m

l)

*

*

* * *

a

a

b

c

d

a*

b

b c

a

b

a,b

a

b

c

a,b

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. Starsindicate the time points where statistically significant differences among differentincubations were detected. Different alphabetic superscripts indicate a significantdifference between incubations at P < 0.05 within each time point using Tukey's test.

types of aerobic heterotrophs reduce the concentrations of toxicaromatics and alkanes around cyanobacteria, which were shown toinhibit photosynthesis, growth and enzyme activity (Megharajet al., 2000). Safonova et al. (1999) demonstrated that the presenceof alkane utilizing bacteria in association with algae and cyano-bacteria restored the growth of sensitive strains exposed toblack oil and stimulated the growth of the tolerant species. Theutilization of cyanobacterial exudates and petroleum compoundsby aerobic heterotrophs in microbial mats results in increased ratesof oxygen consumption, thus reducing the high concentrationsof photosynthetic O2 in mats, which can be toxic to a variety ofmetabolic and biosynthetic pathways (Garcia-Pichel et al., 1999).Using microsensors, the addition of crude oil and its components tomicrobial mats was shown to induce prominent changes in ratesof light respiration, gross and net photosynthesis and pH gradients(Gr€otzschel et al., 2002; Benthien et al., 2004). The respiringbacteria counteract the chemical changes in O2, CO2 and pHinduced by photosynthesis (Wieland and K€uhl, 2006).

Cyanobacteria could feed directly on metabolites (e.g. organicacids) produced upon the degradation of oil components by aerobicheterotrophic bacteria. Alkanes are known to be utilized via theiroxidation to fatty acids that are further oxidized to acetate (Radwanand Al-Hasan, 2000). There are several reports that demonstratedthe heterotrophic growth of several strains of cyanobacteria onorganic compounds like sugars, acetate and glycerol (Rippka, 1972;More et al., 1979; Radwan and Al-Hasan, 2000). Cyanobacteriacould also use the carbon dioxide, formed as the final product ofhexadecane degradation, directly for their photosynthesis.

4.2. The role cyanobacterial exudates in hydrocarbonsdegradation by aerobic heterotrophs

The results of this study showed that cyanobacterial organicexudates play a role in supporting the growth of aerobic heterotrophicbacteria and consequently the degradation of hydrocarbons. Alanineand butanol did not affect the degradation rate of phenanthreneby the GM42 strain, while acetate, pyruvate and glucose exhibiteda stimulatory effect. When phenanthrene and an exudate wereprovided together, it should be expected that that aerobic heterotro-phic bacteria would prefer to utilize easily assimilated, lowmolecularweight, cyanobacterial exudates rather than phenanthrene. A numberof studies have shown that cyanobacterial exudates can be easily

R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e64 63

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, leadingto a significant increase in their numbers, after which they degradehydrocarbons. This explanation is evident from the protein analysiswhich showed around 3e4-fold increase in the bacterial biomasswhen the exudate was added.

Previous studies have demonstrated the significance of cyano-bacterial exudates in accelerating rates of hydrocarbon degradation.Glucose and lipids were shown to enhance degradation of alkanes inpolluted soils from Kuwait (Radwan et al., 2000, 2001). Kirkwoodand his coworkers demonstrated a species-specific effect of cyano-bacterial exudates from four strains on the biodegradation ratesof phenol and dichloroacetate by Pseudomonas and Ancylobacter,respectively (Kirkwood et al., 2006). Exudates fromAphanocapsa andPseudoanabaena spp. repressed the degradation of phenol butenhanced the degradation of dichloroacetate although all exudatessupported the growth of the bacterial strains. The authors attributedthe inhibition of phenol degradation to a toxic rather thana competitive effect of the exudates. In rhizospheres of higher plants,hydrocarbon-degrading bacteria were found in high densitiesbenefiting from the root exudates which include easily assimilatedcompounds like sugars and amino acids (Lee et al., 1995; Radwanet al., 1998). Indeed, the addition of organic fertilizers that containfatty acids, alcohols and sugars was successfully used in the biore-mediation of several polluted sites worldwide including the wellknown Exxon Valdez oil spill in 1989 (Rivet et al., 1993; Bragg et al.,1994; Al-Hadhrami et al., 1996). The use of cyanobacteria in thecleanup of pollution may circumvent the costly use of these fertil-izers by directly supplying the degrading bacteria with necessaryorganics to increase their biomass and with fixed nitrogen that canbe limited in marine environments (Ward and Brock, 1976).

In conclusion, the aerobic heterotrophs-cyanobacterial mat con-sortia offer a cost-effective and promising system for bioremediatingoil-polluted coastal sites. Our study demonstrated that cyanobacteriagrew better in the presence of the associated aerobic heterotrophicbacteria and could provide themwith necessary organics for efficientdegradation activities. Further investigations should extrapolate ourobservations in culture experiments to study this relationship inintact mats and under field conditions. The use of simple organicscompounds like acetate in accelerating biodegradation rates in oil-polluted sediments should be tested in the field. However, it shouldbe noted that these compounds have to be added as a starter atconcentrations enough to induce growth of oil-degrading bacteriawithout enriching specific bacterial populations.

Acknowledgment

I would like to thank Henk Jonkers and Dirk de Beer from theMax-Planck Institute, Bremen, Germany for fruitful discussions andsuggestions. I would also like to thank Florin Musat for his intro-duction to gas chromatography (GC) and J€urgen K€oster for preparingthe organo-clay complexes. Special thanks go to Michael Barry forhis help in the statistical analysis. Natuschka Lee and Derek Robertsare thanked for reviewing the paper. This research was financiallysupported by the Deutsche Forschungsgemeinschaft (grant BE 2167/4) and by the Max-Planck Society.

References

Abed, R.M.M., Safi, N.M.D., K€oster, J., de Beer, D., El-Nahhal, Y., Rullk€otter, J., GarciaPichel, F., 2002. Microbial diversity of a heavily polluted microbial mat and itscommunity changes following degradation of petroleum compounds. Appliedand Environmental Microbiology 68, 1674e1683.

Abed, R.M.M., Al-Thukair, A., de Beer, D., 2006. Bacterial diversity of a cyanobacterialmat degrading petroleum compounds at elevated salinities and temperatures.FEMS Microbiology Ecology 57, 290e301.

Abed, R.M.M., Burhanuddin, Z., Al-Thukair, A., de Beer, D., 2007. Phylogeneticdiversity and activity of aerobic heterotrophic bacteria from a hypersalineoil-polluted mat. Systematic and Applied Microbiology 30, 319e330.

Abed, R.M.M., K€oster, J., 2005. The direct role of aerobic heterotrophic bacteriaassociated with cyanobacteria in the degradation of oil compounds. Interna-tional Biodeterioration and Biodegradation 55, 29e37.

Al-Hadhrami, M.N., Lappin-Scott, H.M., Fisher, P.J., 1996. Effects of the addition oforganic carbon sources on bacterial respiration and n-alkane biodegradation ofOmani crude oil. Marine Pollution Bulletin 32, 351e357.

Amann, R.I., Ludwig, W., Schleifer, K.-H., 1995. Phylogenetic identification and in situdetection of individual microbial cells without cultivation. MicrobiologicalReviews 59, 143e169.

Anderson, K.L., Tayne, T.A., Ward, D.M., 1987. Formation and fate of fermentationproducts in hot-spring cyanobacterial mats. Applied and EnvironmentalMicrobiology 53, 2343e2352.

Barathi, S., Vasudevan, N., 2001. Utilization of petroleum hydrocarbons by Pseudo-monas fluorescens isolated from a petroleum-contaminated soil. EnvironmentInternational 26, 413e416.

Bateson, M.M., Ward, D.M., 1988. Photoexcretion and fate of glycolate in a hot springcyanobacterial mat. Applied and Environmental Microbiology 54, 1738e1743.

Bauld, J., Brock, T.D., 1974. Algal excretion and bacterial assimilation in hot springalgal mats. Journal of Phycology 10, 101e106.

Benthien, M., Wieland, A., de Oteyza, T.G., Grimalt, J.O., K€uhl, M., 2004. Oil-contami-nated effects on a hypersaline microbial mat community (Camargue, France) asstudied with microsensors and geochemical analysis. Ophelia 58, 135e150.

Bragg, G.R., Prince, R.C., Harner, E.J., Atlas, R.M., 1994. Effectiveness of bioremedia-tion for the Exxon V aldez oil spill. Nature 368, 413e418.

Cohen, Y., 2002. Bioremediation of oil by marine microbial mats. InternationalMicrobiology 5, 189e193.

Decho, A., 1990. Microbial exopolymer secretions in ocean environments: their role(s) in food webs and marine processes. Oceanography and Marine Biology: AnAnnual Review 28, 73e153.

Eggink, G., Engel, H., Meijer, W., Otten, J., Kingma, J., Witholt, B., 1988. Alkaneutilization in Pseudomonas oleovorans. Structure and function of the regulatorylocus alkR. The Journal of Biological Chemistry 263, 13400e13405.

Epping, E.H.G., Khalili, A., Thar, R., 1999. Dynamics of photosynthesis and respirationin an intertidal biofilm. Limnology and Oceanography 44, 1936e1948.

Flemming, H.C., Wingender, J., 2001. Relevance of microbial extrapolymericsubstances (EPSs) e part I: structural and ecological aspects. Water Science andTechnology 43, 1e8.

Garcia-Pichel, F., K€uhl, M., N€ubel, U., Muyzer, G., 1999. Salinity dependent limitationof photosynthesis and oxygen exchange in microbial mats. Journal of Phycology35, 184e195.

Gauthier, M.J., Lafay, B., Christen, R., Fernandez, L., Acquaviva, M., Bonin, P.,Bertrand, J.C., 1992. Marinobacter hydrocarbonoclasticus gen. nov., sp. nov., a newextremely halotolerant, hydrocarbon-degrading marine bacterium. InternationalJournal of Systematic Bacteriology 42, 568e576.

Gr€otzschel, S., K€oster, J., Abed, R.M.M., de Beer, D., 2002. Degradation of petroleummodel compounds immobilized on clay by a hypersaline microbial mat.Biodegradation 13, 273e283.

Heijthuijsen, J., Hansen, T.A., 1986. Interspecies hydrogen transfer in co-cultures ofmethanol-utilizing acidogens and sulfate-reducing or methanogenic bacteria.FEMS Microbiology Ecology 38, 57e64.

Hellebust, J., 1965. Excretion of some organic compounds by marine phytoplankton.Limnology and Oceanography 10, 192e206.

Huu, N.B., Denner, E.B.M., Ha, D.T.C., Wanner, G., Stan-Lotter, H., 1999. Marinobacteraquaeolei sp. nov., a halophilic bacterium isolated from a Vietnamese oilproducing well. International Journal of Systematic Bacteriology 49, 367e375.

Jonkers, H.M., Abed, R.M.M., 2003. Identification of aerobic heterotrophic bacteriafrom the photic zone of a hypersaline microbial mat. Aquatic Microbial Ecology30, 127e133.

J�rgensen, B.B., Nelson, D.C., Ward, D.M., 1992. Chemotrophy and decomposition inmodernmicrobial mats. In: Schopf, J.W., Klein, C. (Eds.), The Proterozoic Biosphere:a Multidisciplinary Study. Cambridge University Press, Cambridge, pp. 287e293.

Kirkwood, A.E., Nalewajko, C., Fulthorpe, R.R., 2006. The effects of cyanobacterialexudateson bacterial growth and biodegradation of organic contaminants.Microbial Ecology 51, 4e12.

Lee, K., Tremblay, G.H., Cobanli, S.E., 1995. Bioremediation of oiled beach sediments:assessment of in organic and organic fertilizers. In: Proceedings of the 1991 OilSpill Conference. American Petroleum Institute, Washington, DC, USA,pp. 107e113.

Leys, N.M.E., Ryngaert, A., Bastiaens, L., Verstraete, W., Top, E.M., Springael, D., 2004.Occurrence and phylogenetic diversity of Sphingomonas strains in soilcontaminated with polycyclic aromatic hydrocarbons. Applied and Environ-mental Microbiology 70, 11944e11955.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement withthe Folin-Phenol reagents. The Journal of Biological Chemistry 193, 265e275.

McGowan, L., Herbert, R., Muyzer, G., 2004. A comparative study of hydrocarbondegradation by Marinobacter sp., Rhodococcus sp. and Corynebacterium sp.isolated from different mat systems. Ophelia 58, 271e281.

Megharaj, M., Singleton, I., McClure, N.C., Naidu, R., 2000. Influence of petroleumhydrocarbon contamination on microalage and microbial activities in

R.M.M. Abed / International Biodeterioration & Biodegradation 64 (2010) 58e6464

a long-term contaminated soil. Archives of Environmental Contamination andToxicology 38, 439e445.

More, J.E., William, M.M., Smith, A.J., 1979. Features of organic growth substrateutilization by cyanobacteria. In: Nichols, J.M. (Ed.), Proceedings of the ThirdInternational Symposium on Photosynthetic Prokaryotes. University of Liver-pool, Oxford, p. B49.

Musat, F., Harder, J., Widdel, F., 2006. Study of nitrogen fixation in microbialcommunities of oil-contaminated marine sediment microcosms. EnvironmentalMicrobiology 8, 1834e1843.

Muyzer, G., Teske, A., Wirsen, C.O., Jannasch, H.W., 1995. Phylogenetic relationshipsof Thiomicrospira species and their identification in deep-sea hydrothermal ventsamples by denaturing gradient gel electrophoresis of 16S rDNA fragments.Archives of Microbiology 164, 165e172.

Nold, S.C., Ward, D.M., 1996. Photosynthate partitioning and fermentation in hotspring microbial mat communities. Applied and Environmental Microbiology62, 4598e4607.

de Oteyza, T.G., Grimalt, J.O., 2004. Molecular composition of the gas chromatog-raphy amenable fractions of Maya crude oil. A reference oil for microbialdegradation experiments. Ophelia 58, 233e242.

Pruesse, E., Quast, C., Knittel, K., Fuchs, B.M., Ludwig, W., Peplies Jr., , Gl€ockner, F.O.,2007. SILVA: a comprehensive online resource for quality checked and alignedribosomal RNA sequence data compatible with ARB. Nucleic Acids Research 35,7188e7196.

Quinn, G.P., Keough, M.J., 2002. Experimental Design and Data Analysis for Biolo-gists. University Press, Cambridge.

Radwan, S.S., Al-Hasan, R.H., 2000. Oil pollution and cyanobacteria. In:Whitton, B.A.,Potts, M. (Eds.), The Ecology of Cyanobacteria. Kluwer, The Netherlands,pp. 307e319.

Radwan, S.S., Al-Awandhi, H., Sorkhoh, N.A., El-Nemr, I.M., 1998. Rhizospherichydrocarbon utilizing microorganisms as potential contributors to phy-toremediation for the oily Kuwaiti desert. Microbiological Research 153,247e251.

Radwan, S.S., Al-Mailem, D., El-Nemr, I., Salamah, S., 2000. Enhanced remediation ofhydrocarbon contaminated desert soil fertilized with organic carbons. Inter-national Biodeterioration and Biodegradation 46, 129e132.

Radwan, S.S., Al-Aawadi, H.A., Khanafer, M., 2001. Effects of lipids on n-alkaneattenuation in media supporting oil utilizing microorganisms from the oilyArabian Gulf coasts. FEMS Microbiology Letters 198, 99e103.

Radwan, S.S., Al-Hasan, R.H., Salamah, S., Al-Dabbous, S., 2002. Bioremediation ofoily seawater by bacteria immobilized in biofilms coating macroalgae. Inter-national Biodeterioration and Biodegradation 50, 55e59.

Rippka, R., 1972. Photoheterotrophy and chemoheterotrophy among unicellularblue-green algae. Archives of Microbiology 87, 93e98.

Rippka, R.,1988. Isolation and purification of cyanobacteria. In: Packer, L., Glazer, A.N.(Eds.), Methods in Enzymology: Cyanobacteria. Academic, New York, New York,pp. 3e27.

Rivet, L., Mille, G., Basseres, A., Ladousse, A., Gerin, C., Acquaviva, M., Bertrand, J.-C.,1993. n-Alkane biodegradation by a marine bacterium in the presence of anoleophilic nutriment. Biotechnology Letters 15, 637e640.

Safonova, E.T., Dmitrieva, I.A., Kvitko, K.V., 1999. The interaction of algae withalcanotrophic bacteria in black oil decomposition. Resources, Conservation andRecycling 27, 193e201.

Sagard�ıa, F., Rigau, J.J., Mart�ınez-Lahoz, A., Fuentes, F., Waldemar, C., Flores, W., 1975.Degradation of benzothiophene and related compounds by a soil Pseudomonas in anoileaqueous environment. Applied and Environmental Microbiology 29, 722e725.

S�anchez, O., Diestra, E., Esteve, I., Mas, J., 2005. Molecular characterization of an oildegrading cyanobacterial consortium. Microbial Ecology 50, 580e588.

S�anchez, O., Ferrera, I., Vigues, N., de Oteyza, T.G., Grimalt, J., Mas, J., 2006. Role ofcyanobacteria in oil biodegradation by microbial mats. International Biodete-rioration and Biodegradation 58, 186e195.

Stal, L.J., 1995. Physiological ecology of cyanobacteria in microbial mats and othercommunities. New Phytologist 131, 1e32.

Stal, L.J., van Gemerden, H., Krumbein, W.E., 1984. The simultaneous assay ofchlorophyll and bacteriochlorophyll in natural microbial communities. Journalof Microbiological Methods 2, 295e306.

Stal, L.J., Moezelaar, R., 1997. Fermentation in cyanobacteria. FEMS MicrobiologicalReviews 21, 179e211.

Tian, L., Ma, P., Zhong, J.-J., 2002. Kinetics and key enzyme activities of phenanthrenedegradation by Pseudomonas mendocina. Process Biochemistry 37, 1431e1437.

Ward, D.M., Brock, T.D., 1976. Environmental factors influencing the rate ofhydrocarbon oxidation in temperate lakes. Applied and Environmental Micro-biology 31, 764e772.

Widdel, F., Bak, F., 1992. Gram-negative mesophilic sulfate reducing bacteria. In:Balows, A., Tr€uper, H.G., Dworkin, M., Harder, W., Schleifer, K.H. (Eds.), TheProkaryotes. Springer, New York, pp. 3353e3378.

Wieland, A., K€uhl, M., 2006. Regulation of photosynthesis and oxygen consumptionin a hypersaline cyanobacterial mat (Camargue, France) by irradiance,temperature and salinity. FEMS Microbiology Ecology 55, 195e210.

Yakimov, M.M., Gentile, G., Bruni, V., Cappello, S., D'Auria, G., Golyshin, P.N.,Giuliano, L., 2004. Crude oil-induced structural shift of coastal bacterialcommunities of rod bay (Terra Nova Bay, Ross Sea, Antarctica) and character-ization of cultured cold adapted hydrocarbonoclastic bacteria. FEMS Microbi-ology Ecology 49, 419e432.

Yakimov, M.M., Timmis, K.N., Golyshin, P.N., 2007. Obligate oil-degrading marinebacteria. Current Opinions in Biotechnology 18, 257e266.

Yen, K.M., Karl, M.R., Blatt, L.M., Simon, M.J., Winter, R.B., Fausset, P.R., Lu, H.S.,Harcourt, A.A., Chen, K.K., 1991. Cloning and characterization of a Pseudomonasmendocina KR1 gene cluster encoding toluene-4-monooxygenase. Journal ofBacteriology 173, 5315e5327.

Ying, X.I.A., Hang, M.I.N., Zhen-mei, L.U., Yang-fang, Y.E., 2004. Characterization andphylogenetic analysis of a phenanthrene-degrading strain isolated from oilcontaminated soil. Journal of Environmental Sciences 16, 589e593.