Environment International 51 (2013) 59–72

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Review

Mixotrophic cyanobacteria and microalgae as distinctive biological agents for organicpollutant degradation

Suresh R. Subashchandrabose a, Balasubramanian Ramakrishnan a,b, Mallavarapu Megharaj a,⁎,Kadiyala Venkateswarlu a,c, Ravi Naidu a

a Centre for Environmental Risk Assessment and Remediation, University of South Australia, SA5095, Australia, and Cooperative Research Centre for Contamination Assessment and Remediation ofEnvironment, PO Box 486 Salisbury South, SA5106, Australiab Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, Indiac Department of Microbiology, Sri Krishnadevaraya University, Anantapur 515055, India

⁎ Corresponding author at: Environmental BiotechnoMawson Lakes Campus, Mawson Lakes, South Australi

E-mail address: megharaj.mallavarapu@unisa.edu.au

0160-4120/$ – see front matter © 2012 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.envint.2012.10.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 15 June 2012Accepted 24 October 2012Available online xxxx

Keywords:CyanobacteriaMicroalgaeMixotrophyBiological agentsOrganic pollutantsBiodegradation

Millions of natural and synthetic organic chemical substances are present in both soil and aquatic environments.Toxicity and/or persistence determine the polluting principle of these substances. The biological responses tothese pollutants include accumulation and degradation. The responses of environments with organic pollutantsare perceptible from the dwindling degradative abilities of microorganisms. Among different biologicalmembers, cyanobacteria and microalgae are highly adaptive through many eons, and can grow autotrophically,heterotrophically ormixotrophically.Mixotrophy in cyanobacteria andmicroalgae can providemany competitiveadvantages over bacteria and fungi in degrading organic pollutants. Laboratory culturing of strict phototrophicalgae has limited the realization of their potential as bioremediation agents. In the natural assemblages,mixotrophic algae can contribute to sequestration of carbon, which is otherwise emitted as carbon dioxide tothe atmosphere under heterotrophic conditions by other organisms.Molecularmethods andmetabolic and geno-mic information will help not only in identification and selection of mixotrophic species of cyanobacteria andmicroalgae with capabilities to degrade organic pollutants but also in monitoring the efficiency of remediationefforts under the field conditions. These organisms are relatively easier for genetic engineering with desirabletraits. This review presents a new premise from the literature that mixotrophic algae and cyanobacteria aredistinctive bioremediation agents with capabilities to sequester carbon in the environment.

© 2012 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602. Soil algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

2.1. Diversity and biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602.2. Phototrophy versus heterotrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612.3. Mixotrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

3. Cyanobacterial/Microalgal degradation of organic pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623.1. Phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633.2. Oil, petroleum and hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643.3. Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653.4. Polyaromatic hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663.5. Polychlorinated biphenyls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

4. Genetic engineering of cyanobacteria/microalgae for enhanced biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

logy, Centre for Environmental Risk Assessment and Remediation, University of South Australia, X Building, Room X1-14,a, SA5095, Australia. Tel.: +61 8 8302 5044; fax: +61 8 8302 3057.(M. Megharaj).

rights reserved.

60 S.R. Subashchandrabose et al. / Environment International 51 (2013) 59–72

1. Introduction

Around 10 million species of cyanobacteria and eukaryotic algaeexist on the Earth (Guiry and Guiry, 2012). They are often consideredto prefer aquatic environments, but they grow under numerousnon-aqueous terrestrial habitats including soils, rocks, caves, modernbuildings, and even living animals and plants (Hoffmann, 1989). Theubiquity of algae in these habitats, besides freshwater, estuaries,and oceanic environments, and evidence of their presence in numer-ous stromatolites of the Archaean and the Mesozoic era (Riding,2011) give credence to the theory that they are one of the first organ-isms to appear on the Earth. These organisms are considered to havecontributed to the oxygenation of atmosphere and ocean over theProterozoic era (Jorgensen, 2001). Algae contribute to the functioningof even the non-aqueous ecosystems by their phototrophic nutritionproducts, generation of organic matter from inorganic substances,various biologically active compounds which influence other mem-bers of the communities, and serving as a source of food for bacteriaand invertebrates. Filamentous heterocyst-forming genera and somenon-heterocystous cyanobacteria are capable of fixing atmosphericnitrogen. In many parts of the deserts worldwide, the filamentouscyanobacteria are found to initialize the first step of colonization,due to their ability to withstand high temperatures, radiation, andlow water potential (Zaady et al., 2000). These photosyntheticallyactive organisms, even if they form a thin-layer of an extensivecover on soils can contribute significantly to carbon fixation. Withtheir growth on the surface or at a depth of up to several centimeters,the activities of algae enhance soil formation, water retention, stabili-zation, increased availability of nutrients for plant growth and reduc-tion in soil erosion.

Soils are a major natural reservoir of gene pool of many life-formsincluding those of cyanobacteria and microalgae. Thin-layer of soilcovering the surface of the Earth supports the survival of mostland-based life-forms (Doran et al., 1996). In terms of carbon (C),the soils harbor about 1500–1600 Pg in the upper first meter, whichis two-fold more than that of above ground C pool (Jobbágy andJackson, 2000). About 60 Pg of atmospheric C is photosynthesizedby various biota including cyanobacteria and microalgae, and this Cfixation contributes significantly to the organic C content of soils. Atpresent, the atmospheric C pool is increasing at the expense ofother C pools such as geologic, pedologic and biotic pools. Especially,since the onset of industrial revolution, the reliance on fossil fuels hasincreased and led to the disturbances in the global C cycle with ensu-ing climate change effects. Intensive agricultural activities also accel-erate the loss of soil C to the atmospheric C pool. Sequestration of C bythe net removal of CO2 from the atmosphere into the terrestrial poolwhich includes both soil and biotic C, and the geological C pool offerspromise as a mitigation option to the adverse effects of industrial rev-olution as well as intensive agricultural activities on the globalC cycle. In this context, photosynthetic activities of algae can makesignificant impact in soil C pool too. The modern civilization whichis termed ‘the Carbon civilization’ or the C-Era (Lal, 2007) has alsoled to the manufacturing of numerous C-based synthetic chemicalsubstances using fossil fuels as the base substance. While the fossilfuels (crude oil, natural gas and coal) are the products of deep burialof fossilized remains of plants and animals, some of these organiccompounds, both natural and synthetic, are toxic while others arehazardous to various forms of life. Paradoxically, these chemical sub-stances reach soils when soils are used for disposal of industrialwastes or these chemical substances are accidentally spilled over.Even the catabolic capabilities of heterotrophic microorganisms todegrade the naturally occurring organic chemical substances in soilsare now seriously challenged by the introduction and accumulationof these synthetic organic chemical substances, which can be toxicand hazardous too (Megharaj et al., 2011; Watanabe, 2001). Thenon-target effects of organic pollutants on autotrophic microalgae

and cyanobacteria have been reviewed by many researchers (Padhy,1985; Ramakrishnan et al., 2010, 2011; Venkateswarlu, 1993).

Algae, bacteria, and fungi possess catabolic genes for degradingmany pollutants in soils (Pinyakong et al., 2000; Potin et al., 2004;Semple et al., 1999). Many bacterial species and fungal species tosome extent have been investigated and applied as bioremediationagents. Bacteria as bioremediation agents provide many advantagesdue to faster acclimation and growth rates, higher surface to volumeratio, natural capabilities for horizontal gene transfer for many cata-bolic enzymes, and ease with which these organisms can be geneti-cally engineered. Nevertheless, they require higher energy and/or Ccosts, and nutritional sources. More importantly, complete minerali-zation of organic pollutants by heterotrophic bacteria and fungiproves to be disadvantageous since the fixed carbon finally reachesthe atmospheric C pool, which is increasing alarmingly due to theuse of fossil fuels for other human activities. The innate potential ofcyanobacteria and microalgae for bioremediation has not been fullyrealized by the environmentalists. Recently, the phycoremediationby microalgae has been attempted for various pollutants (Mallick,2002; Mehta and Gaur, 2005; Olguín, 2003). Cyanobacterial/microalgal culture for bioremediation can overcome some of themajor limitations associated with bacteria and fungi that require car-bon and other nutrients in stoichiometric balance for growth anddegradation of pollutants. Based on the progress on researches relat-ed to cyanobacterial/algal degradation of organic pollutants, we pres-ent a premise in this review that mixotrophic cyanobacteria and algaewith dual abilities of renewable energy capture by CO2 fixationthrough photosynthesis and degradative effect on organic pollutantscan be distinctive bioremediation agents.

2. Soil algae

2.1. Diversity and biomass

Cyanobacteria, taxonomically grouped under the Gram-negativeprokaryotes by microbiologists, and as a Division in Plant Kingdomby botanists (Stanier et al., 1971), are found in diverse forms ofunicells of 1–2 μm in diameter to filaments of even 10 cm, and canexist in habitats ranging from oceans to estuaries, freshwater bodies,and non-aqueous terrestrial ecosystems. Among prokaryotes, photo-synthetic capability is present in cyanobacteria, Firmicutes, Chloroflexi,Chlorobi and proteobacteria (Gupta, 2003). Plants and some uni-cellular protists are secondarily photosynthetic due to the presenceof chloroplasts that have originated from cyanobacteria. Whilecyanobacteria can carry out oxygenic photosynthesis, all other photo-synthetic bacteria perform anoxygenic photosynthesis. In addition,some of cyanobacterial species are also capable of asymbioticnitrogen fixation and contribute to the nitrogen economy of soils.Microalgae are unicellular microscopic phytoplanktonic specieswhich include cyanobacteria, diatoms, dinoflagellates and green fla-gellates, and are of sizes from1–2 μm to 2 mm (Hallegraeff, 2002).Based on the International Code of Botanical Nomenclature (Greuteret al., 1994), the phycologists consider microalgae to be of both eu-karyotic and prokaryotic cell types (Mur et al., 1999). The DNA se-quence data suggest that algal phylogeny encompasses ten majorphyla (prokaryotic Cyanophyta and Prochlorophyta, and the eukaryot-ic Glaucophyta, Euglenophyta, Cryptophyta, Haptophyta, Dinophyta,Heterocontophyta (including diatoms, brown algae), Rhodophyta(red algae), and Chlorophyta (green algae)). They are also ubiquitousand comprise a substantial proportion of microbial biomass in soils. Inthe present review, both cyanobacteria and microalgae are consid-ered and referred to as algae. Numerically, the algal biomass in tem-perate lands is estimated to be from 0 to 108 cells g−1 soil (dry wt.)(Lukešová, 2001) or from 0 to 108 cells m−2 (Lukešová andHoffmann, 1996). Boul et al.(1972) suggested a mean value of 10 kgalgal biomass, on hectare basis, in a temperate soil. No such estimates

61S.R. Subashchandrabose et al. / Environment International 51 (2013) 59–72

are available for other soil types including those in tropical areasprobably because there are no standard methods for enumeratingalgal biomass. Nevertheless, enormous diversity of these organismsin different ecosystems is evident from the morphological and molec-ular sequence data-based studies (Lewis and Lewis, 2005; Řehákováet al., 2011). Traditional enrichment protocols or culture-basedmethods may miss a number of species of low abundance.Amaral-Zettler et al. (2009) suggested that these taxa with speciesof low abundance belong to the so-called microbial ‘Rare Biosphere.’Some of the species of microbial ‘Rare Biosphere’ can grow abundant-ly when the environmental conditions change and can carry out spe-cific functions (Caron and Countway, 2009).

The algal species are highly adaptive and can grow up to a depth ofa few centimeters in soil, and from arid to saline conditions. In fact,their preference to aquatic environments is despite the limitationon the availability of CO2 due to low CO2 diffusion rates in water bod-ies, which these algae overcome by carbon concentrating mecha-nisms. Additionally, these oxygenic phototrophic organisms in soilsor microbial mats encounter hypoxic or anaerobic conditions. Underthese conditions, the O2-dependent biosynthetic pathways will beimpaired due to poor evolution of O2 even though photophosphoryla-tion may provide energy to survive in the light. In comparison to thesessile higher plants, algae have faster growth rates and capacities tocolonize many surfaces, which attenuate competition for resources(Stephens et al., 2010).

Algae are well adapted to survive desiccation too. In the desert, bo-real and arctic ecosystems, they form an important component of bio-logical crusts. These crusts exhibit poikilohydric character, welladapted to the nature and the timing of water supply (Zhao et al.,2009). In many countries, these algae have been introduced as soil con-ditioners (Metting, 1981) and as biofertilizers (Venkataraman, 1979).Algal response to chemical substances in the environments can varyfrom doom to bloom. Even the application of chemical fertilizers for ag-ricultural purpose shows transient and permanent effects on algal com-munities in many soils (Paoletti et al., 1992). Kuzyakhmetov (1998)showed that the lengthy periods of intense application of chemical fer-tilizers led to reductions in algal diversity and its abundance.

2.2. Phototrophy versus heterotrophy

Photosynthesis in algae enables them to harness light energy intofree chemical energy for metabolic purposes. Among the prokaryotes,only cyanobacteria can carry out oxygenic photosynthesis sincethey contain two different reaction centers (Fe–S type (PSI) andpheophytin-quinone type (PSII)). Photosynthesis is an important reg-ulator of atmospheric CO2 and ribulose-1,5-bisphosphate carboxylaseis the rate limiting enzyme in photosynthesis. The phylogenetic rela-tionships based on the large subunit of the ribulose-bisphosphate car-boxylase gene (rbcL) show the evolutionary lineages amongphotoautotrophs (Fig. 1). The evolutionary hardiness of cyanobacteriais attributed to their abilities to perform photosynthesis and respira-tion simultaneously in the same compartment (Vermaas, 2001). Thealgae can use only up to 9% of incoming solar energy. Theoretically,microalgae can produce 280 tons of dry biomass ha−1 year−1 withthe sequestration of 513 tons of CO2 (Bilanovic et al., 2009). Sincethe CO2 fixation efficiency of microalgae is about 10–50 times fasterthan that of higher plants, they can grow much faster (Wang et al.,2008). Algal biomass has only a part of the carbon fixed while manyC-containing compounds of volatile organic substances, polysaccha-rides, hormones, organohalogens and others are released extracellu-larly. In soils, the availability of carbon is an important limitingfactor for both autotrophs (in terms of CO2) and heterotrophs (asfixed carbon compounds). Besides, the availability of light may alsolimit the autotrophic growth. Both light intensity and temperatureare the two major determinants of algal growth rate and productivity(Richmond, 1992). Temperature during day exerts major effect on

productivity, and the cessation of growth after midday is attributedto photoinhibition. These limitations make many photosyntheticalgae to obtain C in a heterotrophic manner. In nature, algal auto-trophs with facultative heterotrophy or heterotrophs that are faculta-tively autotrophic can coexist.

The dichotomy of autotrophic and heterotrophic algae is largelydue to the human need for the conceptual clarity. For decades,heterotrophic growth of many algae is known (Droop, 1974). Hetero-trophic energy generation in cyanobacteria is due to the oxidativepentose phosphate (OPP) pathway, the glycolytic pathway and tricar-boxylic acid (TCA) cycle. Economic advantage of heterotrophicgrowth over the phototrophic growth in mass-producing microalgaehas been noted by Borowitzka (1999). Ogbonna et al. (2000) reportedthat the efficiency of nutrient removal by Rhodobacter sphaeroides andChlorella sorokiniana was higher under aerobic dark heterotrophicconditions. The environmental benefits of many heterotrophic algaeinclude their employment as biological agents for treating wastewa-ters from municipal, agricultural or industrial activities (Pittmanet al., 2011). Not all organic chemical substances in the polluted orunpolluted environments can be used since some are known to inhib-it algal growth (Chen, 1996). In the soil environments, the microbialcommunities have complex interactions involving carbon fixation aswell as excretion, and nutrient cycling, whose repercussions for coex-istence of all the members within a community are yet to be clearlyunderstood.

2.3. Mixotrophy

Mixotrophy is defined as a growth regime in which CO2 and organiccarbon are simultaneously assimilated. The assimilation of organic car-bon can be through either phagotrophy or osmotrophy. For the purposeof organic pollutant degradation, mixotrophs employing osmotrophyfor organic carbon chemical substances are relevant. Since the concen-tration of dissolved inorganic carbon (DIC) is less in many water envi-ronments, the majority of phytoplankton obtains DIC by osmotrophy,which actually facilitates autotrophic assimilation. Biochemically, bothphotosynthesis and organic carbon catabolism (respiration) can haveopposing influences on each other. Without forming a unique group,algal species of different physiological guises belonging to differenttaxonomic groups have the capabilities to grow mixotrophically.There are many physiological trade-offs, which are due to the limitedcell surface area, the need for transporter sites for both organic and in-organic sources, the internal cellular components for both autotrophyand heterotrophy, and increased metabolic costs (Litchman et al.,2007; Raven, 1997). Crane and Grover (2010) opined that mixotrophsas generalists utilize resources which are shared with two specialistssuch as autotrophs and heterotrophs.

The first report on mixotrophic microorganisms was provided wayback in 1917 (Pascher, 1917). Mixotrophy, which can offer competi-tive advantage over strict phototrophs and heterotrophs, has beenobserved from oligotrophic habitats to eutrophic estuaries (Jones,2000). Until recently, mixtrophic algae in the nature and their ecolog-ical importance have received less attention, largely because of rigorrequired in doing scientific experiments in the laboratory with purecultures of algae. Low light and/or nutrient deficiency are reportedto encourage mixotrophy (Stoecker et al., 2006). These conditionsoccur commonly in the polluted environments, with availability oflight limited to the surface and nutrients out of stoichiometric bal-ance. Species diversity and equally diverse array of pollutants andtheir degraded products make not only their characterization butalso in understanding the role of mixotrophic algae in pollutant deg-radation difficult.

Photoautotrophic (in the light), heterotrophic (on glucose) andmixotrophic (simultaneously in the light and on glucose) growths ofSpirulina sp. were reported by Chojnacka and Noworyta (2004). Themixotrophic culture of Spirulina sp. had the highest specific growth

Rhodothermus marinus(8568548)

Microalgae

Cyanobacteria

Nephroselmis olivacea (801942)

Chlorella vulgaris (809164)

Leptosira terrestris (5383822)

Chlamydomonas reinhardtii (2717040)

Scenedesmus obliquus (4099835)

Solanum lycopersicum (3950383)

Nicotiana tabacum (800513)

Psilotum nudum (2545153)

Anthoceros formosae (2553411)

Cyanophora paradoxa (801657)

Gloeobacter violaceus (2600557)

Thermosynechococcus elongatus(1011208)

Synechococcus elongatus (3773598)

Nostoc sp. (1105116)

Anabaena variabilis (3683580)

Thalassiosira pseudonana (7447414)

Odontella sinensis (801798)

Gracilaria tenuistipitata var. liui(2944080)

Porphyra yezoensis (3978792)

Rhodobacter sphaeroides(7354137)

Rhodopseudomonas palustris(3907929)

52

99

9976

99

99

53

97

99

9999

72

99

99

99

99

53

0.05

7

Higher plants

Diatoms

Macroalgae

Photosynthetic bacteria

Fig. 1. Phylogeny of RuBisCo large subunit.

62 S.R. Subashchandrabose et al. / Environment International 51 (2013) 59–72

rate of 0.055 h−1. Photoinhibition at a light intensity above 50 W m−2

was observed in the photoautotrophic culture. Such photoinhibitory ac-tion of light was not observed in themixotrophic culture, whichmay bedue to the protective influence of glucose or shift in photoinhibitorylight intensity. Physical (light) and chemical (organic chemical sub-stance) growth substrates makemixotrophic specific growth rate to fol-low multiplicative growth kinetics (Chojnacka and Noworyta, 2004).Practical aspects such as growth kinetics and biomass/bioproduct yieldof mixotrophs are often neglected while screening for species withmixotrophy or demonstrating the use of different substrates (Garcı́aet al., 2005). According to Chen (1996) and Zhang et al. (1999) themixotrophic culture is a dual limiting process. Low light intensities orlow organic carbon substrate concentrations as well as high light inten-sities or high carbon substrate concentrations may inhibit cell growth.Although Marquez et al. (1993) opined that the specific growth rate ofmixotrophs is a simple combination of autotrophic and heterotrophicgrowth rates, it is not necessarily so for many algal species (Chojnackaand Noworyta, 2004). Interestingly, Foster and Chrzanowski (2012)proposed a simple defined medium for growth and maintenance ofOchromonas danica, a eukaryotic alga credited with the meta-cleavagepathway for aromoatic ring degradation of phenol for the first time bySemple and Cain (1996), and the medium enables the studies of itsmixotrophic metabolism under controlled conditions.

Algal cultures under mixotrophic conditions can provide highgrowth rates and biomass with photosynthetic metabolites (Leeand Lee, 2002). Even in the traditionally viewed heterotrophicwaste-treatment systems, cyanobacteria promote bacterial degradationof organic contaminants by oxygen supply or alternative organicsubstrates for bacterial growth or antimicrobial substances (Kirkwood

et al., 2005). Pittman et al. (2011) suggested that organic chemical sub-stances of wastewaters can support mixotrophic growth of algae. Vari-ous byproducts of industrial processes such as acetate, ethanol, andglycerol can support the mixotrophic growth (Heredia-Arroyo et al.,2010; Lee, 2007; Tolga et al., 2010). Although excess CO2 can enhancealgal photosynthetic productivity (Sforza et al., 2010), metabolism oforganic substrates can be blocked under mixotrophic conditions(Sforza et al., 2012). Heterotrophic bacteria need various organic nutri-ents for growth and degrading pollutants. Algal capacity to growmixtrophically during periods of low nutrient concentrations andtheir tolerance to extreme environmental conditions can be a competi-tive advantage over heterotrophs or autotrophs as bioremediationagents. A good strategy to degrade organic pollutants in soil environ-ments is to enrich and isolate the pollutant-degrading mixotrophicalgae, because of their dual capabilities of CO2 assimilation and utiliza-tion of pollutants as organic carbon substances.

3. Cyanobacterial/Microalgal degradation of organic pollutants

In the Chemical Abstracts Service (CAS) Registry System, there aremore than 66 million organic and inorganic substances, with about12,000 new substances being added daily (CAS, 2012). Severalmillions of natural and synthetic compounds are formed with carbonas a constituent element. Human endeavor to synthesize numerousorganic compounds is wrought with the paradox of saving manylives and providing economic benefits to many others while acuteand chronic toxicity of some of these chemical substances makemany others including plants and animals to suffer (Adeola, 2004).Understanding the environmental fate of these chemical substances

63S.R. Subashchandrabose et al. / Environment International 51 (2013) 59–72

is a challenge to human intelligence. Adverse effects and persistentnature of these chemical substances are beginning to be understoodnow. Many a time, chemical substances travel far and wide. Thesechemical substances not only travel through trade channels but alsoacross the atmosphere. Through cycles of volatilization and conden-sations, persistent organic pollutants (POPs) undergo evaporationand atmospheric cycling in warmer climates while condensationand deposition occur in colder climates. The ‘grasshopper effect’ ofPOPs makes them travel all over the Earth (Koziol and Pudykiewicz,2001). When they are degraded slowly, they persist in the environ-ment or get accumulated in organisms. About 25% of organic chemicalsubstances have two or more enantiomers and chirality even deter-mines bioactivity and toxicity (Williams, 1996). Factors such as thestructure and concentration of chemical substances, pH, temperatureand nutrient availability of the medium, cell size, number andphysiological capabilities of microorganisms influence the uptake,bioconcentration and degradation of many chemical pollutants. Or-ganic pollutants that are potential targets for algal degradation in-clude phenolics, pesticides, polyaromatic hydrocarbons (PAHs), andpolychlorinated biphenyls (PCBs), and these chemical substancesare used in industries (Table 1) and agriculture (Table 2). Scientificevidence on the degradation of organic pollutants by microalgaeand cyanobacteria, especially on the capabilities of mixotrophs, lagsfar behind that of bacteria.

3.1. Phenolics

Phenol and its derivatives, which consist of a hydroxyl group(−OH) bonded directly to an aromatic hydrocarbon, are categorizedunder alcohol and more of acidic in nature due to tight coupling ofaromatic ring with oxygen and a relatively loose bond between oxy-gen and hydrogen. Phenolics are a group of dangerous toxic organicpollutants that are toxic to all the living organisms even at lower con-centrations (Pimentel et al., 2008). At higher concentrations, it is verydifficult to remove them from the environment, even by using phys-ical and chemical techniques (Araña et al., 2001). Algal strains are ca-pable of metabolizing phenol in the environment and the mechanistic

Table 1Cyanobacterial/Microalgal degradation of organic pollutants of industrial origin.

Organic pollutant Metabolic by-products

Phenol Pyruvate and CO2

Tributyltin (TBT) Dibutyltin (DBT),Monobutyltin (MBT)

Benzo[a]pyrene (BaP) cis-4,5-, 7,8-, 9,10- and 11,12-BaP-dihydrodiols (under gand 6,12-quinone (under white light)

Phenanthrene (PHE) 1-, 2-, 3-, and 9-Hydroxy- phenanthrene and two DihydNaphthalene 1-Naphthol

1-Naphthalenesulfonic acid 1-Hydroxy-2- naphthalene- sulfonic acid, 1-Naphtholand 1-Naphthyl β-D-glucopyranoside

1-Methylnaphthalene 1-Hydroxymethylnaphthalene

2-Methylnaphthalene 2-Hydroxymethylnaphthalene

2,4,6-Trinitrotoluene Hydroxylamino dinitrotoluene 2-2′ Azy-TeNT, 2-4′ Azy-Dibenzofuran Four Monohydroxylated dibenzofurans and two DihydroDibenzo-p-dioxin 2-Hydroxydibenzo-p-dioxin and two Dihydroxylated diBisphenol Monohydroxy bisphenolBisphenol A Bisphenol A-mono-O-β-D-glucopyranoside (BPAGlc)

Bisphenol A Bisphenol A-mono-O-β-D-galactopyranoside (BPAGal)Biphenyl 4-HydroxybiphenylDimethyl phthalate Phthalic acidSinapic acid 4-Hydroxy-3,5- dimethoxybenzoic acid, 4-Hydroxy-3,5-

benzaldehyde and 4-Hydroxy-3,5-dimethoxy benzylic aBenzotropolone

Azo compounds Aromatic amine

dynamic energy budget model proposed by Lika and Papadakis(2009) for aerobic degradation suggests that inhibition may occur inthe presence of growth-enhancing carbon source like glucose, due tocompetition for oxygen. Report of Klekner and Kosaric(1992b) showedthat Chlorella sp. metabolized 1000 mg L−1 of 2,4-dimethyl phenol toan isomer of dimethyl benzenediol, and with an algal cell concentrationof 4 g L−1, complete degradation was achieved. The Chlorella sp. alsodechlorinated 200 mg L−1 of 2-chlorophenol while Scenedesmus sp.degraded 190 mg L−1 of 2,4-dinitrophenol (2,4-DNP) after five daysof adaptation (Klekner and Kosaric, 1992a). When grown in the pres-ence of 2,4-dimethyl phenol, 2-chlorophenol and 2,4-dichlorophenol,Chlorella sp. effectively degraded 2,4-dichlorophenol, relative to othertwo phenols. Inducible intracellular enzymes like polyphenol oxidaseand laccase enzymes are involved in the algal metabolism of phenol.Megharaj et al. (1992) showed that mixotrophic (photoheterotrophic)growth of microalgal culture alleviated the toxic effect of phenolic pol-lutants, compared to either phototrophic or heterotrophic growth con-ditions. Addition of glucose to culture medium alleviated the toxicity ofPNP and MNP to Chlorella vulgaris (Megharaj et al., 1988). Mixotrophicgrowth conditions improved the ability of microalgae to mineralizephenolic compounds (Tikoo et al., 1997).

The eukaryotic alga,O. danica, can grow heterotrophically in theme-dium containing o- or p-cresols, 2,5-, 2,6-, 3,4-, and 3,5-xylenols(Semple, 1998; Semple and Cain, 1995, 1997). With the expressionof phenol monooxygenase and catechol 2,3-dioxygenase in themeta-cleavage pathway for phenol catabolism, O. danica can convertphenol to pyruvate and CO2, and catechol to 2-hydroxy muconatesemialdehyde and 4-oxalocrotonate as the key intermediates. Carbonassimilation from phenol was evident from the presence of 14C in pro-tein, nucleic acid and lipid contents, when O. danica was grown in themedium containing [U14-C]phenol (Semple and Cain, 1996).Microalgae,Ankistrodesmus braunii and Scenedesmus quadricauda, degraded differ-ent phenols such as catechol, tyrosol, hydroxytyrosol, p-hydroxybenzoic acid, ferulic acid, p-coumaric acid, synaptic acid, caffeic acidand vanillic acid, by about 70% of 400 mg phenolic compounds mL−1

within 10 days (Pinto et al., 2002). Light and dark regimes influencethe degradation potential of algae. When grown in light, Chlorella fusca

Cyanobacterium/Microalga(Reference)

Ochromonas danica (Semple and Cain, 1996)Chlorella vulgaris Chlorella sp. (Tsang et al., 1999)Chlorella miniata (Tam et al., 2002)

old light)1,6-, 3,6- Selanastrum capricornutum (Warshawsky et al., 1988)S. capricornutum (Schoeny et al., 1988)

roxylated PHEs S. capricornutum (Chan et al., 2006)Agmenellum quadruplicatum (Cerniglia et al., 1979)Chlorella vulgaris (Todd et al., 2002)Scenedesmus obiquus (Kneifel et al., 1997)

A. quadruplicatum Oscillatoria sp., Anabaena sp.(Cerniglia et al., 1983)A. quadruplicatum, Oscillatoria sp. Anabaena sp.(Cerniglia et al., 1983)

TeNT and 4-4′ Azy-TeNT Anabaena sp. (Pavlostathis and Jackson, 1999)xylated dibenzofurans Ankistrodesmus sp. (Todd et al., 2002)

benzo-p-dioxin Scenedesmus sp. (Todd et al., 2002)Chlorella fusca (Hirooka et al., 2005)Pseudokirchneriella subcapitata, Scenedesmus acutusCoelastrum reticulatum (Nakajima et al., 2007)Scenedesmus quadricauda (Nakajima et al., 2007)Oscillatoria sp. (Cerniglia et al., 1980)Closterium lunula (Yan and Pan, 2004)

dimethoxylcohol

Stichococcus bacillaris (DellaGreca et al., 2003)

Ankistodesmus braunii (DellaGreca et al., 2003)Chlorella vulgaris (Jinqi and Houtian, 1992)

Table 2Organic chemical substances of agricultural importance as influenced bycyanobacterial/microalgal degradation.

Organic chemical Metabolic product Cyanobacterium/Microalga(Reference)

DDT DDD and DDE Aulosira fertilissima(Lal et al., 1987)Chlorococcum sp.Anabaena sp.Nostoc sp.(Megharaj et al., 2000)

γ-Hexachlorocyclohexane(Lindane)

γ-Pentachlorocyclohexane1,2,4-Trichlorobenzene

Anabaena sp.Anabaena sp. (pRL634)(Kuritz and Wolk, 1995)

Methyl parathion p-Nitrophenolp-Aminophenoland nitrite

Chlorella vulgarisScenedesmus bijugatusNostoc linckia,N. muscorumOscillatoria animalisPhormidium foveolarum(Megharaj et al., 1994)

o,o-Dimethylo-p-nitrosophenylthiophosphate ando,o-Dimethylo-p-aminophenylthiophosphate

Anabaena sp.(Barton et al., 2004)

Metflurazon NorflurazonDesmethyl derivative

Chlorella fusca(Thies et al., 1996)

Norflurazon Desmethyl derivative Chlorella fusca(Thies et al., 1996)

Fluometuron Desmethyl fluometuronTrifluoromethylphenylurea

Ankistrodesmus cf.Nannoselene, Selenastrumcapricornutum(Zablotowicz et al., 1998)

Atrazine Diethyl atrazine Ankistrodesmus sp.Selenastrum sp.(Zablotowicz et al., 1998)

α-Endosulfan Endosulfan sulfate,β-Endosulfan, andEndosulfan ether

Scenedesmus sp.Chlorococcum sp.

Endosulfan diolEndosulfan aldehyde

Scenedesmus sp.(Sethunathan et al., 2004)

Diclofop-methyl (DM) Diclofop (DC)4-(2,4-Dichlorophenoxy)-phenol (DP)

Chlorella vulgarisC. pyrenoidosaScenedesmus obliquus(Cai et al., 2007)

Dichlorprop-methyl(2,4-DCPPM)

2,4-dichlorprop(2,4-DCPP)

Chlorella pyrenoidosa,C. vulgarisScenedesmus obliquus(Li et al., 2008)

Fenamiphos Fenamiphos phenolFenamiphossulfone phenolFenamiphossulfoxide phenol

PseudokirchneriellasubcapitataChlorococcum sp.(Cáceres et al., 2008a)

64 S.R. Subashchandrabose et al. / Environment International 51 (2013) 59–72

removed 100% of o-nitrophenol (ONP), 90% of 2,4-DNP, 85% of biphe-nyl, 77% of p-nitrophenol (PNP), and 60% of m-nitrophenol (MNP)while the percentage removal was marginally lesser in dark(Hirooka et al., 2003). Likewise, the cyanobacterium Anabaenavariabilis removed 100% of ONP and PNP, 95% of 2,4-DNP, 51% of2,4,6-trinitrophenol and 23% of bisphenol when grown in light, andtheir removal percentages were lesser in the dark. In the case ofmono-nitrophenols and mono-methylphenols, the position of NO2

−

and CH3+ on phenolic ring determines the biodegradation efficiency.

For mono-nitrophenols, the biodegradation efficiencies by the greenalga, Scenedesmus obliquus, were in the order: ONP>MNP>PNP,while it was 2-methyl-phenolb3-methyl-phenolb4-methyl-phenolfor themono-methylphenols removal (Papazi and Kotzabasis, 2008).

Bisphenol, an endocrine disruptor, was photodegraded rapidly inthe presence of C. vulgaris at 6.5×109 cells L−1 (Peng et al., 2006).The fresh water green microalga, C. fusca, completely degraded 80 μM

bisphenol within 168 h under continuous illumination (Hirooka et al.,2005). The alga converted the estrogenic bisphenol into a non-estrogenic monohydroxy bisphenol. The conversion of toxic pollutantinto less toxic or nontoxic compound is an important bioremediationstrategy when there is no possibility of complete mineralization of aparticular pollutant. The freshwater microalgae (Pseudokirchneriellasubcapitata, Scenedesmus acutus, Coelastrum reticulatum andS. quadricauda) metabolized 2 mg bisphenol L−1 within eight days.The FAB–MS and H-NMR analysis of the catabolic products of bisphenolshowed the presence of bisphenol glycosides (bisphenol α-mono-O-β-D-glucopyranoside (BPAGlc) by P. subcapitata, S. acutus, andC. reticulatum and bisphenol α-mono-O-β-D-galactopyranoside(BPAGal) by S. quadricauda), both of which are non-estrogenic(Nakajima et al., 2007).

Removal of halide groups from halophenols during algal degrada-tion requires higher energy. Halogen substituent at meta position inthe phenol ring requires more energy for its removal when comparedto para or ortho position. Energy requirement for dehalogenation ofchlorophenol was highest, followed by bromophenol and iodophenolin that order. The presence of additional carbon source will enhancegrowth rate and phenol degradation by algae. The microalgae,S. obliquus, degraded halogenated phenols normally but growth aswell as catabolic activity increased in the presence of 5 g glucose L−1

and 120 μmol/m2/s of light intensity (Papazi and Kotzabasis, 2007).The C. fusca var. vacuolata removed 23% of 40 μM 2,4-dichlorophenol(DCP) within 120 h in the presence of light. On the other hand, thepresence of 1 μM of the photosynthetic inhibitor, 3-(3,4-dichlorophenol)-1,1-dimethyl urea (DCMU), or growth in the absence of light,the removal of DCP was completely inhibited, clearly indicating the di-rect correlation between photosynthesis and the removal of phenol(Tsuji et al., 2003). Such relationship between photosynthesis and pol-lutant degradation is advantageous when algae are employed as biore-mediation agents.

Lima et al. (2004) demonstrated that the consortium of microalgaecomprising axenic cultures of C. vulgaris and Coenochloris pyrenoidosaremoved 50 mg pentachlorophenol(PCP)L−1 in the presence of lightwithin five days. However, growth in the absence of light and in thepresence of PCP was retarded resulting in very less removal of PCP.In contrast, Pinto et al. (2003)reported the rapid removal of low mo-lecular weight phenols from olive mill wastewater when thephenol-resistant algae (Ankistrodesmus braunii and S. quadricauda)were grown in dark. Almost 100% removal of hydroxytyrosol, cate-chol, ferulic acid and synapic acid was observed with these algaewhen grown under light or dark. Other phenolics like tyrosol,4-hydroxybenzoate, p-coumaric acid, caffeic acid and vanillic acidwere better removed when these cultures were grown in dark. C.pyrenoidosa, a potential degrader of PNP, when grown along with C.vulgaris in a ratio of 3:1 removed PNP within two days at the rate ofapproximately 16.5 mg L−1 d−1. But, C. pyrenoidosa required fourdays to remove 50 mg L−1 of PNP completely (Lima et al., 2003).The cyanobacterium, A. variabilis converted 2,4-DNP to2-amino-4-nitrophenol (2-ANP) that accumulated in the medium(Hirooka et al., 2006). But, when grown along with A. cylindrica, theremoval of 2-ANP was also completed. Selection of cultures that candegrade phenolics, not only the pollutant but also its intermediarymetabolite, under mixotrophic conditions is very crucial for develop-ing bioremediation strategy with algal cultures.

3.2. Oil, petroleum and hydrocarbons

Oil and petroleum form the major pollutants due to the wide-spread usage of fossil fuels for automobiles and motors in industriesand agriculture. Way back in 1975, Walker and co-workers isolatedthe petroleum-degrading alga (Prototheca zopfii) from Colgate Creekin Baltimore which degraded motor oil and crude oil (Walker et al.,1975b). Although the chemical mixtures of motor and crude oil are

65S.R. Subashchandrabose et al. / Environment International 51 (2013) 59–72

exclusively diverse, P. zopfii degraded greater percentages of aromatichydrocarbons in motor oil and saturated hydrocarbons in crude oil(Walker et al., 1975a). The Oscillatoria quadripunctulata (a cyanobac-terium) absorbed the dissolved solids from petrochemical effluentswhich are rich in phenols, sulfides, aromatic compounds, biocidesetc., and subsequently reduced the concentration of total dissolvedsalts (TDS) from 411 mg L−1 to 277 mg L−1 within 96 h, whichamounts to almost 40% removal of TDS (Joseph and Joseph, 2001).

The S. obliquus utilized 1-naphthalenesulfonic acid as the sole sourceof sulfur and converted it to 1-hydroxy-2-naphthalenesulfonic acid,1-naphthol and 1-naphthyl β-D-glucopyranoside (Kneifel et al., 1997).The metabolites started appearing in the medium after fifth day ofincubation of alga with 100 mg 1-naphthalenesulfonic acid L−1. For-mation of the main metabolite, 1-hydroxy-2-naphthalenesulfonicacid was due to NIH shift, while the transformation of 1-naphthylβ-D-glucopyranoside from 1-naphthol was through the process ofconjugation. A diatom, Naviculla sp., removed 5.5 mg L−1 of4-methylcyclohexaneacetic acid, a model naphthenic acid, within14 days (Headley et al., 2008). Both S. obliquus and Nitzschia linearisgrew in a medium containing 0.1% crude oil and removed n-alkanesand PAHs (Gamila and Ibrahim, 2004). Within six weeks of incubation,S. obliquus removed around 88% of eight different types of n-alkanes andapproximately 80% of fifteen PAHs. The PAHs utilized by the algainclude naphthalene, 1-methyl naphthalene, 2-methyl naphthalene,acenaphthene, phenanthrene, fluorene, pyrene, anthracene,fluoranthene, chrysene, benzo(b)fluoranthene, benzo(a)anthracene,benzo(k)fluoranthene, and benzo(a)pyrene. The performance ofN. linearis was almost similar to that of S. obliquus, but showed morepreference to the removal of PAHs than n-alkanes. Ibrahim andGamila (2004) also established the efficiency of natural isolates of fresh-water phytoplankton assemblages in removing the crude oil. The as-semblages comprised of two cyanobacteria, Oscillatoria brevis andMicrocystis aeruginosa, and seven green algae Spirogyra mirabilis,Ulothrix subtilissima, Mougeatia scalaris, Pediastrum clathratum,S. quadricauda, Tetraedron minimum and Ankistrodesmus acicularis. Thepresence of oil prolonged growth phase of these algae, and led to higherbiomass production and removal of more PAHs (70–75%) thann-alkanes within three weeks.

3.3. Pesticides

Intensive agriculture with dependence on agrochemicals such asfertilizers and pesticides (herbicides and insecticides) has increasedglobal food production but at the same time, polluted the environ-ment considerably. Biological removal of these chemicals involvingseveral microorganisms including algae had been well established(Cáceres et al., 2010; Singh andWalker, 2006). Algal cell size, density,morphology and activities play an important role in the uptake andremoval of pesticides. High surface area to biovolume ratio of algaeprovides greater potential for sorption and subsequent interactionwith pesticides. Generally, algae utilize pesticides when the concen-trations of chemical substances are nontoxic. Butler et al. (1975) ob-served that Chlorella sp., Monoraphidium sp., Actinastrum sp., Koliellasp., Scenedesmus sp., and Nitzschia sp. degraded only 1 ppm of carba-ryl and diazinon, and 0.01 ppm of methoxychlor and 2,4-D. Theorganophosphorous insecticides like monocrotophos and quinalphoswere degraded by two unicellular green algae (C. vulgaris andScenedesmus bijugatus) and three cyanobacteria (Synechococcuselongatus, Phormidium tenue and Nostoc linckia) within 30 dayswhen these pesticides ranged from 5 to 50 ppm (Megharaj et al.,1987). The cyanobacteria, Anabaena sp. and Aulosira fertilissima accu-mulated DDT, fenitrothion and chlorpyrifos (Lal et al., 1987). Thus,Anabaena sp. removed 1568 ppm DDT, 3467 ppm fenitrothion and6779 ppm chlorpyrifos, while A. fertilissima accumulated 1429 ppmDDT, 6651 ppm fenitrothion and 3971 ppm chlorpyrifos; both theorganisms metabolized DDT to DDD and DDE.

Microalgae (C. vulgaris and S. bijugatus) and cyanobacteria(N. linckia, Nostoc muscorum, Oscillatoria animalis and Phormidiumfoveolarum) degraded methyl parathion, an organophosphorus insec-ticide, and utilized it as a source of phosphorus (Megharaj et al.,1994). Within 30 days of incubation, O. animalis and P. foveolarumcompletely degraded 20 μg mL−1 methyl parathion as well as PNP,its hydrolysis product N. muscorum completely oxidized the nitrogroup of PNP to nitrite within fifteen days. Both N. muscorum andA. fertilissima degraded monocrotophos (100 ppm), malathion(75 ppm), dichlorovos (50 ppm) and phosphomidon (25 ppm)(Subramanian et al., 1994). But, C. vulgaris tolerated toxic concentra-tions of carbofuran when grown mixotrophically with glucose or ace-tate (Megharaj et al., 1993). Also, A. fertilissima was more efficient inutilizing the pesticides as the phosphorus source. Anabaena sp. de-graded highly toxic lindane, a chlorinated aliphatic insecticide, toγ-pentachlorocyclohexene. The genetically modified Anabaena sp.,possessing Pseudomonas paucimobilis gene that controls the firststep of lindane catabolism degraded the parent compound to1,2,4-trichlorobenzene (Kuritz and Wolk, 1995). Another recombi-nant Anabaena sp., containing bacterial fcbABC operon of Arthrobacterglobiformis, dehalogenated 4-halobenzoate. The green algae(Chlamydomonas sp., Chlorella sp., Pediastrum sp., and S. quadricauda)removed more atrazine relative to the diatoms (Cyclotella gamma,Cyclotella meneghiniana, Synedra acus and Synedra radians) (Tanget al., 1998). Megharaj et al. (2000) isolated five algal strains com-prising the three genera, Chlorococcum sp., Anabaena sp. and Nostocsp. from DDT-contaminated soil and demonstrated that the algaewere highly efficient in catabolizing the DDT into DDD and DDE. DDDwas the major metabolite of DDT metabolism by diazotrophiccyanobacteria while DDE was the major metabolite in the case ofgreen alga.

Algal transformation of herbicides may possibly be catalyzed bydealkylating enzymes like cytochrome P450. Some algae like Isochrysisgalbana,Dunaliella tertiolecta, Phaeodactylum tricornutum, P. subcapitata,and Synechococcus sp. accumulated atrazine (Weiner et al., 2004). Someof the factors like surface area, cellular biovolume of the algae, and con-centration of atrazine in the solution determine the algal capacity to up-take the herbicide. C. vulgaris accumulated the triazine group ofherbicides (83% of 0.75 μM atrazine and 93% of 0.75 μM terbutryn)within 12 h (González-Barreiro et al., 2006). Chlorella saccharophilaaccumulated an organophosphorus insecticide, pyridaphenthion, withmaximal algal bioconcentration of 441.5 mg pyridaphenthion per kgof its biomass within five days (Jonsson et al., 2001). Strains ofM. aeruginosa, Anabaena cylindrica, A. flos-aquae and A. spiroides degradedthe toxic phenylurea herbicide (fluometuron) (El-Rahman Mansy andEl-Bestawy, 2002). Among these cyanobacterial strains, M. aeruginosaand Anabaena cylindrica degraded 97% of fluometuron at 1.4 mg m L−1

within a day. A gradual increase in herbicide concentration also enhancedthe biodegradation capabilities of algae, indicating that the biodegrada-tion offluometuron is species-dependent, and biodegradation capabilitiesincreased with increasing exposure time.

Methyl parathion, a toxic organophosphorous insecticide, wastransformed by Anabaena sp. through a reductive process (Bartonet al., 2004). Methyl parathion was converted to O,O-dimethylO-p-nitrosophenylthiophosphate and was subsequently reduced.Both Scenedesmus sp. and Chlorococcum sp. at cell densities of1550×106 and 600×106 cells mL−1, respectively, convertedα-endosulfan, an endocrine disrupting insecticide, to endosulfan sul-fate, endosulfandiol, β-endosulfan, endosulfan aldehyde and endosul-fan ether (Sethunathan et al., 2004). Strains of C. vulgaris,C. pyrenoidosa, and S. obliquus degraded the herbicide, diclofop-methyl (DM) by readily absorbing and subsequently hydrolyzing toyield diclofop (DC) inside the algal cells (Cai et al., 2007). When DCwas further catabolized to 4-(2,4-dichlorophenoxy)-phenol (DP), DPwas more toxic than DM or DC (Cai et al., 2009). El-Bestawy et al.(2007) demonstrated that cyanobacterial isolates (Oscillatoria sp.,

66 S.R. Subashchandrabose et al. / Environment International 51 (2013) 59–72

Synechococcus sp., Nodularia sp., Nostoc sp., Cyanothece sp.,Synechococcus sp., M. aeruginosa and A. cylindrica) from an Egyptianlake possessed the ability to degrade lindane either individually orin combination. Lindane was completely degraded by these isolateswithin seven days; but the intermediary metabolites formed duringthe lindane catabolism were not detected after the fourth day, indi-cating the complete mineralization of the pollutant.

Zhang et al. (2012) isolated a nitrogen-fixing cyanobacteriumAnabaena azotica118 from Chinese paddy soils, which can degradelindane at an initial concentration 0.2 mg L−1. González et al.(2012) demonstrated that lindane-resistant microalgal mutantsarise by spontaneous mutations, which can grow at concentrationshigher than 5 mg lindane L−1. These resistant microalgal cells canbe effective bioremediation agents for lindane or other chlorinatedpollutants. A freshwater alga (P. subcapitata) and a terrestrial alga(Chlorococcum sp.) accumulated and transformed an organophospho-rous insecticide/nematicide, fenamiphos, to its oxides fenamiphossulfoxide and fenamiphos sulfone and then to respective phenolsfenamiphos phenol, fenamiphos sulfone phenol, and fenamiphos sulf-oxide phenol (Cáceres et al., 2008a; Fig. 2). However, in an anotherstudy comprising five soil microalgae and five soil cyanobacteria itwas found that all the organisms readily oxidize fenamiphos tofenamiphos sulfoxide which is then transformed into fenamiphos sulf-oxide phenol (Cáceres et al., 2008b). Cáceres et al. (2008a) also showed

Fig. 2. Metabolism of fenamiphos by

that the transformed phenolic compounds were more toxic than theparent compound. This kind of an adverse effect has to be overcomeby employing other microorganisms which can further metabolize thetoxic products of fenamiphos. The consortia of algae and bacteria willbe effective under these conditions since bacterial partners can degradethe algal metabolized compounds (Subashchandrabose et al., 2011).

Huang et al. (2012) demonstrated that toxic effects and biodegra-dation of R-(−)-and S-(+)-benalaxyl were enantioselective inS. obliquus. Zhang et al. (2011) reported that fluroxypr (4-amino-3,5-dichloro-6-fluoro-2-pyridyloxy acetic acid)-meptyl, an importantherbicide stimulated the growth of Chlamydomonas reinhardtii at0.05–1.00 mg L−1 but inhibited at 0.75–1.00 mg L−1. Higher concen-tration of fluroxypyr led to significant production of reactive oxygenspecies. As a consequence, peroxide accumulation and DNA degrada-tion occurred. Both accumulation and degradation of fluroxypyrwere found to occur simultaneously in the microalgae. The biphasicdose response (Hormesis) of algae to organic pollutants will be eco-logically beneficial to make other members of microbial communitiesto adapt to higher levels of pollutants.

3.4. Polyaromatic hydrocarbons

Polyaromatic hydrocarbons (PAHs), which consist of fused aro-matic rings without any substituents, are released from the oil and

cyanobacteria and microalgae.

A

B

Fig. 3. Proposed pathways for cyanobacterial degradation of PAHs. A) Phenanthrene byAgmenellum quadruplicatum PR-6 (Narro et al., 1992), B) Anthracene by Phormidiumtenue (Kumar et al., 2009).

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coal industries due to incomplete combustion (Liu et al., 2001). ThePAHs are pollutants of serious concern because they are carcinogenic,mutagenic, and teratogenic to humans and animals (Dejmek et al.,2000). The environmental pollution by PAHs and the scope for micro-bial remediation had been reviewed extensively (Haritash andKaushik, 2009; Samanta et al., 2002; Thavamani et al., 2011, 2012).Although the bacterial (Kanaly and Harayama, 2000) and fungal(Barclay et al., 1995) degradation of PAHs are known for long time,algal utilization of PAHs has been established relatively recently.

Metabolismof BaP, a carcinogenic PAH, by Selanastumcapricornutumis through dioxygenase activity, which is the key pathway in bacteria toactivate dioxygen (Warshawsky et al., 1988). Likewise, S. capricornutummetabolized BaP to cis vicinal dihydrodiols like cis-11,12-dihydro-11,12-dihydroxybenzo(a)pyrene, cis-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and cis-4,5-dihydro-4,5-dihydroxybenzo(a)pyrene. The stereo-specificity of BaP dihydrodiols was identified as cist isomers using fluo-rescence spectroscopy, HPLC and liquid scintillation spectrometry(Lindquist andWarshawsky, 1985b). The catabolism of PAHs producedseveral intermediarymetabolic compounds. Inmost cases, the interme-diates are biologically active and retain the toxicity. Conjugation of me-tabolites will detoxify the compounds and increase the water solubilityof these metabolites. For instance, S. capricornutum detoxified 71.1%of the catabolic products of BaP, and 11,12-diol mainly remained inthe 28.9% of non-conjugated metabolites of BaP (Lindquist andWarshawsky, 1985a; Warshawsky et al., 1990).

Schoeny et al. (1988) found that S. capricornutum, S. acutus andAnkistrodesmus braunii converted BaP to mainly 3,6-quinones in thepresence of white light resulting in inhibition of growth, but gold lightenhanced the growth as well as catabolism of the pollutant producingBaP-diols. All the three green algae removed approximately 98% ofBaP from the medium. But, the white light had no negative impact onC. reinhardtii (green alga), Ochromonas malhamensis (yellow-greenalga), Euglena gracilis (euglenoid alga), and Anbaenaflos-aquae (cyano-bacterium). The algal degradation of BaP is thus dependent on thelight energy absorbed and emitted, and the best suitable light sourcefor metabolism of BaP by S. capricornutum was the gold light. Amongvarious quinones produced during the incubation under white light,menadione, danthron, phenanthrene-quinone and hydroquinonewere nontoxic, but 3,6-quinone was highly toxic to S. capricornutum(Warshawsky et al., 1995).

Seven algal species such as Chlamydomonas sp., Chlorella minister,C. vulgaris, Scenedesmus platydiscus, S. quadricauda, Synechocystis sp.,and S. capricornutum removed higher concentrations of pyrene (Leiet al., 2002). Initial removal of pyrene by all these organisms was bypassive physicochemical bioabsorption. Only S. capricornutum absorbed65% of pyrene into the cell wall material, and transformed it completelyby seventh day. Probably, the degree of bioaccumulation and biotrans-formation of pyrene is species-specific, depending on the concentrationof algal biomass. Todd et al. (2002) screened algae for their efficacy inmetabolizing naphthalene and two diaryl ethers, dibenzofuran anddibenzo-p-dioxin, and observed that Ankistrodesmus sp. converteddibenzofuran to four monohydroxylated dibenzofurans and twodihydroxylated dibenzofurans, Scenedesmus sp. transformed dibenzo-p-dioxin to 2-hydroxydibenzo-p-dioxin and two dihydroxylateddibenzo-p-dioxin, and C. vulgaris catabolized naphthalene to 1-naphthol.

Under autotrophic growth conditions, Chlorella protothecoides re-moved only 20% of anthracene whereas growth under heterotrophicconditions removed about 33.53% (Yan et al., 2002). At both the tro-phic conditions, this alga accumulated 80% of anthracene. When theinitial cell density was 1×107 cells mL−1, S. capricornutum removed96% of phenanthrene, 100% of fluoranthene, and 100% of pyrenewithin four days (Chan et al., 2006), metabolized phenanthrene to1-, 2-, 3-, and 9-hydroxyphenanthrene and two dihydroxylated phen-anthrenes, fluoranthene to monohydroxylated fluoranthene and twodihydroxylated fluoranthenes, and pyrene to monohydroxylatedpyrene and two dihydroxylated pyrenes. The chemical preference

for removal by this alga was in the descending order: pyrene>fluoranthene>phenanthrene. Some of the common factors involvedin the algal removal of PAHs are surface area: volume ratio, lipid con-tent, biomass concentration, algal species specificity, presence ofPAHs in the mixture and extent of toxicity exerted by PAHs. Narroet al. (1992) demonstrated the metabolism of phenanthrene to phen-anthrene trans 9,10-dihydrodiol and 1-methoxyphenanthrene by amarine cyanobacterium, Agmenellum quadruplicatum PR-6 (Fig. 3A).During cyanobacterial degradation of anthracene by P. tenue, Kumar etal. (2009) observed the initial accumulation of anthracene-1,2-dionewhich was subsequently converted to 8-hydroxy anthracene-1,2-dione and 10-hydroxy anthracene-1,2-dione (Fig. 3B).

Heterotrophic green alga (Prototheca zopfii) immobilized in poly-urethane foam removed a mixture of phenanthrene and pyrene andmetabolized n-alkanes (Ueno et al., 2008). Accumulation of PAHs inthe matrix did not prevent the consumption of n-alkanes. This dualrole by the immobilized algae may have potential applications inthe environmental clean-up process for mixed contaminants. Twoalgal isolates (Skeletonema costatum and Nitzschia sp.) from the Jiu-long River Estuary Mangrove Nature Reserve in China degradedphenanthrene and fluoranthene (Hong et al., 2008). More phenan-threne was degraded by S. costatum when it was in the mixturealong with fluoranthene than it was provided alone. But, Nitzschiasp. degraded more phenanthrene in the mixture and also when itwas alone; degradation of fluroranthene by both the algae was lessercompared to that of phenanthrene.

3.5. Polychlorinated biphenyls

Polychlorinated biphenyls (PCBs) are a class of chlorinated deriv-atives of aromatic organic compounds with 1 to 10 chlorine atoms

68 S.R. Subashchandrabose et al. / Environment International 51 (2013) 59–72

attached to biphenyl which is a molecule composed of two benzenerings. The empirical formula of PCBs is C12H10-xClx (Phillips, 1986).PCBs are also referred to different trade names such as Aroclor,Phenoclor and Kanechlor (Mullins et al., 1984). PCBs are highly per-sistent and toxic with greater affinity towards the lipids of animalsresulting in their biomagnification in the food chain. Though microbi-al degradation of PCBs has been established (Bevinakatti andNinnekar, 1992; Boyle et al., 1992), limited information on algal ca-tabolism of PCBs is available. The algal bioconcentration of hydropho-bic PCBs depends on physico-chemical properties of the compoundsand physiology, exudates and density of algae. Variations in totallipids at different growth stages and restricted membrane permeabil-ity in algae, and hydrophobicity of PCBs and structure of PCB conge-ners affect bioaccumulation (Stange and Swackhamer, 1994). Theuptake rate constants for PCB-15, PCB-52 and PCB-153 in C.pyrenoidosa increased with hydrophobicity and varied between 200and 710,000 L−1 kg−1 d−1 (Sijm et al., 1998). The sorption coefficientsof PCBs to algal exudates varied between 80 and 1200 L kg−1. Jabuschand Swackhamer (2004) observed accumulation of PCBs such as2-chlorobiphenyl, 2,49-dichlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl,2,2′,6,6′-tetrachlorobiphenyl, 2,3,4,5-tetrachlorobiphenyl, 3,39,4,49-tetrachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, 2,2′,3,3′,4,4′-hexachlorobiphenyl, 2,2′,4,4′,5,5′-hexachlorobiphenyl, 2,2′,4,4′,6,6′-hexachlorobiphenyl, 3,3′,4,4′,5,5′-hexachlorobiphenyl, 2,2′,3,3′,4,4′,5,5′-octachlorobiphenyl and decachlorobiphenyl in subcellular loca-tions of C. reinhardtii. Using 14C labeled PCBs for bioaccumulation by algalcellswith orwithout cellwall, itwas shown that the accumulation of PCBsin thylakoids was slower than the total cellular uptake, and adsorptioninto the cell wall was less than 10% of total accumulation. Certain factorslike log bioconcentration factors (BCF) and log octanol–water partitioncoefficients (Kow), and dissolved organic carbon (DOC) may play crucialrole in algal bioconcentration of hydrophobic PCBs. Interestingly, manyalgal species have been reported to show biosorptive potential for differ-ent organic chemical substances including pesticides (Table 3).

4. Genetic engineering of cyanobacteria/microalgae for enhancedbiodegradation

Genetic engineering helps to make rapid progress in introducing adesired trait into the target organism. Engineering algae genetically

Table 3Biosorptive potential of cyanobacteria and microalgae for some organic chemicalsubstances.

Organic chemical Cyanobacterium/Microalga

BCFa or % Reference

Fenitrothion(10 μg mL−1)

Anabaena sp. 48–347 BCF (Lal et al., 1987)

Chlorpyrifos(10 μg mL−1)

18–678 BCF

DDT (0.5 μg mL−1) 792–3136 BCFFenitrothion(10 μg mL−1)

Aulosirafertilissima

271–560 BCF (Lal et al., 1987)

Chlorpyrifos(10 μg ml−1)

397–194 BCF

DDT (0.5 μg mL−1) 629–2857 BCFPyrene (0.1 mg L−1) C. vulgaris

Scenedesmusquadricauda

83.8%41.0%

(Lei et al., 2002)

Anthracene(2.5 mg L−1)

Chlorellaprotothecoides

80% (Yan et al., 2002)

PCBs congeners(404 mg L−1)

Chlamydomonasreinhardtii

Log BCF4.8–5.7

(Jabusch andSwackhamer, 2004)

Naphthalene(2 mg L−1)

Prototheca zopfii 20% (Ueno et al., 2008)

Phenanthrene(2 mg L−1)

80%

Pyrene (2 mg L−1) 80%

a Bioconcentration factor.

still remains at its infancy (León-Bañares et al., 2004; Stevens andPurton, 1997), especially because of the hurdle posed by codon biasfor expression of foreign genes in microalgae (Heitzer et al., 2007).Nevertheless, the availability of cloning vectors, transposons,methods of mutagenesis and genomic sequences provides ample op-portunities to employ microalgae and cyanobacteria with desiredtraits (Koksharova and Wolk, 2002). Despite these difficulties, trans-genic microalgae have received attention in various biotechnology in-dustries (Walker et al., 2005). Nuclear transformation for metaboliccontrol and chloroplast transformation for high levels of protein ex-pression (León-Bañares et al., 2004; Rosenberg et al., 2008) havebeen adequately demonstrated by many researchers. Genome se-quencing of algae will provide more information on proteins, meta-bolic regulation, evolution and ecological relationships. Moreimportantly, the successful commercialization of algae depends notonly in species selection but also in engineering genetically for effi-cient degradation of pollutants. Being a versatile representative of eu-karyotic and prokaryotic photosynthetic organisms, cyanobacteriaand microalgae are excellent model organisms to understand themechanism of tolerance to pollutants (Siripornadulsil et al., 2002).Novel pollutant degrading genes from bacteria can be transferredinto the mixotrophic algae for effective expression, rather than pho-toautotrophs. Earlier reports suggest the successful transfer of bacte-rial gene to photoautotrophic algae. LinA gene of P. paucimobilis UT26was transferred to Anabaena sp. PCC7120 and the lindane degrada-tion efficiency of this cyanobacterium increased even in the absenceof nitrate (Kuritz et al., 1997). Increase in lindane degradation wasalso noticed in Nostoc ellipsosporum transformed with linA gene(Kuritz et al., 1997). Rajamani et al. (2007) demonstrated that highertolerance of transgenic microalgae to pollutants can be exploited forbioremediation. Future efforts to transfer desired traits intomixotrophs may bring more successful applications of algae for biore-mediation. The social acceptance to the release of transgenic microor-ganisms into the environment is limited because of their unknownconsequences in the environment. However, research into the risk as-sessment of GEMs in terms of ecological, ethical and social aspectswill alleviate the perceived risks thereby paving the way to theiruse in remediation of contaminants in the environment in the nearfuture (Megharaj et al., 2011; Tiedje et al., 1989).

What is important now is to develop molecular strategies for iden-tifying efficient mixotrophic algae from the complex microbial com-munities in the polluted environments. Earlier reports suggest thatthere are distinct changes in biochemical activities and involvementof unique gene products. Yang et al. (2002) demonstrated that theshift from photoautotrophic to photomixotrophic conditions did notaffect expression pattern of transcripts and of proteins while an in-creased activity of key enzymes in Calvin cycle was detected inSynechocystis sp. PCC 6803. Allosteric inhibition of glucose-6-phosphate dehydrogenase by ribulose 1,5-diphosphate was reportedto repress OPP pathway in the presence of light (Pelroy et al., 1972).On the contrary, Knowles and Plaxton (2003) and Kahlon et al.(2006) showed that high glucose-6-phosphate dehydrogenase activ-ity in the photomixotrophically grown cells of Synechocystis sp.suggesting the co-existence of the Calvin cycle and the OPP pathway.Hihara and Ikeuchi (1997) reported from the mutant studies that thefunctional pmgA gene is essential for the photomixotrophic growth ofthis alga. While there is no significant homology with known genes,the amino acid sequences suggest the soluble nature of pmgA product.Takahashi et al. (2008) suggested that pmgA regulates the carbonflow between the Calvin cycle and the OPP pathway. The gene pmgAhas been reported in the strains of S. elongatus, Geobacter violaceus,M. aeruginosa, Nostoc sp. and Cyanothece sp. (Fig. 4). Apparently,this gene is conserved in algae, enabling those strains to followmixotrophic mode of nutrition. Although the structure of pmgA geneproduct can be predicted, its exact function remains to be elucidated.It will be interesting to know from transcriptomic and proteomic

Cyanobacteria

83 Streptomyces flavogriseus (11371205)

Rhodospirillum photometricum (12211249)

Bacillus megaterium (12083330)

Cyanothece sp. (6168379)

Nostoc sp. (1107253)

Microcystis aeruginosa (5864390)

Synechococcus elongatus (3199599)

Gloeobacter violaceus (2600808)

Rhodothermus marinus (8568996)

Pseudomonas putida (6109987)

Stenotrophomonas maltophilia(6394732)

Mycobacterium vanbaalenii (4648530)

Rhodopseudomonas palustris (4361602)

Rhodobacter capsulatus (9006140)

17

6313

28

43

41

75

90

81

70

0.2

Fig. 4. Phylogenetic tree for anti-sigma regulatory factor and photomixotrophic growth-like protein.

69S.R. Subashchandrabose et al. / Environment International 51 (2013) 59–72

analyses whether eukaryotic algae possess similar unique genes ortheir products conferring mixotrophy. The high throughput tech-niques such as transcriptomics and proteomics (Shim et al., 2011)will be not only useful in analyzing algal mixotrophism and in findingout molecular markers for rapid identification but also in estimatingabundance of mixotrophs, which will aid in monitoring their activi-ties in the polluted environments.

5. Conclusions

Man-made chemical substances, without adequate knowledge ontheir fate, occurrence as mixtures and functioning can be more perva-sive and insidious in the environment. There are many knowledgegaps with reference to pollution in soil or water environments, con-tamination of food and feed, and in general populations includingplants, animals, and humans. Unawareness or uncertainty about thereasons for and the costs of treating pollution necessitates the needfor collecting more scientific data on the biological response to pollut-ants and for devising strategies to remediate the polluted sites. Toxic-ity, distribution, or slow biodegradation in the environment makesmany organic chemical substances the worst among the pollutants.Traditional engineering methods of disposal such as landfilling andcombustion by open burning have serious limitations.

Cyanobacteria and microalgae respond to many organic pollutantsby various ways, ranging from bioaccumulation to biodegradation.They can grow in very low water potential, in arid environments, andcan tolerate high salinity and grow in hyper saline ponds. Because oftheir versatile metabolism and their capacity to switch rapidly fromone mode to another, microalgae, more importantly mixotrophicalgae can be successfully employed for remediating the pollutant envi-ronments. Either physiological adaptation (Fogg, 2001) or genetic adap-tation (González et al., 2012; López-Rodas et al., 2009) makes algae

bloom or perish under high levels of pollutants. The ecological advan-tage of employingmixotrophic cyanobacteria andmicroalgae for biore-mediation is that decreasing concentration of pollutant will have noadverse effect on their growth as opposed to declining heterotrophicbacterial growth. What is requisite now is selection of highly adaptive,mixotrophic algaewith dual purpose of effective degradation of organicpollutants and simultaneous sequestration of atmospheric CO2. Onlyunder limitation of multiple resources that favors the generalists,mixotrophs will dominate over the specialist nutritional types(Katechakis and Stibor, 2006), phototrophs or heterotrophs which areoften selected under the laboratory conditions. Despite the fact thatmixotrophic algae can be widespread in the polluted areas, the impor-tance of mixotrophy to pollutant degradation has received less atten-tion. Molecular methods can aid in identification and selection ofcyanobacterial andmicroalgal species with desirable mixotrophic, met-abolic capabilities from polluted environments. The abilities to captureenergy and sequester C through photosynthesis, and degrade organicpollutants by mixotrophic cyanobacteria and microalgae could offermore competitive advantages in bioremediation efforts.

Acknowledgments

SRS acknowledges UniSA for providing UPS scholarship and CRCCARE for PhD scholarship. BR andKV thank the Government of Australia(Department of Education, Employment and Workplace Relations) forthe Endeavour Research Fellowship and Endeavour Executive Award,respectively.

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