Consortia of cyanobacteria/microalgae and bacteria: Biotechnological potential

Biotechnology Advances 29 (2011) 896–907

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Biotechnology Advances

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Research review paper

Consortia of cyanobacteria/microalgae and bacteria: Biotechnological potential

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

a Centre for Environmental Risk Assessment and Remediation, University of South Australia, SA 5095, Australiab Cooperative Research Centre for Contamination Assessment and Remediation of Environment, PO Box 486 Salisbury South, SA 5106, Australiac Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, Indiad Department of Microbiology, Sri Krishnadevaraya University, Anantapur 515055, India

⁎ Corresponding author at: Centre for Environmental3057.

E-mail address: [email protected]

0734-9750/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.biotechadv.2011.07.009

a b s t r a c t
a r t i c l e i n f o
Article history:Received 14 January 2011Received in revised form 14 June 2011Accepted 3 July 2011Available online 23 July 2011

Keywords:ConsortiaCyanobacteria/microalgaeBacteriaPollutant removalOrganic pollutantsMetalsNutrient removal

Microbial metabolites are of huge biotechnological potential and their production can be coupled withdetoxification of environmental pollutants and wastewater treatment mediated by the versatile microor-ganisms. The consortia of cyanobacteria/microalgae and bacteria can be efficient in detoxification of organicand inorganic pollutants, and removal of nutrients from wastewaters, compared to the individualmicroorganisms. Cyanobacterial/algal photosynthesis provides oxygen, a key electron acceptor to thepollutant-degrading heterotrophic bacteria. In turn, bacteria support photoautotrophic growth of the partnersby providing carbon dioxide and other stimulatory means. Competition for resources and cooperation forpollutant abatement between these two guilds of microorganisms will determine the success of consortiumengineering while harnessing the biotechnological potential of the partners. Relative to the introduction ofgene(s) in a single organism wherein the genes depend on the regulatory- and metabolic network for properexpression, microbial consortium engineering is easier and achievable. The currently available biotechno-logical tools such as metabolic profiling and functional genomics can aid in the consortium engineering. Thepresent review examines the current status of research on the consortia, and emphasizes the construction ofconsortia with desired partners to serve a dual mission of pollutant removal and commercial production ofmicrobial metabolites.

Risk Assessment and Remediation, University of South Au

(M. Megharaj).

l rights reserved.

© 2011 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8972. Stromatolites and microbial mats: ancient to modern communities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8983. Interactions between cyanobacteria/microalgae and bacteria: extent of relatedness . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8984. Cyanobacteria/microalgae: pollutant removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8995. Consortium of cyanobacteria/microalgae for pollutant removal: proof-of-principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 899

5.1. Organic pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8995.2. Metal pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9015.3. Nutrient removal from wastewaters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 901

6. CO2 capture and sequestration by the consortia: mitigation of greenhouse gas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9037. Microbial solar cells: the futuristic consortium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9038. Microbial community engineering: construction of the consortium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9039. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 904Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 904References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 905

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897S.R. Subashchandrabose et al. / Biotechnology Advances 29 (2011) 896–907

1. Introduction

Cyanobacterial andmicroalgalmetabolites such as proteins, fatty acids(eicosapentaenoic acid), steroids, carotenoids, phycocolloids (agar,carrageenan, and alginate), lectins, mycosporine-like amino acids,halogenated compounds, and polyketides are of huge biotechnologicalpotential (Cardozo et al., 2007). Species of Nostoc, Arthrospira (Spirulina)and Aphanizomenon have been used as food and a source of proteins since2000 years ago (Jensen et al., 2001). Use of algae has been extended to thetreatment of wastewaters, energy generation, and even as the photosyn-thetic gas exchangers for space travel (Spolaore et al., 2006). Thesystematics of cyanobacteria is generally on the classification schemesbased on cell or colony shape (Rippka et al., 1979; Oren, 2011), but theevolutionary basis includes these organisms under one of the ten groupstermed ‘Eubacterial Phyla’ (Woese et al., 1985). Included in the definitionof plants aremicroalgaewhich are eukaryotic unicellular andmicroscopicwith size ranging from 1/1000 of a mm to 2 mm and include species ofdiatoms, dinoflagellates and green flagellates (Hallegraeff, 1991). Basedon the Tamura–Nei model (Tamura and Nei, 1993), the 16S rRNAsequences of cyanobacteria (oxygenic photosynthetic bacteria), hetero-trophicbacteria andeukaryoticmicroalgal plastids suggest the sharingof acommon ancestor (Fig. 1). These organisms have been isolated, selected,mutated, and genetically engineered for effective bioremediation oforganic or recalcitrant pollutants, achieving enhanced rates of degrada-tion, and ensuring better survival and colonization in the polluted areas(Koksharova andWolk, 2002; Ramakrishnan et al., 2010; Venkateswarlu,1993). ‘Industrial sustainability’ now aims at achieving sustainableproduction and requires the need of incorporating ‘designs for environ-ment’ into many production processes. Many ancient as well as modernbiotechnological techniques are used in treating wastewaters andpollution in the environment. Further research and advances are

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Oscillato

Arthrosp

AnabaenNostoc sp

GloeobacOchromoNannoch

Chlorella

CorynebaMycobacRhodocoNocardia

SphingomAgrobactDesulfatiBdellovibCampyloNautilia AchromoAlcaligen

PseudomStenotrop

LeptospirBorrelia Cristispir

SpirochaTreponem

Chlamyd

ScenedesBacillus PaenibacClostridiAcidamin

Streptom

Fig. 1. Phylogenetic tree showing evolutionary relationships among cyanobacteria (oxy(constructed by using MEGA4 (Tamura et al., 2007) based on maximum likelihood method

necessary to improve the benefits from these biotechnologies. To abateindustrial pollution, to enhance profitability and sustainability, and touncouple economic growth from adverse environmental impact, biore-mediation technologies are required (Gavrilescu and Chisti, 2005).

In nature, most microalgae and cyanobacteria are found inassociation with other aerobic or anaerobic microorganisms. Thebacterial assemblages are known to influence the development ordecline of algal blooms (Fukami et al., 1997). Even the long-termlaboratory algal cultures have maintained symbiotic relationship withbacteria (Park et al., 2008). The molecular oxygen from algalphotosynthesis is used as an electron acceptor by bacteria to degradeorganic matter. Carbon dioxide (CO2) from the bacterial mineraliza-tion completes the photosynthetic cycle. The symbiotic interactions ofmicroalgae and bacteria form the basis of the biological oxygendemand (BOD) removal in the wastewater treatment ponds, firstreported by Oswald et al. (1953). Depletion of ammonium, nitrate andphosphate by the algal growth is advantageous for nutrient removalfrom wastewaters. The principle of self-oxygenation by naturalsystems can be effectively employed for remediation of manypollutants (Muñoz and Guieysse, 2006) since the conventionalengineering technologies suffer from high costs for oxygen supply,incomplete utilization of natural resources, creation of secondarypollutants, and technical impracticability in some situations. Thebiodegradation processes involving the consortium of cyanobacteria/microalgae and bacteria will be an ideal self-sustaining system that ischeaper and technically superior. In nature, there are many evidencesof microbial communities of cyanobacteria ormicroalgae and bacteria,either fossilized or living together. What is pertinent now is to gaininsights on the interactions and organization from the ancientstromatolites and modern cyanobacterial mats, and to apply moleculartechniques to select the desired microbial members for engineering

ria sancta (EU196639)

ira platensis (FJ839360)

a aphanizomenoides (AB551453)

. (FR798938)

ter violaceus (FR798924)

nas distigma (AY702136)

loropsis granulata (AY702166)

vulgaris (D11347)

cterium striatum (JF342700)

terium vanbaalenii (NR_029293)

ccus erythropolis (AF230876)

alba (EU249584)

onas japonica (AB428568)

erium tumefaciens (HQ916822)

bacillum aliphaticivorans (NR_025694)

rio sp. JS5 (AF084859)

bacter troglodytis (HQ864828)

abyssi (AM937002)

bacter denitrificans (FM999734)

us sp. (AY296718)

onas putida (HQ315887)

homonas maltophilia (AB194327)

a meyeri (HQ709385)

lusitaniae (NR_036806)

a sp. (U42638)

eta sp. (AY337318)

a denticola (NR_036899)

omonas reinhardtii (FJ458262)

mus obliquus (AF394206)

cereus (HQ316117)

illus alginolyticus (HQ236042)

um aciditolerans (DQ114945)

ococcus fermentans (X78017)

yces alboflavus (EF178699)

Cyanobacteria

α

δ

ε

β

γ

Spirochetes

Proteo-bacteria

Firmicutes

Actinobacteria

Plastids ofMicroalgae

genic photosynthetic bacteria), other eubacteria and eukaryotic microalgal plastidswith 34 representative 16S rRNA gene sequences).

898 S.R. Subashchandrabose et al. / Biotechnology Advances 29 (2011) 896–907

consortium of self-sustaining systems with dual mission of pollutantremoval and metabolite production (Fig. 2). The present reviewhighlights thepotential of cyanobacteria/microalgae–bacterial consortiaas the self-sustained system for (a) detoxification of environmentalpollutants and removal of nutrients, and (b) production of metabolites/by-products of huge commercial value, coupled with the mitigation ofgreenhouse gas CO2.

2. Stromatolites and microbial mats: ancient to moderncommunities

Stromatolites are internally laminated organosedimentary struc-tures, evolved from microbial mats over time. The ancient stromat-olites are considered to be the only form of life for longer periods inthe earth's history. Probably as the first photosynthetic communities,these stromatolites proliferated in the shallow zone of the oceans,with the consumption of CO2 and production of O2 and H2. Thestromatolites of the Warrawoona Group in Western Australia areabout 3.43 billion year old. Allwood et al. (2007) were of the opinionthat the stromatolites of the Strelley Pool Chert, Pilbara Craton are thebiological milestones on the beginning of life, biodiversity, anddevelopment of different capabilities. Trapping and binding, and/orprecipitation of minerals via microbial or abiotic processes wouldhave resulted in the fossil stromatolite formations and they are amongthe earliest evidence for life on the Earth. There are evidences formodern stromatolites, and those found in the Ruidera Pools NaturalPark of Spain are comprised of cyanobacteria, frequently the species ofLeptolyngbya, and bacterial species of Firmicutes, Bacteriodetes,Proteobacteria, Actinobacteria, Acidobacteria, Planctomycetes andChloroflexi (Santos et al., 2010).

Microbial (cyanobacterial) mats show morphological similarity toancient stromatolites. Some of the microbial mats are laminatedheterotrophic and autotrophic communities vertically stratified,dominated by cyanobacteria, microalgae like diatoms, and anoxygenic

Consortia of Cyanobacteria/Microalgae and Pollutant-degrading

Bacteria

Nutrient removal Removal of organic- and metal pollutants

Biomass

Feed for Animals, Poultry, and

Aquaculture

Biofertilizer

Pigments

Nutraceuticals

Biofuels: Biodiesel, Hydrogen, and

Electricity

Agricultural wastewaters Industrial wastewaters

Fig. 2. Value-addition of the consortia of cyanobacteria/microalgae–bacteria afterbioremediation.

phototrophic bacteria and sulfate-reducing bacteria. These mats havecomplex relationships with trophically-related bacterial groups andthe physiologies of habitat-forming (edificatory) cyanobacteria. Thesecyanobacterial mats were creditedwith self-cleaning properties in theArabian Gulf Coasts (Sorkhoh et al., 1992). ‘Symbiosis,’ which wasdefined as two or more differently named organisms living togetherby de Bary (1879), is an ecological adaptation to life in manyoligotrophic habitats. The symbionts of stromatolites or mats andtheir functioning are more difficult to identify by standardmicroscopyor traditional culture-based microbiological methods due to thecomplexity of the mixed assemblage.

The classic view of microbial mats suggests that the layering ofdifferent microbial members is due to the sequence of metabolicreactions determined by gradients of light and redox potential. Themicrobial populations are required to carry out metabolic reactionsfor gaining redox energy at rates faster than the equivalent chemicalreactions. The metabolic rates (community production per unit mass)of microbial members within these mats are higher than that of rainforests (Jørgensen, 2001). Within the microbial mats, physical effects(dissolution, precipitation, volatilization, and fixation of elements),chemical processes (hydrolysis, condensation, biosynthesis, biotrans-formation and biodegradation), and spatial translocations (mediated bytransport driven by concentration gradients and physical processes)occur due to the coupling of several redox reactions. Information on theorganization and functioning of modern stromatolites and microbialmats can help in the process optimization since the loading anddegradation of pollutants are also affected by these changes.

3. Interactions between cyanobacteria/microalgae and bacteria:extent of relatedness

Cyanobacteria, the cosmopolitan photosynthetic eubacteria, releasea variety of organic molecules, which include low molecular weightcompounds and extrapolymeric substances composed of proteins, lipidsand nucleic acids, mannitol and arabinose as the excretion products,glycolate under hyperoxic and alkaline conditions, and acetate,propionate, lactate and ethanol as fermentation products. All thesemolecules serve as bacterial growth substrates. In the cyanobacterialmats, different heterotrophic bacterial populations are specialized in theuse of specific exudates (Abed et al., 2007). Recently, Abed (2010) foundthat the co-culturing of Pseudomonas related GM41 strain and thecyanobacterium Synechocystis PCC6803, both isolated from the cyano-bacterial mat, led to an 8-fold increase in the cyanobacterial biomass.The cyanobacterial exudates not only serve as an endogenous source ofgrowth substrates to bacteria but also influence the rate of bacterialdegradation (repression or enhancement) of aromatic and aliphaticcontaminants (Kirkwood et al., 2006).

Interactions between autotrophic algae andheterotrophic bacteria canbe cooperative or competitive. Certain bacteria accompany microalgae,even under the conditions of laboratory cultivation as unialgal cultures(Borisova and Nogina, 2000). This has led to the scientific concernswhether microalgae are axenic or not (Radwan and Al-Hasan, 2000). Theunialgal culture may represent the natural algal–bacterial consortiumformed between microalgae and their associated bacteria. The closecontact between two aerobic bacteria (Pseudomonas diminuta andP. vesicularis isolated from laboratory algal cultures) and microalgae(Scenedesmus bicellularis and Chlorella sp.) had led to the stimulatorygrowth of algal cultures (Mouget et al., 1995). Even the compoundsreleased by a Chlorella sp. after exposure to water treatment chemicalssupported better growth of Escherichia coli (Bouteleux et al., 2005). Incontrast, an antagonistic relationship was observed between algae andLeptothrix ochracea in the iron-rich streams (Sheldon andWellnitz, 1998).

Microalgae produce sheaths (of trilaminar organization) whichconsist of carbohydrate, protein and metal cations that are related toformation of algal cell aggregation, wherein bacteria are associatedwith (Croft et al., 2006). Indirect adhesion of bacterial symbionts on


the sheath and the direct adhesion onto the algal cell surface mayreduce diffusion distance and permit rapid and efficient exchange ofsubstrates (Park et al., 2008). Althoughmany photosynthetic algae areconsidered to be completely autotrophic, they require (i) vitamins likebiotin, thiamine and cobalamine as growth factors as the cobalamineauxotrophs are widespread (Croft et al., 2006), and (ii) bacterialsiderophores for growth under iron-deficient conditions (Butler, 1998).Co-inoculation of bacterial strains isolated from the long-term labora-tory algal cultures had resulted in better algal growth than that of algaealone.

Closeproximity, chemotaxis,mobility andadhesionwithalgal host areimportant forbacterial association. Primarily, theaerobicheterotrophsusethe photosynthetically-produced carbon compounds whose accumula-tion can inhibit algal photosynthesis (Bateson andWard, 1988). In naturalsystems, the algal release of dissolved organic carbon ranges from zero to80% of photosynthates and it is around 6 to 16% in the microalgalphotoreactors (Hulatt and Thomas, 2010). Photosynthesis which is areversible set of reactions is inhibited by excessive dissolved oxygen. Inthe enclosed photoreactors, dissolved oxygen supersaturation can be ashigh as 400%, which can inhibit microalgal growth (Kumar et al., 2010).The dissolved O2, a photosynthetic by-product, can lower the netphotosynthetic carbon fixation by favoring the ribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco) activity. But, the bacterial consumptionof O2 lowers the O2 tension within the microenvironment of algal cells,leading to more favorable conditions for algal growth (Mouget et al.,1995). Under normal growth conditions, the photosynthetic rate is 4–7folds higher than the algal respiration rate. Hence, the bacterialconsumption of O2 is an important means of bacteria-mediated algalgrowth enhancement, and similar effects are shown for N2 fixation bycyanobacterial heterocysts. Algae and cyanobacteria can use CO2 forphotosynthesis, produced by bacterial mineralization, and sugars, acetateand glycerol for their heterotrophic growth. However, the algal growthcan also inhibit bacterial activity by releasing toxicmetabolites, increasingthe temperature, and keeping high O2 levels (Skulberg, 2000).

4. Cyanobacteria/microalgae: pollutant removal

Cyanobacteria which possess dinitrogen-fixing capabilities andmicroalgae which probably account for up to 27% of total soil microbialbiomass are widespread in soil and aquatic ecosystems (Burns andHardy, 1975; McCann and Cullimore, 1979). These organisms not onlyaid in detecting pollution (Megharaj et al., 1989) but also transformmany pollutants in the environment. They have been implicated in themetabolism of certain organophosphate insecticides such as mono-crotophos and quinalphos (Megharaj et al., 1987) andmethyl parathion(Megharaj et al., 1994). The alterations in the species composition ofalgae and cyanobacteria in the soil with the long-term contamination ofDDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) can serve as use-ful bioindicators of pollution (Megharaj et al., 2000). Cyanobacteria(particularly dinitrogen-fixing species of Anabaena and Nostoc) prefer-entially transformedDDT to DDD (1,1-dichloro-2,2 bis(p-chlorophenyl)ethane) while green algae (Chlorococcum spp.) converted DDT to DDE(1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene). Toxicity and transfor-mation of pollutants by cyanobacteria or microalgae may changedepending on the species. Cáceres et al. (2008a) observed transforma-tion of an organophosphorous pesticide, fenamiphos (ethyl 4-methylthio-m-tolyl isopropyl phosphoramidate) to its primary oxida-tion product, fenamiphos sulfoxide (FSO) by five different species ofcyanobacteria and five of green algae. Both the pesticide and itsmetabolites (FSO, fenamiphos sulfone, fenamiphos phenol, fenamiphossulfoxide phenol and fenamiphos sulfone phenol) bioaccumulated inthe terrestrial alga Chlorococcum sp. while only metabolites wereaccumulated in the aquatic alga Pseudokirchneriella subcapitata (Cácereset al., 2008b). The abilities to transform or degrade pollutants bycyanobacteria andmicroalgae are gainfully exploited in bioremediationtechnologies of many polluted systems.

The presence of cyanobacterial mats in the Arabian Gulf Coastsafter oil pollution had received enough attention because of theirpotential to degrade hydrocarbons (Sorkhoh et al., 1992). Bacteriaisolated from the blue-greenmats of cyanobacteria growing at the oilycoasts of Kuwait degraded, though not rapidly, most of thehydrocarbons present in the oil. Alcanotrophic bacteria in combina-tion with certain microalgae and cyanobacteria degraded black oilthough oil is toxic to the algae (Safonova et al., 1999). The strains ofmicroalgae (Stichococcus sp.,Chlorella sp. and Scenedesmus quadricauda)and cyanaobacteria (Nostoc sp., and Phormidium sp.) are capable ofgrowing without any apparent toxic effect in medium containing 1%black oil and bacterial protectants. The bacteria protected the algae fromtoxic effect of the oil, while the cyanobacteria provide the bacteria withO2 and other extracellular carbon-rich exudates. Abed and Köster(2005), and Abed (2010) observed that the ability of Oscillatoria sp. andother cyanobacterial mats to degrade hydrocarbons in oil was not oftheir own but was due to the association of aerobic heterotrophicbacteria.

The microbial mats of cyanobacteria or microalgae are naturally-occurring immobilized microbial systems (Radwan et al., 2002), andthe water–sediment interface supports cyanobacterial mats inestuaries, lagoons or sheltered sandy beaches. In most aquatic andterrestrial environments, O2 is one of the limiting factors for microbialhydrocarbon degradation. The hydrocarbon-utilizing bacteria associatedwith cyanobacterial mats obtain O2 from the photosynthesis, probablynitrogenous and phosphorus compounds, vitamins, and protection frombeing washed out or diluted. According to de Oteyza et al. (2006), theconstructed cyanobacterial mats degraded hydrocarbons of lowervolatility (C24–C30 n-alkanes or carbazoles) better than those with lowmolecularweighthydrocarbons (n-alkaneswithchain lengthshorter thann-pentadecane or n-heptadecane, regular isoprenoid hydrocarbons withchain length lower than C16 or C18 or lower molecular weightnaphthalenes). The roles of cyanobacteria include the provision of oxygenby photosynthesis for the breakdown of aliphatic and aromaticcompounds, fixed nitrogen, and organic exudates by photosynthesisand fermentation. The ability to metabolize a range of cyanobacterialphotosynthetic and fermentative exudates can get around the need oforganic or inorganic fertilizer application for thehydrocarbondegradationby the hydrocarbon-degrading bacteria. These microbial mats withhydrocarbon-degrading bacteria offer many advantages since neitherbioaugmentation with pollutant-degrading bacteria or biostimulationwith fertilizers for the naturally-occurring bacteria is feasible due todilution effects in the polluted water bodies.

5. Consortium of cyanobacteria/microalgae for pollutant removal:proof-of-principle

Mixed populations (co-culture or consortia) can perform functionsthat are difficult or even impossible for individual strains or speciesand those which require multiple steps (Brenner et al., 2008). Livingtogether may provide robustness to environmental fluctuations,stability for the members, ability to share metabolites and weatherperiods of nutrient limitations, and resistance to invasion by otherspecies. There have been many proof-of-principle studies on theconsortia of cyanobacteria/microalgae–bacteria for pollutantdegradation.

5.1. Organic pollutants

The O2 production capacity, tolerance to pollutants, and ability tosupport pollutant degradation with varied efficiencies by the bacterialsymbiont can differ among the algal/cyanobacterial species (Table 1).For example, the algal–bacterial microcosms comprising of thesalicylate-degrading Ralstonia basilensis, the phenol-degrading Acine-tobacter haemolyticus and the phenanthrene-degrading Pseudomonasmigulae and Sphingomonas yanoikuyae, and the green alga Chlorella

Table 1Degradation of organic chemical pollutants by consortia of cyanobacteria/microalgae and bacteria.

Cyanobacterium/microalga Bacterium Pollutant and its removal efficiency Reference

Oscillatoria strain OSC Proteobacteria naturallyassociated with Oscillatoria sp.

n-Octadecane 40%pristine 50%phenanthrene 50%dibenzothiophene 80%(1 mg ml−1 organo clay complexcontaining 16.68 (%) of petroleum compounds)

Abed and Köster (2005)

Organics present in Synechocystis sp. PCC6803 Pseudomonas-related strain GM41 Phenanthrene 0.8 μg day−1 (0.15 mM) Abed (2010)Cyanobacterial mats Acinetobacter calcoaceticus Nocardioforms Oil 63.2% (0.5% v/v) Al-Awadhi et al. (2003)Pseudoanabaena PP16 Pseudomonas sp. P1 Phenol 95% (1 mM) Kirkwood et al. (2006)Chlorella sorokiniana 211/8k Ralstonia basilensis Sodium salicylate 1 mmol l−1 day (5 mM)a Guieysse et al. (2002)C. sorokiniana Pseudomonas migulae Phenanthrene 24.2 g m−3 h−1 (200–500 mg l−1) Muñoz et al. (2003a)C. sorokiniana R. basilensis Salicylate 100% (5 mM) Borde et al. (2003)C. sorokiniana Ralstonia sp. Sodium salicylate 19 mg l−1 h−1 (800 mg l−1) Muñoz et al. (2003b)C. sorokiniana R. basilensis Sodium salicylate 87 mg l−1 h−1 (1 g l−1) Muñoz et al. (2004)C. sorokiniana Acinetobacter haemolyticus Phenol 89% (4.25 mM) Borde et al. (2003)C. sorokiniana P. migulae

Sphingomonas yanoikuyaePhenanthrene 15% (1.7 mM) Borde et al. (2003)

C. sorokiniana Comamonas sp. Acetonitrile 0.44 g l−1 day−1 (1 g l−1) Muñoz et al. (2005a)C. sorokiniana Bacterial consortia Acetonitrile 12.8 mg l−1 h−1 (1 g l−1) Muñoz et al. (2005b)C. sorokiniana P. migulae Phenanthrene 36 mg l−1 h−1 (5 g l−1) Muñoz et al. (2005c)C. sorokiniana R. basilensis Sodium salicylate 76% (500 mg l−1) Muñoz et al. (2006)C. sorokiniana IAM C-212 Microbacterium sp. CSSB-3 Propionate 100% (125 mg l−1) Imase et al. (2008)C. sorokiniana 211/8k R. basilensis Sodium salicylate 100–74% (2 g l−1) Muñoz et al. (2009)Chlorella vulgaris R. basilensis Sodium salicylate 14 mg l−1 h−1 (800 mg l−1) Muñoz et al. (2003b)Bolivian microalga strain R. basilensis Sodium salicylate 18 mg l−1 h−1 (800 mg l−1) Muñoz et al. (2003b)Chlorella sp.Scenedesmus obliquusStichococcus sp.Phormidium sp.

Rhodococcus, Kibdelosporangium aridum Phenols 85% (0.48 mg l−1)oil 96% (40 mg l−1)

Safonova et al. (2004)

Selanastrum capricornutum UTEX 1648 Mycobacterium sp. RJGII 135 BaP 73% (14C-BaP (8 μg/0.5 μ Ci) Warshawsky et al. (2007)S. capricornutum UTEX 1648 S. yanoikuyae strain B1 BaP 94% (14C-BaP (8 μg/0.5 μ Ci) Warshawsky et al. (2007)Phormidium sp.Oscillatoria sp.Chroococcus sp.

Burkholderia cepacia Diesel 99.5% (0.6% v/v) Chavan and Mukherji (2008)

Phormidium sp.Oscillatoria sp.Chroococcus sp.

B. cepacia Total petroleum hydrocarbon99% (diesel 0.6% v/v)

Chavan and Mukherji (2010)

Scenedesmus obliquus GH2 Sphingomonas sp. GY2BB. cepacia GS3CPseudomonas GP3APandoraea pnomenusa GP3B

Straight chain alkanes 100%alkylcycloalkanes and alkylbenzenes 100%naphthalene, fluorene and phenanthrene100% (crude oil 0.3% v/v)

Tang et al. (2010)

a Values in parentheses are the initial concentrations added.


sorokiniana were efficient in protecting the green alga from the toxicconcentrations and in the removal (up to 85%) of these threepollutants (Muñoz et al., 2003a). The consortium of C. sorokinianaand R. basilensis strain was reported to degrade sodium salicylate in acontinuous stirred tank reactor. But, Selenastrum capricornutum andAnabaena catenula were completely inhibited by salicylate at500 mg L−1. The salicylate-degrading R. basilensis when grown withsalicylate-resistant alga, C. sorokiniana, rapidly metabolized thechemical at a rate of 19 mg L−1 h−1 (Muñoz et al., 2003b). Both theO2 generation rate, which is proportional to the algal density and themutual shading, and the O2 consumption due to algal respirationinfluence the degradation of salicylate (Guieysse et al., 2002).

Photosynthesis-enhanced biodegradation of toxic aromatic pollutantscan occur in the algal–bacterial microcosms in a one-stage treatment. Thedegradation of N-containing organic compounds can bemore efficient bythe algal–bacterial consortia than bacteria alone asmicroalgae can readilyassimilate NH4

+ released. The consortium of C. sorokiniana and theacetonitrile-degrading Comamonas sp. completely degraded acetonitrilein the column photobioreactor (Muñoz et al., 2005a). But, higherconcentrations of acetonitrile resulted in the inhibition of microalgalactivity due to the combination of high pH and high NH4

+. Hirooka et al.(2003) used the cyanobacterial mixed culture of Anabaena variabilis andAnabaena cylindrica to remove 2,4-dinitrophenol (2,4-DNP) from indus-trial wastewater, without accumulating 2-amino-4-nitrophenol (2-ANP),the degradation product which is a potent mutagen. Although A.variabilis alone had the ability to remove 2,4-DNP at the concentrations

of 5–150 μM with a light and dark cycle, 2-ANP was otherwiseaccumulated in the culture medium.

Polyaromatic hydrocarbons (PAHs) are common constituents ofcombustion residues. PAHs of only one, two, or three rings can bedegraded completely by microorganisms while four and five ringPAHs are recalcitrant. Benzo[a]pyrene (BaP), a five-ring PAH, can bedegraded by the green alga, S. capricornutum strain UTEX 1648, usingdioxygenases with the formation of BaP sulfate ester and glucoseconjugates, while Mycobacterium sp. strain RJGII and S. yanoikuyaestrain B1 can partially degrade BaP (Warshawsky et al., 2007). Thecombination of three organisms was more useful for degrading BaPthan individual species. In natural systems, microbial consortia, ratherthan individual species, are useful for degrading mixtures ofpollutants (Ramakrishnan et al., 2011).

Naturally-occurring cyanobacterialmats are found to aid hydrocarbondegradation. Abed and Köster (2005) demonstrated the presence ofdiverse, aerobic heterotrophic bacteria with both degradative abilities forhydrocarbons and with abilities for specialized consumption of specificexudates from cyanobacteria (species belonging to the genera Marino-bacter and Alcanivorax) and another guild (species of Marinobacter,Halomonas, Roseobacter and Rhodobacter)with ability to use exudates butnot hydrocarbons. The addition of substrates which are representative ofcyanobacterial exudates had variable effects on phenanthrene degrada-tion; acetate, pyruvate, and glucose enhanced the degradation whilealanine and butanol had no effect on the phenanthrene-degrading strainGM 42 (Abed, 2010). By increasing volume of the sheath in C. sorokiniana


with the addition of CaCl2 and constructing a complexwith themicroalgaand a propionate-degrading bacterium (strain PDS1), Imase et al. (2008)demonstrated the enhanced algal growth in a medium containing highconcentration (1.2 g L−1) of propionate besides complete bacterialdegradation of propionate. Methyl tert-butyl ether (MTBE) degradationby the microalgal–bacterial symbiotic system containing a mixed cultureof Methylibium petroleiphilum PMI and Chlorella ellipsoidea with theoptimal ratio of initial cell population of bacteria to algae of 100:1 washigher than the pure cultures of bacteria (Zhong et al., 2011).

Industrial approaches such as filtration, centrifugation and micro-straining are not economical and suitable for removal of algal biomass.The bioflocculent algal–bacterial biomass can help to separate algalbiomass by gravity sedimentation which can circumvent the need forflocculation, filtration or centrifugation required in the conventionaland high rate algal ponds (Gutzeit et al., 2005). Extracellularpolymeric substances and other factors (e.g. content of calcium)influence the formation and stability of flocculent algal–bacterialbiomass. The bacterial exopolymer production can result in theincrease of aggregation possibilities of algal–bacterial consortium,serve as stabilizers of already existing aggregates and efficientlyincrease sedimentation. Muñoz et al. (2009) used the biofilmphotobioreactor using C. sorokiniana–R. basilensis consortium immo-bilized onto foamed-glass beads carriers (Poraver®) and onto reactorwall for treating salicylate contaminant in wastewater.

5.2. Metal pollutants

Cell walls of microalgae and cyanobacteria are composed ofpolysaccharides and carbohydrates that have negatively-charged(amino, carboxyl, hydroxyl or sulfide) groups. Most metals arebound to the negatively-charged ligand groups, which is the basisfor metal removal from wastewaters. Beside this mechanism of metaladsorption onto cell surfaces and extracellular polysaccharides,uptake into cells, incorporation into vacuoles or aragonite (CaCO3)structures, and precipitation on the cell surface or internally canoccur. But, heavy metals are potent inhibitors of photosynthesis asthey can replace or block the prosthetic metal atoms in the active sitesof certain enzymes. Likewise, the acidic functional groups of bacterialcell walls can also bind significant concentrations of aqueous cations,which can affect the speciation, distribution and mobility of thosecations (Ginn and Fein, 2008). Algae growing in wastewaters mayprovide a simple, long-term strategy for removal of metal pollutants(Table 2). To this end, Kalin et al. (2004) described a three-stepprocess for the detoxification of uranium from wastewaters. Initially,the ligands in algal cell walls efficiently remove U(VI) fromwastewaters followed by the removal of U-algal particulates fromthe water column to the sediments. Subsequently, the dead algal cells

Table 2Heavy metal removal from wastewater by consortia of cyanobacteria/microalgae and bacte

Cyanobacterium/microalga

Bacterium Source ofwastewater

Spirulina platensis Sulfate-reducing bacteria Tannery effluent

Chlorella sp.Scenedesmus obliquusStichococcus sp.Phormidium sp.

Rhodococcus sp.Kibdelosporangium aridum

Oil polluted wastewater

C. sorokiniana (biomass) R. basilensis (biomass) Bristol medium with poll

Algae from wastewatertreatment plant (biomass)

Bacteria from wastewatertreatment plant (biomass)

Artificial metal solutionscontaining cadmium and

a Values in parentheses are the initial concentrations added.

provide carbon, nitrogen and phosphorus to the heterotrophicbacteria for the final reduction of U(VI) to U(IV).

The algal–bacterial consortium comprising of C. sorokiniana andR. basilensis was found to metabolize salicylate with a subsequentremoval of heavy metals from the solutions (Muñoz et al., 2006). Theconsortium removed copper more efficiently than the individualorganism at pH 5.0, and nickel, cadmium and zinc were less efficientlyremoved. Dried biomass from amixture of cyanobacteria and bacteria isused to remove the heavy metals from wastewaters, and metals arerecovered subsequently by desorption. Very efficient removal of copper(≈80%) and cadmium (≈100%) from metal waste with a maximumremoval ratewithin 5 minof contact timewas observedwithdriedmassof a mixed culture of microalgae (Scenedesmus sp., Tetraedron sp.,Chlorella sp., Chlorococcus sp.), cyanobacteria (Chroococcus sp., Pseu-doanbaena sp., Leptolyngbya sp.), diatoms (Navicula sp., Nitzschia sp.,Cyclotella sp.) and bacteria in a biofilter (Loutseti et al., 2009).Physiological adaptation, genetic changes or the succession of sensitivespecies by more tolerant bacteria contribute to metal tolerance inbacterial communities. In response of bacteria to metals, correlationexists between the genetic and physiological structure of bacterialcommunities and the species composition of the algal community, butnot the level of metal pollution. There is a strong and species-specificlinkage between bacterial and algal species (Boivin et al., 2007).

5.3. Nutrient removal from wastewaters

Activated sludge method is probably the first major use ofbiotechnology in bioremediation applications and continues to bean effective technology for pollution containment (Gavrilescu andChisti, 2005). The association of microalga, Chlorella vulgarisHamburg,and activated sludge bacteria improved the performance of wastestabilization ponds, the removal of organic matter, nutrients andpathogens, the content of O2 with no need of aeration and effectiveseparation of alga by sedimentation (Medina and Neis, 2007). Manyimprovements have led to the use of algal–bacterial consortium infacultative ponds and high rate algal ponds (HRAPs). The floccularbiomass of ‘ALBAZOD’ comprising of algae, bacteria, zooplankton anddetritus bound together generally occurs in HRAPs. The environmentaland/or pond operational changes in terms of COD loading rates andretention time led to alterations to gross algal composition and celldimensions of different species in these floccularmaterials (Cromar andFallowfield, 2003).

Various types of bioreactors now offer optimal conditions oftemperature, pH, O2 transfer, mixing, and substrate concentration forefficient cellular metabolism, besides providing the basic function ofcontainment. Oxidation of both organic matter and ammonium wasachieved by the C. sorokiniana-mixed bacterial culture from the

ria.

Metal and itsremoval efficiency

System/reactor used

Reference

Copper 79.2% (500 mg l−1)a

zinc 88.0% (500 mg l−1)iron 100% (500 mg l−1)

High ratealgal pond

Rose et al.(1998)

Copper 62% (0.04 mg l−1)nickel 62% (0.21 mg l−1)zinc 90% (0.10 mg l−1)iron 64% (6.43 mg l−1)manganese 70% (0.20 mg l−1)

Pilot installation Safonova etal. (2004)

utants Copper 57.5% (20 mg l−1) @ pH 5.0 Conical glassreactor

Muñoz et al.(2006)

copperCopper 80% (100 mg l−1)cadmium 100% (100 mg l−1) @ pH 4.0

Continuous flow-through column

Loutseti et al.(2009)

Table 3Removal of nutrients from wastewater by consortia of cyanobacteria/microalgae and bacteria.

Cyanobacterium/microalga Bacterium Source of waste water Nutrients and its removal efficiency System/reactor used Reference

Spirulina platensis Sulfate-reducing bacteria Tannery effluent Sulfate 80% (2000 mg l−1)a High rate algal pond (HRAP) Rose et al. (1998)Chlorella vulgaris Azospirillum brasilense Synthetic wastewater Ammonia 91% (21 mg l−l)

phosphorous 75% (15 mg l−l)Chemostat (Virtis, Gardiner, NY) de-Bashan et al. (2002b)

C. vulgaris Wastewater bacteria Pretreated sewage DOC 93% (230 mg C l−1)nitrogen 15% (78.5 mg l−1)phosphorous 47% (10.8 mg l−1)

Photobioreactor pilot-scale Gutzeit et al. (2005)

C. vulgaris Alcaligenes sp. Coke factory wastewater NH+4 45% (500 mg l−l)

phenol 100% (325 mg l−l)Continuous photobioreactorwith sludge recirculation

Tamer et al. (2006)

C. vulgaris A. brasilense Synthetic wastewater Phosphorous 31.5% (50 mg l−l)nitrogen 22% (50 mg l−l)

Inverted conical glass bioreactor Perez-Garcia et al.(2010)

Chlorella sorokiniana Mixed bacterial culture from anactivated sludge process

Swine wastewater Phosphorous 86% (15 mg l−l)nitrogen 99% (180 mg l−l)

Tubular biofilm photobioreactor González et al. (2008a)

C. sorokiniana Activated sludge bacteria Pretreated piggerywastewater

TOC 86% (645 mg l−l)nitrogen 87% (373 mg l−l)

Glass bottle González et al. (2008b)

C. sorokiniana Activated sludge consortium Pretreated swine slurry TOC 9–61% (1247 mg l−l)nitrogen 94–100% (656 mg l−l)phosphorous 70–90% (117 mg l−l)

Tubular biofilm photobioreactor de Godos et al. (2009a)

C. sorokiniana Activated sludge bacteria Piggery wastewater TOC 47% (550 mg l−1)phosphorous 54% (19.4 mg l−1)NH+

4 21% (350 mg l−1)

Jacketed glass tank photobioreactor de Godos et al. (2010)

Euglena viridis Activated sludge bacteria Piggery wastewater TOC 51% (450 mg l−1)phosphorous 53% (19.4 mg l−1)NH+

4 34% (320 mg l−1)

Jacketed glass tank photobioreactor de Godos et al. (2010)

Microalgae present in tertiary stabilizationpond treating domestic wastewater

Bacteria present in tertiary stabilizationpond treating domestic wastewater

Piggery wastewater COD 58.7% (526 mg l−l)TKN 78% (59 mg l−l)

HRAP de Godos et al. (2009b)

Resident algae in facultative lagoon system Nitrifying bacteria in facultativelagoon system

Dairy wastewater Ammoniacal-NN99% (147 g m−3)fecal enterococci 95% (22,000 g m−3)

Two-pond lagoon system Sukias et al. (2003)

Microalgae from wastewater Indigenous nitrate- and selenium-reducingbacteria

Drainage water Selenium 77% (400 μg l−1)Nitrogen 95% (95 mg l−l)

Advanced integrated wastewater pondsystem

Green et al. (2003)

Algae from wastewater stabilization ponds Activated sludge bacteria from municipalwastewater treatment plant

Synthetic wastewater Ammonia 20% (21 mg l−l) Wastewater stabilization pond Babu et al. (2010)

a Values in parentheses are the initial concentrations added. Abbreviations: DOC = dissolved oxygen concentration; TKN = total Kjeldahl nitrogen; COD = chemical oxygen demand; TOC = total organic carbon.

902S.R.Subashchandrabose

etal./

BiotechnologyAdvances

29(2011)

896–907


activated sludge process in the tubular biofilm photobioreactor(González et al., 2008a). The enclosed biofilm photo-bioreactors areeconomical and have nutrient removal efficiencies of up to 99% NH4

+,86% PO4

3−, and 75% total COD. Wu et al. (2011) proposed a hybridbioreactor which supports heterotrophic and autotrophic microor-ganisms for the removal of high-loading nutrients with efficiencies ofabout 81% for total phosphorus, 74% for total dissolved phosphorus,82% for total nitrogen, 79% for NO3

−-N and 86% for NH4+-N. Removal of

nutrients into biomass (PO43− precipitation or NH3 stripping) add up

to this environmental friendly approach of using the algal–bacterialconsortium for the wastewater reclamation (Table 3). In addition, theresidual biomass can be exploited as green fertilizer due to the slowrelease of nutrients into the soil or as biosorbent for heavymetals. Thespecies of Chlorella and Scenedesmuswhich are used for domestic sewagetreatment are rich in protein, mineral salts, vitamins A and B, and mostamino acids, comparable to the levels found in fish meal and soy bean(Kawai et al., 1984). In a recent study, Bhatnagar et al. (2010) observed apromising fuel alga Chlorella minutissima for cultivation in municipalwaters. Complex relationships amongmicroalgae/cyanobacteria, bacteria,light and pollutant concentrations need to be understood to optimizeparameters for the operation of photobioreactor or HRAPs for thedegradation of different pollutants.

6. CO2 capture and sequestration by the consortia: mitigation ofgreenhouse gas

The purpose of algal cultivation has always depended on thespecific needs which include wastewater treatment, biomass forbiogas generation or use in aquaculture, production of fine chemicalsand extracellular compounds, and even in CO2 fixation (Ho et al.,2010; Ugwu et al., 2008). An initial report on mass cultivation of algaefor CO2 abatement was at Carnegie Institute in Washington (Burlew,1953). CO2 is one of the purported greenhouse gasses (Knight et al.,2009), and, as a consequence of global warming the emissions of CO2

need to be mitigated. Since the CO2 fixation rates of microalgae/cyanobacteria are about 10–50 times faster than terrestrial plants, theuse of these biological agents is considered as one of the effectiveapproaches to fixing CO2 and thereby mitigating possible globalwarming. On mass basis, algal dry biomass of 1 kg requires about1.83 kg of CO2 (Chisti, 2007). Traditionally, the sources of CO2 for thealgal cultivation are: (i) flue gas emitted by the coal fired power plants(typically with 10–20% CO2), (ii) increased CO2 concentration in theclosed photobioreactor (no more than 1.0%), and (iii) atmosphericCO2 (380 ppm) (Wang et al., 2008; Yun et al., 1997). With economicincentives in terms of carbon credits provided under the Kyotoprotocol (Wang et al., 2008), CO2 capture and sequestration by thealgal–bacterial consortiumwill gain more importance in the future. Inthis consortium, the mutual dependence for substrates such as CO2

and oxygen will enhance the efficiencies of CO2 fixation.CO2 mitigation and removal of nutrients into biomass (PO4

3−

precipitation orNH3 stripping)not only contribute to theenvironmentalfriendly approaches to wastewater reclamation but also to the biomassutilization as ‘green fertilizer’ due to the slow release of nutrients intothe soil or as biosorbent for metal removal. Energy savings in supply ofsubstrates and revalorization of algal biomass, a pay-back to theoperation cost make this mitigation option very attractive and valuable.For the purpose of CO2 abatement, the selection of algal strains for theirability to utilize CO2 at higher rates, especially tolerance to elevatedlevels of CO2 and temperature (Chinnasamy et al., 2009), suitablebacterial partners, and appropriate design of bioreactors at the fieldscale are important prerequisites and objectives of future research.

7. Microbial solar cells: the futuristic consortium

Research efforts are now on integrating photosynthesis withmicrobial fuel cells (photoMFCs). Two types of photoMFCs such as (i)

electrocatalytic bioelectrochemical systems that convert hydrogenfrom photosynthesis, and (ii) the sediment-based bioelectrochemicalsystems that convert excreted organics are on the basis of synergisticrelationships between photosynthetic producers (cyanobacteria orplants) and heterotrophic bacteria, widely established in variousecosystems, for example, microbial mats (Rosenbaum et al., 2010). Anew biotechnological system that integrates photosynthetic andelectrochemically active organisms to generate in situ green electricityor chemical compounds (hydrogen, methane, ethanol or hydrogenperoxide) is microbial solar cell (MSC). The basic principles of MSCs areon the premises of photosynthesis, transport of organics to the anode,anodic oxidation of organics by electrochemically active bacteria andcathodic reduction of O2 (Strik et al., 2011). He et al. (2009)demonstrated that the MSCs with phototrophic biofilms containingmembers of Chlorophyta and/or cyanobacteria and mixed microbialpopulations of Firmicutes and a Gammaproteobacterium (closelyrelated to Alkaliliminicola ehrlichii) could convert solar energy toelectricity on the anode of a fuel cell. In the MSC with a photoreactorand an anaerobic digester, photosynthesis by cyanobacteria/microalgaeoccurs in the photoreactor; biogas is produced from the organic matterand transported to the digester. At the anode of theMFC, the remainingorganicmatter is oxidized by the electrochemically active bacteriawhileO2 from the photoreactor is reduced towater at the cathode (Strik et al.,2008). Better utilization of the solar spectrum can occur in theconsortium of algae and bacteria. Not only electricity but a range offuels and useful chemicals are produced in theMSCswhich are based onthe photosynthetic activity of cyanobacteria/microalgae and electro-chemical reactions of heterotrophic bacteria. The future applications ofthis technology can benefit from the isolation of efficient members andengineering of stable microbial consortia within a single reactor.

8. Microbial community engineering: construction of theconsortium

An early report, and subsequent patenting, on the construction ofmicrobial communities of cyanobacteria and bacteria for pollutantdegradation was provided by Bender and Phillips (1994). The thick,gelatinous green mat was constructed from an artificial ecosystem inglass containers consisting of a soil base to provide motile bacteria,filtered tap water, a floating layer of ensiled grass clippings (as a sourceof lactic and acetic acid aswell as amicrobial consortiumof fermentativeanaerobes) and cyanobacteria (Oscillatoria sp.). With the pre-exposureto chlordane, the constructed mat was found to degrade pesticides(carbofuran, chlordane, and paraquat) better than indigenous bacteria(Murray et al., 1997). Paniagua-Michel and Garcia (2003) collected thenatural microbial mats from marine sediments, constructed microbialmats after immobilizing the mat on glass wool, prepared theconstructed mats for adaptation by step-wise additions of water fromeffluent to be treated, and then found those microbial mats to remove97% and 95% of ammoniacal-nitrogen and nitrate-nitrogen from shrimpculture effluents. The constructedmicrobialmats hadfilamentous formsof cyanobacteria (Microcoleus chthonoplastes, Spirulina sp., Oscillatoriasp., Schizothrix sp., Calothrix sp. and Phormidium sp.), green algae(Chlorella sp. and Dunaliella sp.), diatoms (Nitzchia sp. and Navicula sp.)and nitrifying bacteria (Nitrosomonas sp. and Nitrobacter sp.).

Immobilization of algae (species of Chlorella, Scenedesmus, Sticho-coccus and Phormidium) onto capron fibers and bacterial strains(Rhodococcus sp., Kibdelosporangium aridum and two unidentifiedstrains) onto ceramics, capron andwood led to the formation of stableconsortia, preventing them from being washed off, and to the removalof phenols (85%), anionic surface active substances (73%), oil spills(96%), copper (62%), nickel (62%), zinc (90%), manganese (70%) andiron (64%), and the reductions of BOD25 to 97% and COD to 51%,respectively (Safonova et al., 2004). In other constructed cyanobac-terial mats, hydrocarbons of lower volatility (C24–C30 n-alkanes orcarbazoles) were degraded better than the low molecular weight


hydrocarbons (n-alkanes with chain length shorter than n-pentade-cane or n-heptadecane, regular isoprenoid hydrocarbons with chainlength lower than C16 or C18 or lower molecular weight thannaphthalenes) (de Oteyza et al., 2006). The consortium comprisingspecies of Phormidium, Oscillatoria and Chroococcus, and the oil-degrading bacterium, Burkholderia cepacia, developed on a rotatingbiological contactor (RBC), resulted in good total petroleum hydro-carbon removal and settleability of biosolids, without the requirementof a soluble carbon source. The performance efficiency of the reactorfor the treatment of complex non-aqueous phase liquids (NAPLs) andthe relative dominance of the phototrophic microorganisms andbacteria was determined by the N:P ratio (Chavan and Mukherji,2008). Tang et al. (2010) found that the unialgal culture of the oil-tolerant Scenedesmus obliquus GH2 was unsuitable, compared to theaxenic culture, for the construction of the consortium with crude oil-degrading bacteria (Sphingomonas sp. GY2B, B. cepacia GS3C and amixed culture, named GP3).

Themicroalgal or cyanobacterial growth has limitations, despite theefforts to improve by various technological, physical, molecular andenvironmental methods. For the continuous treatment of variable inletconcentrations of pollutants, algal selection is paramount as thepollutants can decrease the photosynthetic activity, leading to processfailure. González and Bashan (2000) proposed the hypothesis of using‘microalgal growth-promoting bacterium (MGPB)’ such as Azospirillumbrasilense to stimulate the microalgal growth by the phytohormonessynthesized by theMGPB cells. The co-immobilization of C. vulgaris andC. sorokiniana with A. brasilense led to increases in the contents ofchlorophyll a and b, lutein and violoaxanthin, lipid content(80–320 μg g−1 dry weight) and the number (5 to 8 different) of fattyacids (de-Bashan et al., 2002a). This provides strong evidence on theinfluences of bacteria onmicroalgal metabolism, species-specific effectsand the need for optimization of a bacterial–algal consortium.Thermodynamic conditions necessitate about 300 bacterial units to analgal unit for CO2 supply (Oronet al., 1979). Imase et al. (2008) proposeda method for constructing artificial communities of Chlorella sp. andsymbiotic microorganisms by increasing the volume of algal sheath byaddition of CaCl2 solution.

Pankratova et al. (2004) observed that a consortium of a cyano-bacterium, Nostoc palusodum Kutz and Rhizobium galegae grown over18 months in a liquid medium for seed treatment of Galega orientaliswas stable and maintained its structure and activity over long-termgrowth with positive effects as a seed inoculant. C. sorokinianacultivated in the slants with bacteria as a consortium was stable evenafter 7 months of sub-culturing (Watanabe et al., 2005). DGGE profilesof bacterial community showed that a consortium comprising of amicroalga, S. obliquus and four oil-degrading bacteria was stable after3 cycles of crude oil degradation, each cycle lasting for 8 days (Suet al., 2011). However, the diversity of species does not alwaysguarantee the survival and success of the engineered consortia thatwill perform better under the fluctuating environmental conditions.The stability of a microbial consortium relies on two importantorganizing features: (i) communication which refers to trading ofmetabolites and exchange of dedicated molecular signals (quorumsensing intraspecific signals, multispecies quorum sensing interspecificcues and cross feeding) within each population or between individuals,and (ii) the division of labor (Brenner et al., 2008; West et al., 2006). Inthe natural or engineered microbial consortia, the chances forcompetition, communication, or collaboration increase with thenumber of interacting agents (West et al., 2007;Wintermute and Silver,2010). Venturi et al. (2010) suggested that the general mechanism forstability is primarily the intercellular signaling in microbial communi-ties. The challenges which are to be met for achieving the stability ofengineered microbial consortium include (i) maintenance of long-termhomeostasis (or long-term extinction), (ii) functionality of consortiadespite horizontal gene transfer, (iii) incorporation of stable changesinto the genomes of microbial members, and (iv) fine-tuning of the

performance of multiple populations (Brenner et al., 2008). Goldmanand Brown (2009) suggested that the ecology and evolution theory hasgreat potential to overcome the problems associatedwith the long-termbehavior and stability of microbial consortia.

Bacteria that are associated earlierwith orwithout algae can interactcooperatively or competitivelywhenbothpartners are brought togetherin a consortium. Coordination amongmicrobialmemberswithin a givenspace and the maintenance of homeostasis will determine the survivaland success of a microbial consortium. Engineering of microbialconsortium should provide opportunities for re-introduction or elim-ination as needed and in which its tasks can be monitored over time,with enhanced functional capabilities (Brenner et al., 2008). Metaboliteprofiling approaches offer unprecedented opportunities to understandthe complex interactions among themicrobial members and to identifythe economically important metabolites. Likewise, metabolic engineer-ing can help to design new cell factories. Due to the large genomesequencing programs for a number of microorganisms, the functionalgenomics (Zhou et al., 2004) can aid in metabolic engineering thatprovides consent to the interactions among all the members of aconsortium. Metabolic profiling, functional genomics, and combinato-rial biochemical approaches including metabolic engineering canextend the concept of molecular farming for the algal–bacterialconsortium for efficient pollutant degradation. Efficient pollutantdegradation by a microbial consortium will gain public acceptancealong with the concurrent production of their useful metabolites orbioelectricity using wastewaters.

9. Conclusion

Photosynthetic oxygenation by microalgae or cyanobacteria andpollutant degradation by bacteria are an attractive proposition forwastewater treatment. Energy savings in O2 supply, process safety (norisk of aerosolization), CO2 mitigation, efficient recycling of nutrients,and revalorization of algal biomass, a pay-back to the plant operationcost, make their utilization very valuable. Further improvements inefficiencies of these biological agents for nutrient removal and/orpollutant degradation may enhance public acceptance of theseconsortia for producing microbial metabolites commercially (Fig. 2)by using polluted waters. For the continuous treatment of variableinlet concentrations of pollutants, algal or bacterial selection isparamount as the pollutants can decrease the survival and degrada-tion efficiency, leading to process failure. High concentrations oforganic pollutants like styrene and phenolic compounds can inhibitthe microbial degrader community while low aqueous solubility ofcompounds such as PAHs limits the biodegradation process. Metabolicroutes as well as bottlenecks formicrobial degradation of pollutants orphotosynthetic oxygenation are extensive, which require the need touse a molecular toolbox (Ramadas and Thattai, 2010). Compared tothe introduction of genes or enzymes in a single organism whichrequire their integrationwithin the regulatory andmetabolic networkfor proper expression (Silva-Rocha and de Lorenzo, 2010), theengineering of microbial consortium can be easier and achievable.Communication for trading metabolites or exchanging dedicatedmolecular signals and the ability for ‘the division of labor’ by acombination of tasks by constituent members are significant to themicrobial community engineering. Better understanding of thenatural assemblages of microbial communities, and engineeringmicrobial consortium with enhanced abilities, can guide us towardthe dual mission of pollutant degradation and commercial productionof metabolites of biotechnological importance (Table 4) and simul-taneous mitigation of CO2 by its photosynthetic fixation.

Acknowledgments

SRS thankfully acknowledges UniSA for UPS scholarship and CRCCARE for PhD scholarship. BR and KV thank the Government of

Table 4Biotechnological potential of consortia of cyanobacteria/microalgae and bacteria.

Cyanobacterium/microalga Bacterium Nature ofassociation

Advantage of association Biotechnological application Reference

Halophilic cyanobacteria Sulfate-reducingbacteria

Natural Dolomite formationCarbonate precipitation

Dolomite to study biogenicfactors of sedimentation,ornamental/horticultural applications

Gerasimenko andMikhodyuk (2009)

Chlorella vulgaris Bacillus pumilusES4

Artificial Enhanced microalgal growth Efficient removal of ammonium andphosphorus from wastewaters

Hernandez et al. (2009)

Chlorella ellipsoidea Brevundimonassp.

Natural Increased microalgal growth Pigment and nutrient production Park et al. (2008)

C. vulgaris UTEX 395Chlorella sorokiniana UTEX 1602

Azospirillumbrasilense

Artificial Increase in microalgal pigment,lipid content and variety,cell size and growth

Great potential in the algalbiofuel industry

de-Bashan et al. (2002a)

Chaetoceros gracilisIsochrysis galbanaPavlova lutheri

Flavobacterium sp. Artificial Increase in growth ofmicroalgae

Microalgae used inhatcheries/aquaculture

Suminto and Hirayama(1997)


Australia (Department of Education, Employment and WorkplaceRelations) for the Endeavour Research Fellowship and EndeavourExecutive Award, respectively.

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Consortia of cyanobacteria/microalgae and bacteria: Biotechnological potential

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