Download Consortia of cyanobacteria/microalgae and bacteria

Biotechnology Advances 29 (2011) 896–907
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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v
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, Australia
Cooperative Research Centre for Contamination Assessment and Remediation of Environment, PO Box 486 Salisbury South, SA 5106, Australia
Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, India
d
Department of Microbiology, Sri Krishnadevaraya University, Anantapur 515055, India
b
c
a r t i c l e
i n f o
Article history:
Received 14 January 2011
Received in revised form 14 June 2011
Accepted 3 July 2011
Available online 23 July 2011
Keywords:
Consortia
Cyanobacteria/microalgae
Bacteria
Pollutant removal
Organic pollutants
Metals
Nutrient removal
a b s t r a c t
Microbial metabolites are of huge biotechnological potential and their production can be coupled with
detoxification of environmental pollutants and wastewater treatment mediated by the versatile microorganisms. The consortia of cyanobacteria/microalgae and bacteria can be efficient in detoxification of organic
and inorganic pollutants, and removal of nutrients from wastewaters, compared to the individual
microorganisms. Cyanobacterial/algal photosynthesis provides oxygen, a key electron acceptor to the
pollutant-degrading heterotrophic bacteria. In turn, bacteria support photoautotrophic growth of the partners
by providing carbon dioxide and other stimulatory means. Competition for resources and cooperation for
pollutant abatement between these two guilds of microorganisms will determine the success of consortium
engineering while harnessing the biotechnological potential of the partners. Relative to the introduction of
gene(s) in a single organism wherein the genes depend on the regulatory- and metabolic network for proper
expression, microbial consortium engineering is easier and achievable. The currently available biotechnological tools such as metabolic profiling and functional genomics can aid in the consortium engineering. The
present review examines the current status of research on the consortia, and emphasizes the construction of
consortia with desired partners to serve a dual mission of pollutant removal and commercial production of
microbial metabolites.
© 2011 Elsevier Inc. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stromatolites and microbial mats: ancient to modern communities . . . . . . . .
Interactions between cyanobacteria/microalgae and bacteria: extent of relatedness
Cyanobacteria/microalgae: pollutant removal . . . . . . . . . . . . . . . . . .
Consortium of cyanobacteria/microalgae for pollutant removal: proof-of-principle .
5.1.
Organic pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Metal pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.
Nutrient removal from wastewaters . . . . . . . . . . . . . . . . . . .
6.
CO2 capture and sequestration by the consortia: mitigation of greenhouse gas . . .
7.
Microbial solar cells: the futuristic consortium . . . . . . . . . . . . . . . . . .
8.
Microbial community engineering: construction of the consortium . . . . . . . .
9.
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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⁎ Corresponding author at: Centre for Environmental Risk Assessment and Remediation, University of South Australia, SA 5095, Australia. Tel.: +61 8 8302 5044; fax: +61 8 8302
3057.
E-mail address: [email protected] (M. Megharaj).
0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2011.07.009
S.R. Subashchandrabose et al. / Biotechnology Advances 29 (2011) 896–907
1. Introduction
Cyanobacterial and microalgal metabolites 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 biotechnological
potential (Cardozo et al., 2007). Species of Nostoc, Arthrospira (Spirulina)
and Aphanizomenon have been used as food and a source of proteins since
2000 years ago (Jensen et al., 2001). Use of algae has been extended to the
treatment of wastewaters, energy generation, and even as the photosynthetic gas exchangers for space travel (Spolaore et al., 2006). The
systematics of cyanobacteria is generally on the classification schemes
based on cell or colony shape (Rippka et al., 1979; Oren, 2011), but the
evolutionary basis includes these organisms under one of the ten groups
termed ‘Eubacterial Phyla’ (Woese et al., 1985). Included in the definition
of plants are microalgae which are eukaryotic unicellular and microscopic
with size ranging from 1/1000 of a mm to 2 mm and include species of
diatoms, dinoflagellates and green flagellates (Hallegraeff, 1991). Based
on the Tamura–Nei model (Tamura and Nei, 1993), the 16S rRNA
sequences of cyanobacteria (oxygenic photosynthetic bacteria), heterotrophic bacteria and eukaryotic microalgal plastids suggest the sharing of a
common ancestor (Fig. 1). These organisms have been isolated, selected,
mutated, and genetically engineered for effective bioremediation of
organic or recalcitrant pollutants, achieving enhanced rates of degradation, and ensuring better survival and colonization in the polluted areas
(Koksharova and Wolk, 2002; Ramakrishnan et al., 2010; Venkateswarlu,
1993). ‘Industrial sustainability’ now aims at achieving sustainable
production and requires the need of incorporating ‘designs for environment’ into many production processes. Many ancient as well as modern
biotechnological techniques are used in treating wastewaters and
pollution in the environment. Further research and advances are
100
72
93
897
necessary to improve the benefits from these biotechnologies. To abate
industrial pollution, to enhance profitability and sustainability, and to
uncouple economic growth from adverse environmental impact, bioremediation technologies are required (Gavrilescu and Chisti, 2005).
In nature, most microalgae and cyanobacteria are found in
association with other aerobic or anaerobic microorganisms. The
bacterial assemblages are known to influence the development or
decline of algal blooms (Fukami et al., 1997). Even the long-term
laboratory algal cultures have maintained symbiotic relationship with
bacteria (Park et al., 2008). The molecular oxygen from algal
photosynthesis is used as an electron acceptor by bacteria to degrade
organic matter. Carbon dioxide (CO2) from the bacterial mineralization completes the photosynthetic cycle. The symbiotic interactions of
microalgae and bacteria form the basis of the biological oxygen
demand (BOD) removal in the wastewater treatment ponds, first
reported by Oswald et al. (1953). Depletion of ammonium, nitrate and
phosphate by the algal growth is advantageous for nutrient removal
from wastewaters. The principle of self-oxygenation by natural
systems can be effectively employed for remediation of many
pollutants (Muñoz and Guieysse, 2006) since the conventional
engineering technologies suffer from high costs for oxygen supply,
incomplete utilization of natural resources, creation of secondary
pollutants, and technical impracticability in some situations. The
biodegradation processes involving the consortium of cyanobacteria/
microalgae and bacteria will be an ideal self-sustaining system that is
cheaper and technically superior. In nature, there are many evidences
of microbial communities of cyanobacteria or microalgae and bacteria,
either fossilized or living together. What is pertinent now is to gain
insights on the interactions and organization from the ancient
stromatolites and modern cyanobacterial mats, and to apply molecular
techniques to select the desired microbial members for engineering
Anabaena aphanizomenoides (AB551453)
Nostoc sp. (FR798938)
Cyanobacteria
Arthrospira platensis (FJ839360)
Oscillatoria sancta (EU196639)
99
100
100
90
68
99
96
84
80
60
100
98
74
99
97
77
100
100
83
100
Gloeobacter violaceus (FR798924)
Ochromonas distigma (AY702136)
Nannochloropsis granulata (AY702166)
Chlorella vulgaris (D11347)
Chlamydomonas reinhardtii (FJ458262)
Scenedesmus obliquus (AF394206)
Bacillus cereus (HQ316117)
Paenibacillus alginolyticus (HQ236042)
Clostridium aciditolerans (DQ114945)
Acidaminococcus fermentans (X78017)
Streptomyces alboflavus (EF178699)
Corynebacterium striatum (JF342700)
Mycobacterium vanbaalenii (NR_029293)
Rhodococcus erythropolis (AF230876)
Nocardia alba (EU249584)
Leptospira meyeri (HQ709385)
Borrelia lusitaniae (NR_036806)
Cristispira sp. (U42638)
68
Spirochaeta sp. (AY337318)
Treponema denticola (NR_036899)
100
Firmicutes
Actinobacteria
Sphingomonas japonica (AB428568)
α
Agrobacterium tumefaciens (HQ916822)
Desulfatibacillum aliphaticivorans (NR_025694)
Bdellovibrio sp. JS5 (AF084859)
Campylobacter troglodytis (HQ864828)
ε
Nautilia abyssi (AM937002)
Achromobacter denitrificans (FM999734)
β
Alcaligenus sp. (AY296718)
Pseudomonas putida (HQ315887)
γ
Stenotrophomonas maltophilia (AB194327)
91
99
Plastids of
Microalgae
δ
Proteobacteria
Spirochetes
Fig. 1. Phylogenetic tree showing evolutionary relationships among cyanobacteria (oxygenic photosynthetic bacteria), other eubacteria and eukaryotic microalgal plastids
(constructed by using MEGA4 (Tamura et al., 2007) based on maximum likelihood method with 34 representative 16S rRNA gene sequences).
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S.R. Subashchandrabose et al. / Biotechnology Advances 29 (2011) 896–907
consortium of self-sustaining systems with dual mission of pollutant
removal and metabolite production (Fig. 2). The present review
highlights the potential of cyanobacteria/microalgae–bacterial consortia
as the self-sustained system for (a) detoxification of environmental
pollutants and removal of nutrients, and (b) production of metabolites/
by-products of huge commercial value, coupled with the mitigation of
greenhouse gas CO2.
2. Stromatolites and microbial mats: ancient to modern
communities
Stromatolites are internally laminated organosedimentary structures, evolved from microbial mats over time. The ancient stromatolites are considered to be the only form of life for longer periods in
the 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. The
stromatolites of the Warrawoona Group in Western Australia are
about 3.43 billion year old. Allwood et al. (2007) were of the opinion
that the stromatolites of the Strelley Pool Chert, Pilbara Craton are the
biological milestones on the beginning of life, biodiversity, and
development of different capabilities. Trapping and binding, and/or
precipitation of minerals via microbial or abiotic processes would
have resulted in the fossil stromatolite formations and they are among
the earliest evidence for life on the Earth. There are evidences for
modern stromatolites, and those found in the Ruidera Pools Natural
Park of Spain are comprised of cyanobacteria, frequently the species of
Leptolyngbya, and bacterial species of Firmicutes, Bacteriodetes,
Proteobacteria, Actinobacteria, Acidobacteria, Planctomycetes and
Chloroflexi (Santos et al., 2010).
Microbial (cyanobacterial) mats show morphological similarity to
ancient stromatolites. Some of the microbial mats are laminated
heterotrophic and autotrophic communities vertically stratified,
dominated by cyanobacteria, microalgae like diatoms, and anoxygenic
Consortia of
Cyanobacteria/Microalgae
and Pollutant-degrading
Bacteria
Agricultural wastewaters
Industrial wastewaters
Nutrient removal
Removal of organic- and
metal pollutants
Biomass
Biofertilizer
Feed for
Animals, Poultry, and
Aquaculture
Pigments
Nutraceuticals
Biofuels:
Biodiesel, Hydrogen, and
Electricity
Fig. 2. Value-addition of the consortia of cyanobacteria/microalgae–bacteria after
bioremediation.
phototrophic bacteria and sulfate-reducing bacteria. These mats have
complex relationships with trophically-related bacterial groups and
the physiologies of habitat-forming (edificatory) cyanobacteria. These
cyanobacterial mats were credited with self-cleaning properties in the
Arabian Gulf Coasts (Sorkhoh et al., 1992). ‘Symbiosis,’ which was
defined as two or more differently named organisms living together
by de Bary (1879), is an ecological adaptation to life in many
oligotrophic habitats. The symbionts of stromatolites or mats and
their functioning are more difficult to identify by standard microscopy
or traditional culture-based microbiological methods due to the
complexity of the mixed assemblage.
The classic view of microbial mats suggests that the layering of
different microbial members is due to the sequence of metabolic
reactions determined by gradients of light and redox potential. The
microbial populations are required to carry out metabolic reactions
for gaining redox energy at rates faster than the equivalent chemical
reactions. The metabolic rates (community production per unit mass)
of microbial members within these mats are higher than that of rain
forests (Jørgensen, 2001). Within the microbial mats, physical effects
(dissolution, precipitation, volatilization, and fixation of elements),
chemical processes (hydrolysis, condensation, biosynthesis, biotransformation and biodegradation), and spatial translocations (mediated by
transport driven by concentration gradients and physical processes)
occur due to the coupling of several redox reactions. Information on the
organization and functioning of modern stromatolites and microbial
mats can help in the process optimization since the loading and
degradation of pollutants are also affected by these changes.
3. Interactions between cyanobacteria/microalgae and bacteria:
extent of relatedness
Cyanobacteria, the cosmopolitan photosynthetic eubacteria, release
a variety of organic molecules, which include low molecular weight
compounds and extrapolymeric substances composed of proteins, lipids
and 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 these
molecules serve as bacterial growth substrates. In the cyanobacterial
mats, different heterotrophic bacterial populations are specialized in the
use of specific exudates (Abed et al., 2007). Recently, Abed (2010) found
that the co-culturing of Pseudomonas related GM41 strain and the
cyanobacterium Synechocystis PCC6803, both isolated from the cyanobacterial mat, led to an 8-fold increase in the cyanobacterial biomass.
The cyanobacterial exudates not only serve as an endogenous source of
growth substrates to bacteria but also influence the rate of bacterial
degradation (repression or enhancement) of aromatic and aliphatic
contaminants (Kirkwood et al., 2006).
Interactions between autotrophic algae and heterotrophic bacteria can
be 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 concerns
whether microalgae are axenic or not (Radwan and Al-Hasan, 2000). The
unialgal culture may represent the natural algal–bacterial consortium
formed between microalgae and their associated bacteria. The close
contact between two aerobic bacteria (Pseudomonas diminuta and
P. vesicularis isolated from laboratory algal cultures) and microalgae
(Scenedesmus bicellularis and Chlorella sp.) had led to the stimulatory
growth of algal cultures (Mouget et al., 1995). Even the compounds
released by a Chlorella sp. after exposure to water treatment chemicals
supported better growth of Escherichia coli (Bouteleux et al., 2005). In
contrast, an antagonistic relationship was observed between algae and
Leptothrix ochracea in the iron-rich streams (Sheldon and Wellnitz, 1998).
Microalgae produce sheaths (of trilaminar organization) which
consist of carbohydrate, protein and metal cations that are related to
formation of algal cell aggregation, wherein bacteria are associated
with (Croft et al., 2006). Indirect adhesion of bacterial symbionts on
S.R. Subashchandrabose et al. / Biotechnology Advances 29 (2011) 896–907
the sheath and the direct adhesion onto the algal cell surface may
reduce diffusion distance and permit rapid and efficient exchange of
substrates (Park et al., 2008). Although many photosynthetic algae are
considered to be completely autotrophic, they require (i) vitamins like
biotin, thiamine and cobalamine as growth factors as the cobalamine
auxotrophs are widespread (Croft et al., 2006), and (ii) bacterial
siderophores for growth under iron-deficient conditions (Butler, 1998).
Co-inoculation of bacterial strains isolated from the long-term laboratory algal cultures had resulted in better algal growth than that of algae
alone.
Close proximity, chemotaxis, mobility and adhesion with algal host are
important for bacterial association. Primarily, the aerobic heterotrophs use
the photosynthetically-produced carbon compounds whose accumulation can inhibit algal photosynthesis (Bateson and Ward, 1988). In natural
systems, the algal release of dissolved organic carbon ranges from zero to
80% of photosynthates and it is around 6 to 16% in the microalgal
photoreactors (Hulatt and Thomas, 2010). Photosynthesis which is a
reversible set of reactions is inhibited by excessive dissolved oxygen. In
the enclosed photoreactors, dissolved oxygen supersaturation can be as
high as 400%, which can inhibit microalgal growth (Kumar et al., 2010).
The dissolved O2, a photosynthetic by-product, can lower the net
photosynthetic carbon fixation by favoring the ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco) activity. But, the bacterial consumption
of 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 4–7
folds higher than the algal respiration rate. Hence, the bacterial
consumption of O2 is an important means of bacteria-mediated algal
growth enhancement, and similar effects are shown for N2 fixation by
cyanobacterial heterocysts. Algae and cyanobacteria can use CO2 for
photosynthesis, produced by bacterial mineralization, and sugars, acetate
and glycerol for their heterotrophic growth. However, the algal growth
can also inhibit bacterial activity by releasing toxic metabolites, increasing
the temperature, and keeping high O2 levels (Skulberg, 2000).
4. Cyanobacteria/microalgae: pollutant removal
Cyanobacteria which possess dinitrogen-fixing capabilities and
microalgae which probably account for up to 27% of total soil microbial
biomass are widespread in soil and aquatic ecosystems (Burns and
Hardy, 1975; McCann and Cullimore, 1979). These organisms not only
aid in detecting pollution (Megharaj et al., 1989) but also transform
many pollutants in the environment. They have been implicated in the
metabolism of certain organophosphate insecticides such as monocrotophos and quinalphos (Megharaj et al., 1987) and methyl parathion
(Megharaj et al., 1994). The alterations in the species composition of
algae and cyanobacteria in the soil with the long-term contamination of
DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) can serve as useful bioindicators of pollution (Megharaj et al., 2000). Cyanobacteria
(particularly dinitrogen-fixing species of Anabaena and Nostoc) preferentially transformed DDT 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 transformation of pollutants by cyanobacteria or microalgae may change
depending on the species. Cáceres et al. (2008a) observed transformation of an organophosphorous pesticide, fenamiphos (ethyl 4methylthio-m-tolyl isopropyl phosphoramidate) to its primary oxidation product, fenamiphos sulfoxide (FSO) by five different species of
cyanobacteria and five of green algae. Both the pesticide and its
metabolites (FSO, fenamiphos sulfone, fenamiphos phenol, fenamiphos
sulfoxide phenol and fenamiphos sulfone phenol) bioaccumulated in
the terrestrial alga Chlorococcum sp. while only metabolites were
accumulated in the aquatic alga Pseudokirchneriella subcapitata (Cáceres
et al., 2008b). The abilities to transform or degrade pollutants by
cyanobacteria and microalgae are gainfully exploited in bioremediation
technologies of many polluted systems.
899
The presence of cyanobacterial mats in the Arabian Gulf Coasts
after oil pollution had received enough attention because of their
potential to degrade hydrocarbons (Sorkhoh et al., 1992). Bacteria
isolated from the blue-green mats of cyanobacteria growing at the oily
coasts of Kuwait degraded, though not rapidly, most of the
hydrocarbons present in the oil. Alcanotrophic bacteria in combination with certain microalgae and cyanobacteria degraded black oil
though oil is toxic to the algae (Safonova et al., 1999). The strains of
microalgae (Stichococcus sp., Chlorella sp. and Scenedesmus quadricauda)
and cyanaobacteria (Nostoc sp., and Phormidium sp.) are capable of
growing without any apparent toxic effect in medium containing 1%
black oil and bacterial protectants. The bacteria protected the algae from
toxic effect of the oil, while the cyanobacteria provide the bacteria with
O2 and other extracellular carbon-rich exudates. Abed and Köster
(2005), and Abed (2010) observed that the ability of Oscillatoria sp. and
other cyanobacterial mats to degrade hydrocarbons in oil was not of
their own but was due to the association of aerobic heterotrophic
bacteria.
The microbial mats of cyanobacteria or microalgae are naturallyoccurring immobilized microbial systems (Radwan et al., 2002), and
the water–sediment interface supports cyanobacterial mats in
estuaries, lagoons or sheltered sandy beaches. In most aquatic and
terrestrial environments, O2 is one of the limiting factors for microbial
hydrocarbon degradation. The hydrocarbon-utilizing bacteria associated
with cyanobacterial mats obtain O2 from the photosynthesis, probably
nitrogenous and phosphorus compounds, vitamins, and protection from
being washed out or diluted. According to de Oteyza et al. (2006), the
constructed cyanobacterial mats degraded hydrocarbons of lower
volatility (C24–C30 n-alkanes or carbazoles) better than those with low
molecular weight hydrocarbons (n-alkanes with chain length shorter than
n-pentadecane or n-heptadecane, regular isoprenoid hydrocarbons with
chain length lower than C16 or C18 or lower molecular weight
naphthalenes). The roles of cyanobacteria include the provision of oxygen
by photosynthesis for the breakdown of aliphatic and aromatic
compounds, fixed nitrogen, and organic exudates by photosynthesis
and fermentation. The ability to metabolize a range of cyanobacterial
photosynthetic and fermentative exudates can get around the need of
organic or inorganic fertilizer application for the hydrocarbon degradation
by the hydrocarbon-degrading bacteria. These microbial mats with
hydrocarbon-degrading bacteria offer many advantages since neither
bioaugmentation with pollutant-degrading bacteria or biostimulation
with fertilizers for the naturally-occurring bacteria is feasible due to
dilution 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 functions
that are difficult or even impossible for individual strains or species
and those which require multiple steps (Brenner et al., 2008). Living
together may provide robustness to environmental fluctuations,
stability for the members, ability to share metabolites and weather
periods of nutrient limitations, and resistance to invasion by other
species. There have been many proof-of-principle studies on the
consortia of cyanobacteria/microalgae–bacteria for pollutant
degradation.
5.1. Organic pollutants
The O2 production capacity, tolerance to pollutants, and ability to
support pollutant degradation with varied efficiencies by the bacterial
symbiont can differ among the algal/cyanobacterial species (Table 1).
For example, the algal–bacterial microcosms comprising of the
salicylate-degrading Ralstonia basilensis, the phenol-degrading Acinetobacter haemolyticus and the phenanthrene-degrading Pseudomonas
migulae and Sphingomonas yanoikuyae, and the green alga Chlorella
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S.R. Subashchandrabose et al. / Biotechnology Advances 29 (2011) 896–907
Table 1
Degradation of organic chemical pollutants by consortia of cyanobacteria/microalgae and bacteria.
Cyanobacterium/microalga
Bacterium
Pollutant and its removal efficiency
Reference
Oscillatoria strain OSC
Proteobacteria naturally
associated with Oscillatoria sp.
Abed and Köster (2005)
Organics present in Synechocystis sp. PCC6803
Cyanobacterial mats
Pseudoanabaena PP16
Chlorella sorokiniana 211/8k
C. sorokiniana
C. sorokiniana
C. sorokiniana
C. sorokiniana
C. sorokiniana
C. sorokiniana
Pseudomonas-related strain GM41
Acinetobacter calcoaceticus Nocardioforms
Pseudomonas sp. P1
Ralstonia basilensis
Pseudomonas migulae
R. basilensis
Ralstonia sp.
R. basilensis
Acinetobacter haemolyticus
P. migulae
Sphingomonas yanoikuyae
Comamonas sp.
Bacterial consortia
P. migulae
R. basilensis
Microbacterium sp. CSSB-3
R. basilensis
R. basilensis
R. basilensis
Rhodococcus, Kibdelosporangium aridum
n-Octadecane 40%
pristine 50%
phenanthrene 50%
dibenzothiophene 80%
(1 mg ml−1 organo clay complex
containing 16.68 (%) of petroleum compounds)
Phenanthrene 0.8 μg day−1 (0.15 mM)
Oil 63.2% (0.5% v/v)
Phenol 95% (1 mM)
Sodium salicylate 1 mmol l−1 day (5 mM)a
Phenanthrene 24.2 g m−3 h−1 (200–500 mg l−1)
Salicylate 100% (5 mM)
Sodium salicylate 19 mg l−1 h−1 (800 mg l−1)
Sodium salicylate 87 mg l−1 h−1 (1 g l−1)
Phenol 89% (4.25 mM)
Phenanthrene 15% (1.7 mM)
C. sorokiniana
C. sorokiniana
C. sorokiniana
C. sorokiniana
C. sorokiniana IAM C-212
C. sorokiniana 211/8k
Chlorella vulgaris
Bolivian microalga strain
Chlorella sp.
Scenedesmus obliquus
Stichococcus sp.
Phormidium sp.
Selanastrum capricornutum UTEX 1648
S. capricornutum UTEX 1648
Phormidium sp.
Oscillatoria sp.
Chroococcus sp.
Phormidium sp.
Oscillatoria sp.
Chroococcus sp.
Scenedesmus obliquus GH2
a
Abed (2010)
Al-Awadhi et al. (2003)
Kirkwood et al. (2006)
Guieysse et al. (2002)
Muñoz et al. (2003a)
Borde et al. (2003)
Muñoz et al. (2003b)
Muñoz et al. (2004)
Borde et al. (2003)
Borde et al. (2003)
Acetonitrile 0.44 g l−1 day−1 (1 g l−1)
Acetonitrile 12.8 mg l−1 h−1 (1 g l−1)
Phenanthrene 36 mg l−1 h−1 (5 g l−1)
Sodium salicylate 76% (500 mg l−1)
Propionate 100% (125 mg l−1)
Sodium salicylate 100–74% (2 g l−1)
Sodium salicylate 14 mg l−1 h−1 (800 mg l−1)
Sodium salicylate 18 mg l−1 h−1 (800 mg l−1)
Phenols 85% (0.48 mg l−1)
oil 96% (40 mg l−1)
Muñoz et al. (2005a)
Muñoz et al. (2005b)
Muñoz et al. (2005c)
Muñoz et al. (2006)
Imase et al. (2008)
Muñoz et al. (2009)
Muñoz et al. (2003b)
Muñoz et al. (2003b)
Safonova et al. (2004)
Mycobacterium sp. RJGII 135
S. yanoikuyae strain B1
Burkholderia cepacia
BaP 73% (14C-BaP (8 μg/0.5 μ Ci)
BaP 94% (14C-BaP (8 μg/0.5 μ Ci)
Diesel 99.5% (0.6% v/v)
Warshawsky et al. (2007)
Warshawsky et al. (2007)
Chavan and Mukherji (2008)
B. cepacia
Total petroleum hydrocarbon
99% (diesel 0.6% v/v)
Chavan and Mukherji (2010)
Sphingomonas sp. GY2B
B. cepacia GS3C
Pseudomonas GP3A
Pandoraea pnomenusa GP3B
Straight chain alkanes 100%
alkylcycloalkanes and alkylbenzenes 100%
naphthalene, fluorene and phenanthrene
100% (crude oil 0.3% v/v)
Tang et al. (2010)
Values in parentheses are the initial concentrations added.
sorokiniana were efficient in protecting the green alga from the toxic
concentrations and in the removal (up to 85%) of these three
pollutants (Muñoz et al., 2003a). The consortium of C. sorokiniana
and R. basilensis strain was reported to degrade sodium salicylate in a
continuous stirred tank reactor. But, Selenastrum capricornutum and
Anabaena catenula were completely inhibited by salicylate at
500 mg L −1. The salicylate-degrading R. basilensis when grown with
salicylate-resistant alga, C. sorokiniana, rapidly metabolized the
chemical at a rate of 19 mg L −1 h −1 (Muñoz et al., 2003b). Both the
O2 generation rate, which is proportional to the algal density and the
mutual shading, and the O2 consumption due to algal respiration
influence the degradation of salicylate (Guieysse et al., 2002).
Photosynthesis-enhanced biodegradation of toxic aromatic pollutants
can occur in the algal–bacterial microcosms in a one-stage treatment. The
degradation of N-containing organic compounds can be more efficient by
the algal–bacterial consortia than bacteria alone as microalgae can readily
assimilate NH4+ released. The consortium of C. sorokiniana and the
acetonitrile-degrading Comamonas sp. completely degraded acetonitrile
in the column photobioreactor (Muñoz et al., 2005a). But, higher
concentrations of acetonitrile resulted in the inhibition of microalgal
activity due to the combination of high pH and high NH4+. Hirooka et al.
(2003) used the cyanobacterial mixed culture of Anabaena variabilis and
Anabaena cylindrica to remove 2,4-dinitrophenol (2,4-DNP) from industrial 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 otherwise
accumulated in the culture medium.
Polyaromatic hydrocarbons (PAHs) are common constituents of
combustion residues. PAHs of only one, two, or three rings can be
degraded completely by microorganisms while four and five ring
PAHs are recalcitrant. Benzo[a]pyrene (BaP), a five-ring PAH, can be
degraded by the green alga, S. capricornutum strain UTEX 1648, using
dioxygenases with the formation of BaP sulfate ester and glucose
conjugates, while Mycobacterium sp. strain RJGII and S. yanoikuyae
strain B1 can partially degrade BaP (Warshawsky et al., 2007). The
combination of three organisms was more useful for degrading BaP
than individual species. In natural systems, microbial consortia, rather
than individual species, are useful for degrading mixtures of
pollutants (Ramakrishnan et al., 2011).
Naturally-occurring cyanobacterial mats are found to aid hydrocarbon
degradation. Abed and Köster (2005) demonstrated the presence of
diverse, aerobic heterotrophic bacteria with both degradative abilities for
hydrocarbons and with abilities for specialized consumption of specific
exudates from cyanobacteria (species belonging to the genera Marinobacter and Alcanivorax) and another guild (species of Marinobacter,
Halomonas, Roseobacter and Rhodobacter) with ability to use exudates but
not hydrocarbons. The addition of substrates which are representative of
cyanobacterial exudates had variable effects on phenanthrene degradation; acetate, pyruvate, and glucose enhanced the degradation while
alanine and butanol had no effect on the phenanthrene-degrading strain
GM 42 (Abed, 2010). By increasing volume of the sheath in C. sorokiniana
S.R. Subashchandrabose et al. / Biotechnology Advances 29 (2011) 896–907
with the addition of CaCl2 and constructing a complex with the microalga
and a propionate-degrading bacterium (strain PDS1), Imase et al. (2008)
demonstrated the enhanced algal growth in a medium containing high
concentration (1.2 g L −1) of propionate besides complete bacterial
degradation of propionate. Methyl tert-butyl ether (MTBE) degradation
by the microalgal–bacterial symbiotic system containing a mixed culture
of Methylibium petroleiphilum PMI and Chlorella ellipsoidea with the
optimal ratio of initial cell population of bacteria to algae of 100:1 was
higher than the pure cultures of bacteria (Zhong et al., 2011).
Industrial approaches such as filtration, centrifugation and microstraining are not economical and suitable for removal of algal biomass.
The bioflocculent algal–bacterial biomass can help to separate algal
biomass by gravity sedimentation which can circumvent the need for
flocculation, filtration or centrifugation required in the conventional
and high rate algal ponds (Gutzeit et al., 2005). Extracellular
polymeric substances and other factors (e.g. content of calcium)
influence the formation and stability of flocculent algal–bacterial
biomass. The bacterial exopolymer production can result in the
increase of aggregation possibilities of algal–bacterial consortium,
serve as stabilizers of already existing aggregates and efficiently
increase sedimentation. Muñoz et al. (2009) used the biofilm
photobioreactor using C. sorokiniana–R. basilensis consortium immobilized onto foamed-glass beads carriers (Poraver®) and onto reactor
wall for treating salicylate contaminant in wastewater.
901
provide carbon, nitrogen and phosphorus to the heterotrophic
bacteria for the final reduction of U(VI) to U(IV).
The algal–bacterial consortium comprising of C. sorokiniana and
R. basilensis was found to metabolize salicylate with a subsequent
removal of heavy metals from the solutions (Muñoz et al., 2006). The
consortium removed copper more efficiently than the individual
organism at pH 5.0, and nickel, cadmium and zinc were less efficiently
removed. Dried biomass from a mixture of cyanobacteria and bacteria is
used to remove the heavy metals from wastewaters, and metals are
recovered subsequently by desorption. Very efficient removal of copper
(≈80%) and cadmium (≈100%) from metal waste with a maximum
removal rate within 5 min of contact time was observed with dried mass
of a mixed culture of microalgae (Scenedesmus sp., Tetraedron sp.,
Chlorella sp., Chlorococcus sp.), cyanobacteria (Chroococcus sp., Pseudoanbaena 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 sensitive
species by more tolerant bacteria contribute to metal tolerance in
bacterial communities. In response of bacteria to metals, correlation
exists between the genetic and physiological structure of bacterial
communities and the species composition of the algal community, but
not the level of metal pollution. There is a strong and species-specific
linkage between bacterial and algal species (Boivin et al., 2007).
5.3. Nutrient removal from wastewaters
5.2. Metal pollutants
Cell walls of microalgae and cyanobacteria are composed of
polysaccharides and carbohydrates that have negatively-charged
(amino, carboxyl, hydroxyl or sulfide) groups. Most metals are
bound to the negatively-charged ligand groups, which is the basis
for metal removal from wastewaters. Beside this mechanism of metal
adsorption onto cell surfaces and extracellular polysaccharides,
uptake into cells, incorporation into vacuoles or aragonite (CaCO3)
structures, and precipitation on the cell surface or internally can
occur. But, heavy metals are potent inhibitors of photosynthesis as
they can replace or block the prosthetic metal atoms in the active sites
of certain enzymes. Likewise, the acidic functional groups of bacterial
cell walls can also bind significant concentrations of aqueous cations,
which can affect the speciation, distribution and mobility of those
cations (Ginn and Fein, 2008). Algae growing in wastewaters may
provide a simple, long-term strategy for removal of metal pollutants
(Table 2). To this end, Kalin et al. (2004) described a three-step
process for the detoxification of uranium from wastewaters. Initially,
the ligands in algal cell walls efficiently remove U(VI) from
wastewaters followed by the removal of U-algal particulates from
the water column to the sediments. Subsequently, the dead algal cells
Activated sludge method is probably the first major use of
biotechnology in bioremediation applications and continues to be
an effective technology for pollution containment (Gavrilescu and
Chisti, 2005). The association of microalga, Chlorella vulgaris Hamburg,
and activated sludge bacteria improved the performance of waste
stabilization ponds, the removal of organic matter, nutrients and
pathogens, the content of O2 with no need of aeration and effective
separation of alga by sedimentation (Medina and Neis, 2007). Many
improvements have led to the use of algal–bacterial consortium in
facultative ponds and high rate algal ponds (HRAPs). The floccular
biomass of ‘ALBAZOD’ comprising of algae, bacteria, zooplankton and
detritus bound together generally occurs in HRAPs. The environmental
and/or pond operational changes in terms of COD loading rates and
retention time led to alterations to gross algal composition and cell
dimensions of different species in these floccular materials (Cromar and
Fallowfield, 2003).
Various types of bioreactors now offer optimal conditions of
temperature, pH, O2 transfer, mixing, and substrate concentration for
efficient cellular metabolism, besides providing the basic function of
containment. Oxidation of both organic matter and ammonium was
achieved by the C. sorokiniana-mixed bacterial culture from the
Table 2
Heavy metal removal from wastewater by consortia of cyanobacteria/microalgae and bacteria.
Cyanobacterium/
microalga
Bacterium
Source of
wastewater
Metal and its
removal efficiency
System/
reactor used
Reference
Spirulina platensis
Sulfate-reducing bacteria
Tannery effluent
High rate
algal pond
Rose et al.
(1998)
Chlorella sp.
Scenedesmus obliquus
Stichococcus sp.
Phormidium sp.
Rhodococcus sp.
Kibdelosporangium aridum
Oil polluted wastewater
Pilot installation
Safonova et
al. (2004)
C. sorokiniana (biomass)
R. basilensis (biomass)
Bristol medium with pollutants
Copper 79.2% (500 mg l−1)a
zinc 88.0% (500 mg l−1)
iron 100% (500 mg l−1)
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)
Copper 57.5% (20 mg l−1) @ pH 5.0
Algae from wastewater
Bacteria from wastewater
treatment plant (biomass) treatment plant (biomass)
a
Values in parentheses are the initial concentrations added.
Conical glass
reactor
Artificial metal solutions
Copper 80% (100 mg l )
Continuous flowcontaining cadmium and copper cadmium 100% (100 mg l−1) @ pH 4.0 through column
−1
Muñoz et al.
(2006)
Loutseti et al.
(2009)
902
Table 3
Removal of nutrients from wastewater by consortia of cyanobacteria/microalgae and bacteria.
Bacterium
Source of waste water
Nutrients and its removal efficiency
System/reactor used
Reference
Spirulina platensis
Chlorella vulgaris
Sulfate-reducing bacteria
Azospirillum brasilense
Tannery effluent
Synthetic wastewater
High rate algal pond (HRAP)
Chemostat (Virtis, Gardiner, NY)
Rose et al. (1998)
de-Bashan et al. (2002b)
C. vulgaris
Wastewater bacteria
Pretreated sewage
Photobioreactor pilot-scale
Gutzeit et al. (2005)
C. vulgaris
Alcaligenes sp.
Coke factory wastewater
A. brasilense
Synthetic wastewater
Continuous photobioreactor
with sludge recirculation
Inverted conical glass bioreactor
Tamer et al. (2006)
C. vulgaris
Chlorella sorokiniana
Swine wastewater
C. sorokiniana
Mixed bacterial culture from an
activated sludge process
Activated sludge bacteria
C. sorokiniana
Activated sludge consortium
Pretreated piggery
wastewater
Pretreated swine slurry
C. sorokiniana
Activated sludge bacteria
Piggery wastewater
Euglena viridis
Activated sludge bacteria
Piggery wastewater
Sulfate 80% (2000 mg l−1)a
Ammonia 91% (21 mg l−l)
phosphorous 75% (15 mg l−l)
DOC 93% (230 mg C l−1)
nitrogen 15% (78.5 mg l−1)
phosphorous 47% (10.8 mg l−1)
NH+4 45% (500 mg l−l)
phenol 100% (325 mg l−l)
Phosphorous 31.5% (50 mg l−l)
nitrogen 22% (50 mg l−l)
Phosphorous 86% (15 mg l−l)
nitrogen 99% (180 mg l−l)
TOC 86% (645 mg l−l)
nitrogen 87% (373 mg l−l)
TOC 9–61% (1247 mg l−l)
nitrogen 94–100% (656 mg l−l)
phosphorous 70–90% (117 mg l−l)
TOC 47% (550 mg l−1)
phosphorous 54% (19.4 mg l−1)
NH+4 21% (350 mg l−1)
TOC 51% (450 mg l−1)
phosphorous 53% (19.4 mg l−1)
NH+4 34% (320 mg l−1)
COD 58.7% (526 mg l−l)
TKN 78% (59 mg l−l)
Ammoniacal-N N 99% (147 g m−3)
fecal enterococci 95% (22,000 g m−3)
Selenium 77% (400 μg l−1)
Nitrogen 95% (95 mg l−l)
Ammonia 20% (21 mg l−l)
Microalgae present in tertiary stabilization Bacteria present in tertiary stabilization
pond treating domestic wastewater
pond treating domestic wastewater
Resident algae in facultative lagoon system Nitrifying bacteria in facultative
lagoon system
Microalgae from wastewater
Indigenous nitrate- and selenium-reducing
bacteria
Algae from wastewater stabilization ponds Activated sludge bacteria from municipal
wastewater treatment plant
a
Piggery wastewater
Dairy wastewater
Drainage water
Synthetic wastewater
Tubular biofilm photobioreactor
Perez-Garcia et al.
(2010)
González et al. (2008a)
Glass bottle
González et al. (2008b)
Tubular biofilm photobioreactor
de Godos et al. (2009a)
Jacketed glass tank photobioreactor
de Godos et al. (2010)
Jacketed glass tank photobioreactor
de Godos et al. (2010)
HRAP
de Godos et al. (2009b)
Two-pond lagoon system
Sukias et al. (2003)
Advanced integrated wastewater pond
system
Wastewater stabilization pond
Green et al. (2003)
Babu et al. (2010)
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.
S.R. Subashchandrabose et al. / Biotechnology Advances 29 (2011) 896–907
Cyanobacterium/microalga
S.R. Subashchandrabose et al. / Biotechnology Advances 29 (2011) 896–907
activated sludge process in the tubular biofilm photobioreactor
(González et al., 2008a). The enclosed biofilm photo-bioreactors are
economical and have nutrient removal efficiencies of up to 99% NH4+,
86% PO43−, and 75% total COD. Wu et al. (2011) proposed a hybrid
bioreactor which supports heterotrophic and autotrophic microorganisms for the removal of high-loading nutrients with efficiencies of
about 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–bacterial
consortium for the wastewater reclamation (Table 3). In addition, the
residual biomass can be exploited as green fertilizer due to the slow
release of nutrients into the soil or as biosorbent for heavy metals. The
species of Chlorella and Scenedesmus which are used for domestic sewage
treatment are rich in protein, mineral salts, vitamins A and B, and most
amino 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 a
promising fuel alga Chlorella minutissima for cultivation in municipal
waters. Complex relationships among microalgae/cyanobacteria, bacteria,
light and pollutant concentrations need to be understood to optimize
parameters for the operation of photobioreactor or HRAPs for the
degradation of different pollutants.
6. CO2 capture and sequestration by the consortia: mitigation of
greenhouse gas
The purpose of algal cultivation has always depended on the
specific needs which include wastewater treatment, biomass for
biogas generation or use in aquaculture, production of fine chemicals
and extracellular compounds, and even in CO2 fixation (Ho et al.,
2010; Ugwu et al., 2008). An initial report on mass cultivation of algae
for 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, the
use of these biological agents is considered as one of the effective
approaches to fixing CO2 and thereby mitigating possible global
warming. On mass basis, algal dry biomass of 1 kg requires about
1.83 kg of CO2 (Chisti, 2007). Traditionally, the sources of CO2 for the
algal cultivation are: (i) flue gas emitted by the coal fired power plants
(typically with 10–20% CO2), (ii) increased CO2 concentration in the
closed photobioreactor (no more than 1.0%), and (iii) atmospheric
CO2 (380 ppm) (Wang et al., 2008; Yun et al., 1997). With economic
incentives in terms of carbon credits provided under the Kyoto
protocol (Wang et al., 2008), CO2 capture and sequestration by the
algal–bacterial consortium will gain more importance in the future. In
this 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 (PO43−
precipitation or NH3 stripping) not only contribute to the environmental
friendly approaches to wastewater reclamation but also to the biomass
utilization as ‘green fertilizer’ due to the slow release of nutrients into
the soil or as biosorbent for metal removal. Energy savings in supply of
substrates and revalorization of algal biomass, a pay-back to the
operation cost make this mitigation option very attractive and valuable.
For the purpose of CO2 abatement, the selection of algal strains for their
ability to utilize CO2 at higher rates, especially tolerance to elevated
levels of CO2 and temperature (Chinnasamy et al., 2009), suitable
bacterial partners, and appropriate design of bioreactors at the field
scale are important prerequisites and objectives of future research.
7. Microbial solar cells: the futuristic consortium
Research efforts are now on integrating photosynthesis with
microbial fuel cells (photoMFCs). Two types of photoMFCs such as (i)
903
electrocatalytic bioelectrochemical systems that convert hydrogen
from photosynthesis, and (ii) the sediment-based bioelectrochemical
systems that convert excreted organics are on the basis of synergistic
relationships between photosynthetic producers (cyanobacteria or
plants) and heterotrophic bacteria, widely established in various
ecosystems, for example, microbial mats (Rosenbaum et al., 2010). A
new biotechnological system that integrates photosynthetic and
electrochemically active organisms to generate in situ green electricity
or chemical compounds (hydrogen, methane, ethanol or hydrogen
peroxide) is microbial solar cell (MSC). The basic principles of MSCs are
on the premises of photosynthesis, transport of organics to the anode,
anodic oxidation of organics by electrochemically active bacteria and
cathodic reduction of O2 (Strik et al., 2011). He et al. (2009)
demonstrated that the MSCs with phototrophic biofilms containing
members of Chlorophyta and/or cyanobacteria and mixed microbial
populations of Firmicutes and a Gammaproteobacterium (closely
related to Alkaliliminicola ehrlichii) could convert solar energy to
electricity on the anode of a fuel cell. In the MSC with a photoreactor
and an anaerobic digester, photosynthesis by cyanobacteria/microalgae
occurs in the photoreactor; biogas is produced from the organic matter
and transported to the digester. At the anode of the MFC, the remaining
organic matter is oxidized by the electrochemically active bacteria while
O2 from the photoreactor is reduced to water at the cathode (Strik et al.,
2008). Better utilization of the solar spectrum can occur in the
consortium of algae and bacteria. Not only electricity but a range of
fuels and useful chemicals are produced in the MSCs which are based on
the photosynthetic activity of cyanobacteria/microalgae and electrochemical reactions of heterotrophic bacteria. The future applications of
this technology can benefit from the isolation of efficient members and
engineering of stable microbial consortia within a single reactor.
8. Microbial community engineering: construction of the
consortium
An early report, and subsequent patenting, on the construction of
microbial communities of cyanobacteria and bacteria for pollutant
degradation was provided by Bender and Phillips (1994). The thick,
gelatinous green mat was constructed from an artificial ecosystem in
glass containers consisting of a soil base to provide motile bacteria,
filtered tap water, a floating layer of ensiled grass clippings (as a source
of lactic and acetic acid as well as a microbial consortium of fermentative
anaerobes) and cyanobacteria (Oscillatoria sp.). With the pre-exposure
to 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 the
natural microbial mats from marine sediments, constructed microbial
mats after immobilizing the mat on glass wool, prepared the
constructed mats for adaptation by step-wise additions of water from
effluent to be treated, and then found those microbial mats to remove
97% and 95% of ammoniacal-nitrogen and nitrate-nitrogen from shrimp
culture effluents. The constructed microbial mats had filamentous forms
of cyanobacteria (Microcoleus chthonoplastes, Spirulina sp., Oscillatoria
sp., 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, Stichococcus and Phormidium) onto capron fibers and bacterial strains
(Rhodococcus sp., Kibdelosporangium aridum and two unidentified
strains) onto ceramics, capron and wood led to the formation of stable
consortia, preventing them from being washed off, and to the removal
of phenols (85%), anionic surface active substances (73%), oil spills
(96%), copper (62%), nickel (62%), zinc (90%), manganese (70%) and
iron (64%), and the reductions of BOD25 to 97% and COD to 51%,
respectively (Safonova et al., 2004). In other constructed cyanobacterial mats, hydrocarbons of lower volatility (C24–C30 n-alkanes or
carbazoles) were degraded better than the low molecular weight
904
S.R. Subashchandrabose et al. / Biotechnology Advances 29 (2011) 896–907
hydrocarbons (n-alkanes with chain length shorter than n-pentadecane or n-heptadecane, regular isoprenoid hydrocarbons with chain
length lower than C16 or C18 or lower molecular weight than
naphthalenes) (de Oteyza et al., 2006). The consortium comprising
species of Phormidium, Oscillatoria and Chroococcus, and the oildegrading bacterium, Burkholderia cepacia, developed on a rotating
biological contactor (RBC), resulted in good total petroleum hydrocarbon removal and settleability of biosolids, without the requirement
of a soluble carbon source. The performance efficiency of the reactor
for the treatment of complex non-aqueous phase liquids (NAPLs) and
the relative dominance of the phototrophic microorganisms and
bacteria was determined by the N:P ratio (Chavan and Mukherji,
2008). Tang et al. (2010) found that the unialgal culture of the oiltolerant Scenedesmus obliquus GH2 was unsuitable, compared to the
axenic culture, for the construction of the consortium with crude oildegrading bacteria (Sphingomonas sp. GY2B, B. cepacia GS3C and a
mixed culture, named GP3).
The microalgal or cyanobacterial growth has limitations, despite the
efforts to improve by various technological, physical, molecular and
environmental methods. For the continuous treatment of variable inlet
concentrations of pollutants, algal selection is paramount as the
pollutants can decrease the photosynthetic activity, leading to process
failure. González and Bashan (2000) proposed the hypothesis of using
‘microalgal growth-promoting bacterium (MGPB)’ such as Azospirillum
brasilense to stimulate the microalgal growth by the phytohormones
synthesized by the MGPB cells. The co-immobilization of C. vulgaris and
C. sorokiniana with A. brasilense led to increases in the contents of
chlorophyll a and b, lutein and violoaxanthin, lipid content
(80–320 μg g−1 dry weight) and the number (5 to 8 different) of fatty
acids (de-Bashan et al., 2002a). This provides strong evidence on the
influences of bacteria on microalgal metabolism, species-specific effects
and the need for optimization of a bacterial–algal consortium.
Thermodynamic conditions necessitate about 300 bacterial units to an
algal unit for CO2 supply (Oron et al., 1979). Imase et al. (2008) proposed
a method for constructing artificial communities of Chlorella sp. and
symbiotic microorganisms by increasing the volume of algal sheath by
addition of CaCl2 solution.
Pankratova et al. (2004) observed that a consortium of a cyanobacterium, Nostoc palusodum Kutz and Rhizobium galegae grown over
18 months in a liquid medium for seed treatment of Galega orientalis
was stable and maintained its structure and activity over long-term
growth with positive effects as a seed inoculant. C. sorokiniana
cultivated in the slants with bacteria as a consortium was stable even
after 7 months of sub-culturing (Watanabe et al., 2005). DGGE profiles
of bacterial community showed that a consortium comprising of a
microalga, S. obliquus and four oil-degrading bacteria was stable after
3 cycles of crude oil degradation, each cycle lasting for 8 days (Su
et al., 2011). However, the diversity of species does not always
guarantee the survival and success of the engineered consortia that
will perform better under the fluctuating environmental conditions.
The stability of a microbial consortium relies on two important
organizing features: (i) communication which refers to trading of
metabolites and exchange of dedicated molecular signals (quorum
sensing intraspecific signals, multispecies quorum sensing interspecific
cues and cross feeding) within each population or between individuals,
and (ii) the division of labor (Brenner et al., 2008; West et al., 2006). In
the natural or engineered microbial consortia, the chances for
competition, communication, or collaboration increase with the
number of interacting agents (West et al., 2007; Wintermute and Silver,
2010). Venturi et al. (2010) suggested that the general mechanism for
stability is primarily the intercellular signaling in microbial communities. The challenges which are to be met for achieving the stability of
engineered microbial consortium include (i) maintenance of long-term
homeostasis (or long-term extinction), (ii) functionality of consortia
despite horizontal gene transfer, (iii) incorporation of stable changes
into the genomes of microbial members, and (iv) fine-tuning of the
performance of multiple populations (Brenner et al., 2008). Goldman
and Brown (2009) suggested that the ecology and evolution theory has
great potential to overcome the problems associated with the long-term
behavior and stability of microbial consortia.
Bacteria that are associated earlier with or without algae can interact
cooperatively or competitively when both partners are brought together
in a consortium. Coordination among microbial members within a given
space and the maintenance of homeostasis will determine the survival
and success of a microbial consortium. Engineering of microbial
consortium should provide opportunities for re-introduction or elimination as needed and in which its tasks can be monitored over time,
with enhanced functional capabilities (Brenner et al., 2008). Metabolite
profiling approaches offer unprecedented opportunities to understand
the complex interactions among the microbial members and to identify
the economically important metabolites. Likewise, metabolic engineering can help to design new cell factories. Due to the large genome
sequencing programs for a number of microorganisms, the functional
genomics (Zhou et al., 2004) can aid in metabolic engineering that
provides consent to the interactions among all the members of a
consortium. Metabolic profiling, functional genomics, and combinatorial biochemical approaches including metabolic engineering can
extend the concept of molecular farming for the algal–bacterial
consortium for efficient pollutant degradation. Efficient pollutant
degradation by a microbial consortium will gain public acceptance
along with the concurrent production of their useful metabolites or
bioelectricity using wastewaters.
9. Conclusion
Photosynthetic oxygenation by microalgae or cyanobacteria and
pollutant degradation by bacteria are an attractive proposition for
wastewater treatment. Energy savings in O2 supply, process safety (no
risk of aerosolization), CO2 mitigation, efficient recycling of nutrients,
and revalorization of algal biomass, a pay-back to the plant operation
cost, make their utilization very valuable. Further improvements in
efficiencies of these biological agents for nutrient removal and/or
pollutant degradation may enhance public acceptance of these
consortia for producing microbial metabolites commercially (Fig. 2)
by using polluted waters. For the continuous treatment of variable
inlet concentrations of pollutants, algal or bacterial selection is
paramount as the pollutants can decrease the survival and degradation efficiency, leading to process failure. High concentrations of
organic pollutants like styrene and phenolic compounds can inhibit
the microbial degrader community while low aqueous solubility of
compounds such as PAHs limits the biodegradation process. Metabolic
routes as well as bottlenecks for microbial degradation of pollutants or
photosynthetic oxygenation are extensive, which require the need to
use a molecular toolbox (Ramadas and Thattai, 2010). Compared to
the introduction of genes or enzymes in a single organism which
require their integration within the regulatory and metabolic network
for proper expression (Silva-Rocha and de Lorenzo, 2010), the
engineering of microbial consortium can be easier and achievable.
Communication for trading metabolites or exchanging dedicated
molecular signals and the ability for ‘the division of labor’ by a
combination of tasks by constituent members are significant to the
microbial community engineering. Better understanding of the
natural assemblages of microbial communities, and engineering
microbial consortium with enhanced abilities, can guide us toward
the dual mission of pollutant degradation and commercial production
of metabolites of biotechnological importance (Table 4) and simultaneous mitigation of CO2 by its photosynthetic fixation.
Acknowledgments
SRS thankfully acknowledges UniSA for UPS scholarship and CRC
CARE for PhD scholarship. BR and KV thank the Government of
S.R. Subashchandrabose et al. / Biotechnology Advances 29 (2011) 896–907
905
Table 4
Biotechnological potential of consortia of cyanobacteria/microalgae and bacteria.
Cyanobacterium/microalga
Bacterium
Nature of
association
Advantage of association
Biotechnological application
Reference
Halophilic cyanobacteria
Sulfate-reducing
bacteria
Natural
Dolomite formation
Carbonate precipitation
Gerasimenko and
Mikhodyuk (2009)
Artificial
Enhanced microalgal growth
Natural
Increased microalgal growth
Dolomite to study biogenic
factors of sedimentation,
ornamental/horticultural applications
Efficient removal of ammonium and
phosphorus from wastewaters
Pigment and nutrient production
Artificial
Increase in microalgal pigment,
lipid content and variety,
cell size and growth
Increase in growth of
microalgae
Great potential in the algal
biofuel industry
de-Bashan et al. (2002a)
Microalgae used in
hatcheries/aquaculture
Suminto and Hirayama
(1997)
Chlorella vulgaris
Bacillus pumilus
ES4
Chlorella ellipsoidea
Brevundimonas
sp.
C. vulgaris UTEX 395
Azospirillum
Chlorella sorokiniana UTEX 1602 brasilense
Chaetoceros gracilis
Isochrysis galbana
Pavlova lutheri
Flavobacterium sp. Artificial
Australia (Department of Education, Employment and Workplace
Relations) for the Endeavour Research Fellowship and Endeavour
Executive Award, respectively.
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