G. KANTHARAJAN

ICAR-CIFE

Course No : AEM 510Course Title : Environmental Biotechnology

Consortia of Microbes for Environmental Protection(concepts, scope and feasibility)

INTRODUCTION

• Microbial consortia are ubiquitous in nature - great importance to humans, fromenvironmental remediation and wastewater treatment to assistance in food digestion.

• Synthetic biologists are honing their ability to program the behavior of individual microbialpopulations, forcing the microbes to focus on specific applications, such as the production ofdrugs and fuels.

• Microbial consortia can perform even more complicated tasks and endure more changeableenvironments than monocultures.

• Represent an important new frontier for synthetic biology.

CONCEPTS OF MICROBIAL CONSORTIA

Consortia concept for bioprocessing applications is supported byobservations in nature.

Naturally occurring ecosystems, optimized by eons of evolution, arealmost exclusively organized as mixed communities.

A group of different species of microorganisms that act together asa community (Encyclopedia of medical concepts).

2 organizing features

1. Members of the consortium communicate with one another. Whether bytrading metabolites or by exchanging dedicated molecular signals, each population orindividual detects and responds to the presence of others in the consortium.

The overall output of the consortium rests on a combination of tasks performed byconstituent individuals or sub-populations.

2. Communication enables the second important

feature which is the division of labor

Ecology as the foundation for engineered consortia

Two ecological theories

1. Resource Ratio Theory (RRT)

- One of the most successful theories in ecology.

- Used both qualitatively and quantitatively to assess outcomes between organisms competing for shared,limiting resources.

- These resource-based interactions can lead to either coexistence or exclusion of competitors.

- Example: how photoautotrophic communities competing for three essential resources (light, nitrogen,phosphorous) can create distinct environmental resource niches which permit coexistence of multiplemicrobes.

- RRT has been adapted to consider the benefits of resource trading in consortia, highlighting conditionswhere coexistence is more competitive than monoculture strategies.

- A super-competitor unit is a consortium that possesses the emergent system property of enhanced resourceutilization and therefore depletes resources more efficiently than the respective monocultures.

2. Maximum Power Principle (MPP)

Initially proposed by Lotka (1922)

A consortium that utilizes multiple substrates in parallel would have a higher metabolic rateand therefore fitness than a monoculture that utilized the same substrates sequentially.

Both RRT and MPP are useful for examining design principles for engineering microbialconsortial interactions for environment management.

Consortial Interaction Motifs

1. DIVISION OF LABOR

At the foundation of many cooperative interactions is division of labor through functional differentiation andspecialization.

It permits parallel or sequential processing of resources

- enhanced productivity, nutrient cycling and stability against perturbation.

- overall resource usage efficiency

- increasing reaction specificity

- reducing the formation of side-products by localizing the reactions to favorable environments.

- permits concurrent optimization of multiple tasks, a trait useful for multistep-processes like degradation of complex material.

2. SYNERGISTIC DIVISION OF RESOURCES

Carbon or energy source are partitioned between community members in a non-competitivemanner based on metabolic functionality.

This template permits parallel processing of substrates and has been used to construct consortiawhich simultaneously ferment pentose and hexose sugars, a functionality that is oftenunattainable in monocultures due to catabolite repression .

Catabolite repression allows bacteria to adapt quickly to a preferred(rapidly metabolisable) carbon and energy source first.

3. COMMENSALISM

One community member’s activity provides an ecological niche for others at no benefit or costto itself.

Commensalism is frequent in biofilms where, for instance, the consumption of oxygen by onecommunity member establishes an oxygen gradient creating microenvironments suitable foranaerobic microbes.

Metabolite exchange: when a producer organism secretes by-products at no benefit or cost toitself which permits sequential consumption by other community members.

4. MUTUALISM

Observed in nature and are defined as relationships that benefit all participants.

In cellular factory applications, mutualism can involve syntrophy, defined here as resource exchanges or cross-feeding.

Mutualistic designs have been utilized in numerous biotechnology studies including consolidated bioprocessingof cellulose coupled with biofuel production.

For instance, it is commonly demonstrated in producer-consumer relationships where an organic acidconsuming community member scavenges inhibitory byproducts from a producer population.

CONCEPTS OF MICROBIAL CONSORTIUM

Mono culture vs Consortium

SCOPE OF MICROBIAL CONSORTIA

Mixed populations can perform complex tasks

• Mixed populations can perform functions that are difficult or even impossible forindividual strains or species.

• Balancing two or more tasks so that they are efficiently completed

• Ability to perform functions requiring multiple steps. Such tasks are possible whendifferent steps are completed by dedicated cell-types.

• Example, cellulolytic microbes make and excrete several different protein components(e.g. scaffolding proteins and enzymes) that assemble into an extracellular cellulosomethat is capable of cellulose degradation

Mixed populations can be robust to changes in environment

Compared with monocultures, communities might be more capable of resisting invasion byother species.

They might be able to weather periods of nutrient limitation better because of the diversity ofmetabolic modes available to a mix of species combined with the ability to share metaboliteswithin the community.

For example, when nutrients become limited, the most prevalent species in a community are notalways the most metabolically active species.

A minority population can become the most active population during nutrient limitation.

Diversity of species in a consortium does not guarantee survival, but it might be that engineeredconsortia will perform most reliably in changeable environments.

Communication organizes function in engineered consortia

• Communication in natural consortia can involve the exchange of dedicated signal moleculeswithin or between single populations.

- Exchange of acyl-homoserine lactone (acyl-HSL) signaling molecules - inGram-negative species

- Small peptides - in Gram-positive species

- Inter-population communication between Gram-positive and Gram-negativespecies, through auto-inducers 2 and 3.

• For example, the member species of a consortium that degrades the herbicide diclofop methylpass intermediate metabolites back and forth in the process of degrading the compound -exchanging metabolic intermediates that either assist or compromise the growth of theirneighbor.

Consortia of Naturally Occurring Species

Naturally occurring consortia have been characterized as well as defined consortia of naturallyoccurring organisms.

The impact of natural consortia was shown to be profound. In one example, a synergetic effect wasduring chalcopyrite leaching with a defined consortia of A. ferrooxidans and A. thiooxidans.

The mixed culture was more efficient at leaching chalcopyrite than the pure cultures. Employment ofheterotrophic acidophiles to remove inhibiting organic compounds that accumulate during growth ledto acceleration of the leaching process.

This was attributed to the increased growth rate of A. ferrooxidans while it was co-cultured with theheterotroph Acidiphilium acidophilum.

Applications And Feasibility In Environment Management

Consortium In Bioremediation Of Pesticide

Diversity makes it possible to break-down a large number of different organic chemicals. Actually microorganisms individually cannot mineralize most hazardous substances.

The simultaneous degradation of the pesticide methyl parathion and chlorpyrifos was tested using a bacterialconsortium obtained by selective enrichment from highly contaminated soils in Moravia (Medellin, Colombia).

Microorganisms identified in the consortium were Acinetobacter sp, Pseudomonas putida,Bacillus sp, Pseudomonas aeruginosa, Citrobacter freundii, Stenotrophomonas sp, Flavobacterium sp, Proteusvulgaris, Pseudomonas sp, Acinetobacter sp, Klebsiella sp and Proteus sp.

In culture medium enriched with each of the pesticides, the consortium was able to degrade 150 mg l−1 of methylparathion and chlorpyrifos in 120 h. When a mixture of 150 mg l−1 of both pesticides was used the percentagedecreased to 72% for methyl parathion and 39% for chlorpyrifos.

• With the addition of glucose to the culture medium, the consortium simultaneously degraded 150 mg l−1 of the pesticides in the mixture (Nancy Pino et al)

Compound Organism Reference

Polychlorinated Arthrobacter sp. B1B and Ralstonia Singer et al., 2000biphenyl (soil) eutrophus H850

BTEX Methanogenic consortia Da Silva andAlvarez, 2004

Chloroethenes Consortium that contains Dehalococcoides Lendvay et al.,2003

Consortium that contains Dehalococcoides Adamson et al.,2003

Consortium that contains Dehalococcoides Major et al., 2002

Chlorobenzenes P. putida GJ31, P. aeruginosa RHO1 and P. Wenderoth et al.,putida F1∆CC 2003

1,1,1-Trichloroethane Butane-utilizing enrichment culture Jitnuyanont et al.,2001

Atrazine Consortia degrading atrazine Goux et al., 2003

Toluene nitrate-reducing genera Azoarcus and Harwood, C.SThauera, iron-reducing Geobacter et.al.,(1997)

Metallireducens

Tolune Pseudomonas putida strain mt-2 Meckenstock et al.,Thauera aromatica strain K172, (1999)

Geobacter metallireducens

Microbial Consortia in Biomining

The impact of microbial consortia in bioleaching, particularly in copper recovery, is widely recognized inliterature and industry.

The “indirect” mechanism assumes that chemoautotrophic iron-oxidizing microorganismslike Acidithiobacillus ferrooxidans or Leptospirillum ferrooxidans generate ferric ions by oxidation of ferrousiron.

Chalcopyrite leaching is particularly sensitive to inactivation by formation of jarosite layers as a function ofredox potential and is thus one of the most recalcitrant ore to leach. These sulfur layers however can beoxidized to soluble sulfate by sulfur-oxidizing bacteria such as Acidithiobacillus caldus or Acidithiobacillusthiooxidans.

Hence naturally occurring consortia of autotrophic iron-oxidizing microbes and sulfur-oxidizing microbeshave been proposed to be symbiotic, potentially mutualistic or at least synergetic in substrate use.

Mixed Bacteria Consortium for Treating Dyeing Wastewater

• The organic matter in dyeing wastewater may be degradated more thoroughly and completely due to the co-metabolism between a variety of bacteria.

• Bacteria consortium TJ-1 (In TJ-1 three bacterial strains were identified as Aeromonas caviae, Proteusmirabilis and Rhodococcus globerulus by 16S rRNA gene sequence analysis) which possesses the degradationcapacity of acid orange 7 and a lot of azo dyes wastewater.

• The decolorization rate of TJ-1 is higher than single bacteria which prove that there are interactions among thebacteria.

• After treating AO7 solution 16 h at the concentration of 200 mg/L, the decolorization rate had reached 90%which showed perfect effects.

Consortia-Mediated Bioprocessing of Cellulose to Ethanol

Application of a symbiotic co-culture of a cellulolytic mesophile, C. phytofermentans, and thecellodextrin fermenting yeast, C. molischianaor S. cerevisiae cdt-1 for direct ethanol productionfrom α-cellulose.

Controlled oxygen transfer is used to induce a symbiosis between the two organisms in which theyeast removes oxygen, protecting C. phytofermentans, in return for soluble carbohydratesliberated from cellulose.

The symbiotic co-cultures were stable for almost 2 months, hydrolyzed cellulose under semi-aerobic conditions and produced more ethanol from α-cellulose via SSF - Simultaneoussaccharification and fermentation than C. phytofermentans or S. cerevisiae cdt-1 mono-cultures.

The addition of a moderate level of cellulase 400 mg/L to the co-cultures in SSF experimentsimproved ethanol production two-fold greater than S. cerevisiae cdt-1 mono-culture andapproximately four-fold greater than C. phytofermentans mono-cultures giving a finalconcentration of approximately 22 g ethanol/L after 400 hours.

Microbial Consortium in Bioremediation of Petroleum Product

Petroleum hydrocarbons are not easily degradable.

Individual microorganisms can metabolize only a limited quantity of hydrocarbon substrates. So themixed cultures of microorganisms are required to increase the rate of petroleum biodegradation.

The common bacterial genera exploited for benzene bioremediation are Pseudomonas, Bacillus,Acinetobacter, Gammaproteobacteria, and Marinobacter.

The other bacterial species identified for diesel biodegradation were Pseudomonas aeruginosa andStaphylococcus aureus.

Consortium comprising of three hydrocarbon-degrading bacterial strains viz. B. subtilis DM-04 and P.aeruginosa M and NM can degrade benzene, toluene, and xylene (BTX) compounds, at a significantlyhigher rate as compared to degradation of the same compounds by an individual isolate of theconsortium

CHALLENGES IN ENGINEERING MICROBIAL CONSORTIA

There are significant challenges associated with engineering microbial consortia, and these will requireattention as engineers consider their potential applications.

Many of the challenges are shared with those faced when engineering single microbial populations, some areparticular to controlling the behavior of multiple, interacting populations.

1. Natural microbial communities can maintain homeostasis

members generally do not out-compete one another and do not exhaust the resources in theirenvironments. It is difficult to design either long-term homeostasis or long-term extinction into a syntheticconsortium, because long-term behavior, and even the long-term genetic composition of an engineeredorganism, is unpredictable - their behavior can be monitored over time.

2. In nature, gene transfer between microbes is common. As a result, engineered consortia should functiondespite horizontal gene transfer, or even exploit it.

3. To develop methods for incorporating stable changes into the genomes of microbes that are notcurrently commonly engineered.

species of Clostridia (e.g. Clostridium thermocellum, for which there are no established geneticcloning protocols, and Clostridium acetobutylicum, the protocols for which are difficult and proprietary)live in consortia with other microbes and naturally secrete powerful cellulases.

4. Inherent in engineering consortia is fine-tuning the performance of multiple populations. Techniquessuch as directed evolution that can optimize the behavior of a single population must be extended forapplication to multiple populations and varying environments.

CONCLUSIONS

• Consortia are most likely to occur in nature, engineering defined natural consortia has opened newpossibilities for enhanced environmental management applications.

• We can choose from a wide range of microbes from different geographic locations, there is potentialfor additional, yet unexplored synergetic effects that may arise as these artificially assembledmicrobial consortia would not be encountered in nature.

• The use of consortia assembled from naturally occurring species is furthermore interesting becausethey would not be considered genetically modified and are hence not susceptible to regulatoryprocedures.

REFERENCES

1. M Vinas., M Grifoll., J Sabate and AM Solanas. (2002). Biodegradation of a crude oil by three microbial consortia ofdifferent origins and metabolic capabilities. Journal of Industrial Microbiology and Biotechnology, 28: 252 – 260.

2. Katie Brenner., Lingchong You and Frances H. Arnold. (2012). Engineering microbial consortia: a new frontier insynthetic biology. Trends in Biotechnology, vol. 26 no. 9.

3. Anushree Malik. (2006). ENVIRONMENTAL MICROBIOLOGY – Bioremediation. Centre for Rural Development &Technology, Indian Institute of Technology Delhi, New Delhi. Pp. 1- 28.

4. Trevor R Zuroff., Salvador Barri Xiques and Wayne R Curtis. (2013). Consortia-mediated bioprocessing of cellulose toethanol with a symbiotic Clostridium phytofermentans/yeast co-culture. Biotechnology for Biofuels, 6:59.

5. Karl D. Brune and Travis S. Bayer. (2012). Engineering microbial consortia to enhance biomining and bioremediation.Frontiers in Microbiology. |Volume 3| Article 203| pp. 1-6.

6. X. H. Xie, N. Liu., H. Jiang., L. Y. Zhu. (2014). Construction and Application of Engineered Bacteria forBioaugmentation Decolorization of Dyeing Wastewater: A Review. Journal of Geoscience and EnvironmentProtection, (2) pp 84-88.

7. Nancy Pino., Gustavo Peñuela. (2011). Simultaneous degradation of the pesticides methyl parathiy an isolated bacterialconsortium from a contaminated site. International Biodeterioration & Biodegradation, Volume 65 (6) Pages 827–831.

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