A brief introduction to the microbial consortium and the concepts of the consortium with its environmental applications were given...

1. G. KANTHARAJAN
ICAR-CIFE
Course No : AEM 510
Course Title : Environmental Biotechnology
Consortia of Microbes for Environmental Protection
(concepts, scope and feasibility)

2. INTRODUCTION
• Microbial consortia are ubiquitous in nature - great importance to humans, from
environmental remediation and wastewater treatment to assistance in food digestion.
• Synthetic biologists are honing their ability to program the behavior of individual microbial
populations, forcing the microbes to focus on specific applications, such as the production of
drugs and fuels.
• Microbial consortia can perform even more complicated tasks and endure more changeable
environments than monocultures.
• Represent an important new frontier for synthetic biology.

3. CONCEPTS OF MICROBIAL
CONSORTIA
 Consortia concept for bioprocessing applications is supported by
observations in nature.
Naturally occurring ecosystems, optimized by eons of evolution, are
almost exclusively organized as mixed communities.
A group of different species of microorganisms that act together as
a community (Encyclopedia of medical concepts).

4. 2 organizing features
1. Members of the consortium communicate with one another. Whether by
trading metabolites or by exchanging dedicated molecular signals, each population or
individual detects and responds to the presence of others in the consortium.
The overall output of the consortium rests on a combination of tasks performed by
constituent individuals or sub-populations.
2. Communication enables the second important
feature which is the division of labor

5. 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 multiple
microbes.
- RRT has been adapted to consider the benefits of resource trading in consortia, highlighting conditions
where coexistence is more competitive than monoculture strategies.
- A super-competitor unit is a consortium that possesses the emergent system property of enhanced resource
utilization and therefore depletes resources more efficiently than the respective monocultures.

6. 2. Maximum Power
Principle (MPP)
Initially proposed by Lotka (1922)
A consortium that utilizes multiple substrates in parallel would have a higher metabolic rate
and therefore fitness than a monoculture that utilized the same substrates sequentially.
Both RRT and MPP are useful for examining design principles for engineering microbial
consortial interactions for environment management.

7. Consortial Interaction
Motifs
1. DIVISION OF LABOR
At the foundation of many cooperative interactions is division of labor through functional differentiation and
specialization.
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.

8. 2. SYNERGISTIC DIVISION OF RESOURCES
Carbon or energy source are partitioned between community members in a non-competitive
manner based on metabolic functionality.
This template permits parallel processing of substrates and has been used to construct consortia
which simultaneously ferment pentose and hexose sugars, a functionality that is often
unattainable in monocultures due to catabolite repression .
Catabolite repression allows bacteria to adapt quickly to a preferred
(rapidly metabolisable) carbon and energy source first.

9. 3. COMMENSALISM
 One community member’s activity provides an ecological niche for others at no benefit or cost
to itself.
 Commensalism is frequent in biofilms where, for instance, the consumption of oxygen by one
community member establishes an oxygen gradient creating microenvironments suitable for
anaerobic microbes.
 Metabolite exchange: when a producer organism secretes by-products at no benefit or cost to
itself which permits sequential consumption by other community members.

10. 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 bioprocessing
of cellulose coupled with biofuel production.
 For instance, it is commonly demonstrated in producer-consumer relationships where an organic acid
consuming community member scavenges inhibitory byproducts from a producer population.

11. CONCEPTS OF MICROBIAL CONSORTIUM

12. Mono culture vs Consortium

13. SCOPE OF MICROBIAL CONSORTIA
Mixed populations can perform complex tasks
• Mixed populations can perform functions that are difficult or even impossible for
individual 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 when
different 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 cellulosome
that is capable of cellulose degradation

14. Mixed populations can be robust to changes in
environment
 Compared with monocultures, communities might be more capable of resisting invasion by
other species.
 They might be able to weather periods of nutrient limitation better because of the diversity of
metabolic modes available to a mix of species combined with the ability to share metabolites
within the community.
For example, when nutrients become limited, the most prevalent species in a community are not
always 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 engineered
consortia will perform most reliably in changeable environments.

15. Communication organizes function in
engineered consortia
• Communication in natural consortia can involve the exchange of dedicated signal molecules
within or between single populations.
- Exchange of acyl-homoserine lactone (acyl-HSL) signaling molecules - in
Gram-negative species
- Small peptides - in Gram-positive species
- Inter-population communication between Gram-positive and Gram-negative
species, through auto-inducers 2 and 3.
• For example, the member species of a consortium that degrades the herbicide diclofop methyl
pass intermediate metabolites back and forth in the process of degrading the compound -
exchanging metabolic intermediates that either assist or compromise the growth of their
neighbor.

16. Consortia of Naturally Occurring Species
 Naturally occurring consortia have been characterized as well as defined consortia of naturally
occurring organisms.
 The impact of natural consortia was shown to be profound. In one example, a synergetic effect was
during 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 of
heterotrophic acidophiles to remove inhibiting organic compounds that accumulate during growth led
to acceleration of the leaching process.
This was attributed to the increased growth rate of A. ferrooxidans while it was co-cultured with the
heterotroph Acidiphilium acidophilum.

17. 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 bacterial
consortium 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, Proteus
vulgaris, 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 methyl
parathion and chlorpyrifos in 120 h. When a mixture of 150 mg l−1 of both pesticides was used the percentage
decreased 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)

18. Compound Organism Reference
Polychlorinated Arthrobacter sp. B1B and Ralstonia Singer et al., 2000
biphenyl (soil) eutrophus H850
BTEX Methanogenic consortia Da Silva and
Alvarez, 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 generaAzoarcus and Harwood,C.S
Thauera, iron-reducingGeobacter et.al.,(1997)
Metallireducens
Tolune Pseudomonas putida strain mt-2 Meckenstock et al.,
Thauera aromatica strain K172, (1999)

19. Microbial Consortia in Biomining
 The impact of microbial consortia in bioleaching, particularly in copper recovery, is widely recognized in
literature and industry.
The “indirect” mechanism assumes that chemoautotrophic iron-oxidizing microorganisms
like Acidithiobacillus ferrooxidans or Leptospirillum ferrooxidans generate ferric ions by oxidation of ferrous
iron.
Chalcopyrite leaching is particularly sensitive to inactivation by formation of jarosite layers as a function of
redox potential and is thus one of the most recalcitrant ore to leach. These sulfur layers however can be
oxidized to soluble sulfate by sulfur-oxidizing bacteria such as Acidithiobacillus caldus or Acidithiobacillus
thiooxidans.
Hence naturally occurring consortia of autotrophic iron-oxidizing microbes and sulfur-oxidizing microbes
have been proposed to be symbiotic, potentially mutualistic or at least synergetic in substrate use.

20. Mixed Bacteria Consortium forTreating 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, Proteus
mirabilis and Rhodococcus globerulus by 16S rRNA gene sequence analysis) which possesses the degradation
capacity 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 the
bacteria.
• After treating AO7 solution 16 h at the concentration of 200 mg/L, the decolorization rate had reached 90%
which showed perfect effects.

21. Consortia-Mediated Bioprocessing of Cellulose to
Ethanol
 Application of a symbiotic co-culture of a cellulolytic mesophile, C. phytofermentans, and the
cellodextrin fermenting yeast, C. molischianaor S. cerevisiae cdt-1 for direct ethanol production
from α-cellulose.
 Controlled oxygen transfer is used to induce a symbiosis between the two organisms in which the
yeast removes oxygen, protecting C. phytofermentans, in return for soluble carbohydrates
liberated 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 - Simultaneous
saccharification 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 experiments
improved ethanol production two-fold greater than S. cerevisiae cdt-1 mono-culture and
approximately four-fold greater than C. phytofermentans mono-cultures giving a final
concentration of approximately 22 g ethanol/L after 400 hours.

22. 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 the
mixed 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 and
Staphylococcus 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 significantly
higher rate as compared to degradation of the same compounds by an individual isolate of the
consortium

23. CHALLENGES IN ENGINEERING MICROBIAL
CONSORTIA
 There are significant challenges associated with engineering microbial consortia, and these will require
attention as engineers consider their potential applications.
 Many of the challenges are shared with those faced when engineering single microbial populations, some are
particular 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 their
environments. It is difficult to design either long-term homeostasis or long-term extinction into a synthetic
consortium, because long-term behavior, and even the long-term genetic composition of an engineered
organism, is unpredictable - their behavior can be monitored over time.
2. In nature, gene transfer between microbes is common. As a result, engineered consortia should function
despite horizontal gene transfer, or even exploit it.

24. 3. To develop methods for incorporating stable changes into the genomes of microbes that are not
currently commonly engineered.
species of Clostridia (e.g. Clostridium thermocellum, for which there are no established genetic
cloning 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. Techniques
such as directed evolution that can optimize the behavior of a single population must be extended for
application to multiple populations and varying environments.

25. CONCLUSIONS
• Consortia are most likely to occur in nature, engineering defined natural consortia has opened new
possibilities for enhanced environmental management applications.
• We can choose from a wide range of microbes from different geographic locations, there is potential
for additional, yet unexplored synergetic effects that may arise as these artificially assembled
microbial consortia would not be encountered in nature.
• The use of consortia assembled from naturally occurring species is furthermore interesting because
they would not be considered genetically modified and are hence not susceptible to regulatory
procedures.

26. REFERENCES
1. M Vinas., M Grifoll., J Sabate and AM Solanas. (2002). Biodegradation of a crude oil by three microbial consortia of
different 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 in
synthetic 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 to
ethanol 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 for
Bioaugmentation Decolorization of Dyeing Wastewater: A Review. Journal of Geoscience and Environment
Protection, (2) pp 84-88.
7. Nancy Pino., Gustavo Peñuela. (2011). Simultaneous degradation of the pesticides methyl parathiy an isolated bacterial
consortium from a contaminated site. International Biodeterioration & Biodegradation, Volume 65 (6) Pages 827–831.

27. THANK YOU