Bioaugmentation as a Remediation Technology

1.     INTRODUCTION

Bioaugmentation is the introduction of a group of natural microbial strains or genetically engineered variants to reinforce the treatment ofc ontaminated soil or water or to support other microorganisms that provide the treatment.It is being practiced since many years in a number of areas such as agriculture and forestry, waste water treatment, soil remediation, etc. Microorganisms have been used in the clean-up of many industrial wastes containing pollutants which otherwise would be toxic to the environment. However many industrial wastes can contain manmade chemicals, which due to their unfamiliarity to microorganisms may be resistant to degradation by indigenous microbial populations. This can cause great difficulties in the biological treatment of wastes, as accumulation of such xenobiotics can cause a complete breakdown of the treatment process. In such cases, the indigenous population may be augmented by specialised bacteria, selected to perform a desired task such as the degradation of specific pollutants, in a process known as bioaugmentation. Addition of certain organisms can increase the biological diversity and metabolic activity of an indigenous population. Such an increase in the biological diversity broadens the gene pool available to the population in times of environmental stress.[6]

2.     FACTORS AFFECTING BIOAUGMENTATION

·         Indigenous micro-organisms

The adaptation capacity of indigenous microorganisms is tremendous. They are often better distributed than added microorganisms. The distance between the target compound and microorganism is also important. The added microorganisms are closer to the pollutants recently added and the indigenous organisms are closer to the already existing pollution.

·         Compound characteristics

Pollutant toxicity could inhibit the degradative activity of indigenous microorganisms. The potential of bioaugmentation exists if the microorganisms resist the toxicity. Sometimes dilution of the pollutant may be required to enable bioaugmentation. In some case of soil pollution, dilution by soil washing or bioslurry techniques were required to achieve pollutant degradation before employment of exogenous microorganisms.

·         Physico-chemical environmental characteristics

Environmental conditions play a pivotal role in determining the biological activity, whether of indigenous microorganisms or added microorganisms or cultured indigenous microorganisms. In case of soil, these conditions fall under two general categories: those that reduce the microbial activity such as temperature, humidity and ionic strength; and those that restrict the mass transfer of the compound to the microorganisms such as clay and organic matter content. 

·         Niche adjustment

The parameters that could affect the performance of the microorganisms include niche fitness, steady state microbial concentration and predators. Fitness can be defined by the quantity of microorganisms but for the treatment goals of bioaugmentation, performance is important. Sometimes when one nutrient is in excess and another is in limitation, both lead to an improved performance. The addition of nutrients to optimize the performance of an added microorganism can also lead to the increased development of indigenous microorganisms, which themselves either aid in the treatment process or hinder the process by consuming the added nutrient. In cases where co-metabolism is desired, the consumption of added nutrients by indigenous microorganisms incapable of co-metabolizing the pollutant, results in poor performance.

The difficulties in adjusting the environment or in selecting the microorganisms fit for their target environment have led to the development of techniques for protecting the added microorganisms such as encapsulation. These techniques provide the long term viability of the added microorganisms.

·         Microbial ecology

Microbial ecology is very important in evaluating the potential success of bioaugmentation and the possible advantages over biostimulation. Microorganisms are affected by maintenance energy, the production and resistance to antibiotics and toxic metabolites, predation, etc. 

·         Engineering process design

Engineering design requires an understanding of the parameters that control the bioaugmentation process. Good engineering needs to be applied to augmentation process design at experimental level. [6]

3.     DIFFERENCE BETWEEN BIOAUGMENTATION AND BIOSTIMULATION

There are two basic forms of bioremediation currently being practiced:

·         Bioaugmentation: the microbiological approach or

·         Biostimulation : the microbial ecological approach

The bioaugmentation approach involves addition of highly concentrated and specialized populations of specific microbes into a contaminated site to enhance the rate of contaminant biodegradation in the affected soil or water because the density of contaminant-specific degraders might have been artificially increased. This technique is best suited for sites that:

(i)                 do not have sufficient microbial cells or

(ii)               the native population does not possess the metabolic routes necessary to metabolize the compounds under concern.

On the other hand, in biostimulation approach, emphasis is placed on identifying and adjusting certain physical and chemical factors (such as soil temperature, pH, moisture content, nutrient content etc.) that may be impending the rate of biodegradation of the contaminant by the indigenous microorganism in the affected site. Besides the type and concentration of nutrients, physical and environmental parameters also influence the mineralization rate of hydrocarbons by degrading bacteria. These factors include the chemical composition, physical state and concentration of the crude oil or hydrocarbons; along with the temperature, oxygen availability, salinity, pressure, water activity and pH on the site. [6]

4.      DIFFERENT TECHNOLOGIES OF BIOAUGMENTATION

The remediation genes can be delivered to a contaminated site by the following methods:

·         Cell bioaugmentation

·         Gene bioaugmentation

·         Rhizosphere bioaugmentation

·         Phytoaugmentation

The different technologies for delivering remediation genes to contaminated sites are shown in figure1.

Fig1. Overview of different technologies for delivering remediation genes to contaminated

sites[6]

4.1.         Cell bioaugmentation

The different methods of cell bioaugmentation are discussed below:

4.1.1.      Use of carrier materials for bioaugmentation

Microbial inoculants havebeen applied to the soil as live microorganisms in either a liquidculture or attached to a carrier material. When applying the inoculant to aharsh environment such as soil, it may be desirable to use a carrier materialsince it can provide a protective niche and even temporary nutrition for theintroduced microorganism.Numerous different carrier materials have beenused including biosolids, charcoal-amended soil, clay, lignite, manure, andpeat.

In an experiment, the researchers added a Pseudomonasfluorescensstrain to soil either as a liquid culture, in a sterile soil carrier,or in a nonsterile soil carrier. The bacteria introduced via the sterile soildemonstrated enhanced survival as compared to the other treatments. After28 d, <103 CFU/g of the 107 CFU/g of introduced bacteria remained in the microcosms amended with the liquid inoculant and nonsterile soil inoculant,as compared to >104 CFU of introduced bacteria per gram in microcosmsamended with the sterile soil inoculant.

The ideal characteristics for a carrier material include:

·   providing an adequate environment for cell survivaland growth resulting in a long shelf life and enhanced activity when addedto the environment; being nontoxic to the inoculant microorganisms andthe environment; and allowing targeted introduction of cells and also ameans to contain the introduced microorganisms when control is necessary.

4.1.2.      Bioaugmentation with encapsulated microorganisms

Several materials such as acrylate copolymers, agarose, alginate,gelatine, gellan gum, kappa-carrageenan, polyurethane, and polyvinyl alcoholgel have been used to encapsulate microorganisms for introduction intosoil or water. Alginate is the most commonly used carrierfor bioremediation applications, and has been used with numerous contaminantsincluding chromium, cresol, nitrate, pentachlorophenol, phenanthrene,phenol, phosphate, and 2,4,6-trichlorophenol. Alginate may also have potential for delivery of naked DNA directly into theenvironment for the purpose of gene bioaugmentation.

Theuse of these materials allowsthe microorganisms to be contained in a relatively non-toxic matrix throughwhich gases and liquids can diffuse. Another potential benefit of the encapsulation technology is theability to create microsites with a unique microbial community that worksinteractively to remediate a given compound.Properties of various materials used to encapsulate inoculants are given in table 1.

4.1.3.      Activated soil bioaugmentation

Another approach to cell bioaugmentation is to use activated soil directlyas both the inoculant and carrier without extracting the degraders from the soil. Activated soil is defined as soil that has been exposed to thecontaminant of interest and contains a developed degrader population thatcan eliminate the contaminant. The use of activated soil for bioaugmentationhas the appearance of being less scientific than other methods but has thepotential advantages of:

·   the degraders are not cultured outside of the soil and thus do not lose their ability to compete in the environment as is often observed for lab-cultured strains; and Activated soil also provides many of the benefits of materials such as peat and alginate.

·    Potential inclusion of unculturable degraders that would be missed in attempts to isolate and culture an organism from one site in order to introduce the organism to another site.

Table 1.Properties of Various Materials Used to Encapsulate Inoculants [6]

Material	Description	Notable properties
Alginate	Linear polymer comprised of mannuronic and guluronic acid monomers. Produced by algae and several bacteria. Solidified by cross-linking with Ca2+ ions.	Nontoxic, biodegradable. Commonly used encapsulating material.
Carrageenan	Comprised of galactose monomers that differ in degree of sulfonation. Produced by algae. Extrusion into K+ ions strengthens gel.	Nontoxic, biodegradable. Cell exposure to >35◦C during some encapsulation processes may harm microorganisms.
Polyacrylamide .	Synthetic polymer formed by crosslinking acrylamide monomers using bisacrylamide.	More stable, not readily degradable, but acrylamide monomer is toxic
Polyvinyl alcohol gel	Synthetic gel. Polyvinyl alcohol may be mixed with alginate and cross-linked with Ca2+ ions	Nontoxic, not readily degradable. Forms very elastic gel.

Despite the potential benefits, there can be disadvantages to the useof carriers, encapsulated cells, or activated soils for bioaugmentation. Thesetechnologies are more suited to surface applications due to the probabilitythat microbial encapsulation in, or attachment to, larger particles may furtherimpede their movement through soil or sediment. Depending on theenvironmental conditions, microorganisms, and encapsulating material used,adverse conditions may develop within the capsule, such as the accumulationof toxic compounds or anoxic conditions, which may inhibit or kill theinoculant. It is therefore critical to match the appropriate carrier technologywith the specific conditions of the contaminated site.

4.2.         Gene bioaugmentation

Since introduced microorganisms often do not survive following bioaugmentation, naturally occurring horizontalgene transfer processes has been used for the introduction of remediation genes into a contaminatedsite. Horizontal gene transfer may occur via:

·     Transformation: The uptakeof naked DNA,

·     Transduction: The mediation by bacteriophage,or

·   Conjugation: The physical contact and exchange of genetic material such as plasmids orconjugative transposons between microorganisms

The potential advantages for use of gene bioaugmentation, where theremediation genes are in a mobile form such as a self-transmissible plasmid,over the traditional cell bioaugmentation approaches are:

·    The introductionof remediation genes into indigenous microorganisms that are alreadyadapted to survive and proliferate in the environment; and

No requirementfor long-term survival of the introduced host strain. [6]

4.3.         Bioaugmentation with microbial-derived materials

Another bioaugmentation approach is to add microbial products, such asbiosurfactants or enzymes, directly as an amendment either alone or incombination with a microbial inoculant. Biosurfactants have been used forbioremediation of metal and organic-contaminated material,and they may also have a utility in bioaugmentation applications either toprotect a microbial inoculant from metal toxicity or to increase the amountof organic substrates available for degradation. Enzymes, either purified or encapsulated indead microbial cells, are used for contaminant remediation. The use of these derived-materials mayavoid some of the difficulties often associated with bioaugmentation, suchas the need for survival of live microbial inoculants in harsh field environments.However, there still may be problems with biosurfactant toxicity and effectivenessalong with the potential hazards inherent in delivery ofenzymes to the subsurface while attempting to minimize enzymatic sorptionto soil solids and/or inactivation.[6]

4.4.         Rhizosphere bioaugmentation

A developing approach for bioaugmentation is to add the microbial inoculantto the soil along with a plant that supports the inoculant’s growth.The use of plants for remediation, or phytoremediation, is a relatively newtechnology. Phytoremediation has generated much interest because it is alow-cost technique that also has less of a negative impact on the site thanother remediation methods such as excavation. Phytoremediation is defined as “the direct use of living plants for in situ remediation ofcontaminated soil, sludge, sediments, and ground water through contaminantremoval, degradation, or containment.” Phytoremediation processespotentially include extraction; filtration; stabilization; degradation; and/orevapotranspiration of the contaminant. Additionally, these processes can bemediated by plants and/or plant-associated microorganisms. For example,(1) trichloroethylene (TCE) is taken up and metabolized or transpired bypoplar trees; (2) some metals are changed into more bioavailable formsby microorganisms and then taken up by hyper accumulating plants; and(3) many recalcitrant, organic pollutants are transformed or degraded byplant-associated microorganisms

The selection of specific microorganisms in the rhizosphere has potentialadvantages for bioaugmentation.Specific rhizosphere-competentmicroorganisms that degrade a given contaminant can be added to soil alongwith a plant that supports the growth of these microorganisms. By using theplant-microorganism combination, the microorganism is added to soil along with a niche, the plant root, supporting its growth thus increasing the likelihoodfor the microorganisms’ survival. [6]

4.5.         Phytoaugmentation

Phytoaugmentation is a term used to describe theaddition of remediation genes to a site via an engineered plant that containsthe microbial genes. By incorporation of these genes into plants, it is alsoeasier to control the persistence and spread of genes introduced into the environmentthan via an analogous genetically engineered microorganism. In fact, several genetically engineeredplants, including those engineered with herbicide- or insect-resistance genes,are commonly used in production agriculture. The most common approaches for applying this technology to remediationare to incorporate genes for metal binding/transforming proteins, orfor organic degradation into the plant.

Engineered Arabidopsisthaliana with the bacterial genes for arsenate reductase (arsC) andγ -glutamylcysteinesynthetase (γ –ECS) is an example of phytoaugmentation. Arsenate can potentially be taken upfrom soil by plants in conjunction with phosphate.The theory behind theconstructed system was that more arsenic could be accumulated by the plantif the arsenate taken up by the plant was reduced to arsenite.[6]

Different approaches for use of bioaugmentationas a remediation technology is summarised in table 2.

Table 2.Different approaches for use of bioaugmentationas a remediation technology [6]

Bioaugmentation approach	Organisms used	Contaminants
Cell
Culture	ComamonastestosteroniBR60 RalstoniaeutrophaJMP134 and Pseudomonas strain H1	3-Chlorobenzoate Cadmium and 2,4-dichlorophenoxyaceticacid
Immobilised	Alcaligenesfaecalis Mixed microbial culture Pseudomonas sp. UG14LrFlavobacteriumsp. and Rhodococcus chlorophenolicusPCP-1	Phenol 2,4-dichlorophenol Phenanthrene Pentachlorophenol
Activated soil	Indigenous microorganisms Indigenous microorganisms Indigenous microorganisms	Pentachlorophenol Atrazine 2-, 3-, and 4-Chlorobenzoate
Gene	RalstoniaeutrophaJMP RalstoniaeutrophaJMP134 and E. coli D11 Comamonassp. rN7(R503) Pseudomonas putidaUWC3	2,4-Dichlorophenoxyacetic acid 2,4-Dichlorophenoxyacetic acid Phenol 2,4-Dichlorophenoxyacetic acid
Rhizosphere	Pinussylvestrisand Suillus variegatus Triticumaestivumand Pseudomonas fluorescens Elymusdauricusand Pseudomonas spp. Bromus erectus Huds. and Pseudomonas sp. Strain I4	2,4-Dichlorophenol Trichloroethylene 2-Chlorobenzoate 2,4,6-Trinitrotoluene
Phytoaugmentation	Oryzasativa Arabidopsis thaliana Arabidopsis thaliana Nicotianaglauca Nicotianatabacum Nicotianatabacum	3-Chlorocatechol Methylmercury Arsenic Lead Copper Trinitrotoluene

5.     BIOAUGMENTATION OF ACTIVATED SLUDGE

Bioaugmentation is the application of indigenous or allochthonous wild-type or genetically modified organisms to polluted hazardous waste sites or bioreactors in order to accelerate the removal of undesired compounds. In spite of several successes of small-scale bioaugmentation in activated sludge and other waste treating bioreactors and the low cost, the addition of specialised strains to activated sludge to enhance the removal of pollutants present in the influent is not yet widely applied. This is due to the fact that bioaugmentation of activated sludge is less predictable and controllable than the direct physical or chemical destruction of pollutants. Natural bacterial strains can be used, but the construction of new genetically modified organisms with the potential for enhanced breakdown of organic compounds or specialised in the degradation of different chemical compounds can also be very promising. Bioaugmentation with plasmid-encoded metabolic pathways could therefore be an interesting alternative to the inoculation of strains with chromosomal pathways, because the plasmids could be more easily exchanged between the bacterial species of the sludge and thus provide the microbial community with these useful genes, thereby improving biodegradation. The activated sludge process is operated as a continuous bioreactor with feedback of the biocatalyst, which ensures rapid oxidation of pollutants present in the influent and also stabilises the system against variations in influent composition. Process conditions are regulated and cell growth is minimised in order to obtain flocculation and a highly clarified effluent. Activated sludge mixed liquors generally contain more than 108 bacteria/ml . The formation of well-settling activated sludge flocs is based on the ability of the microbial community to aggregate. [2]

Bioaugmentation may reduce start-up periods, increase the rate of degradation or introduce catabolic properties to an indigenous population where previously none existed. Simple addition of pure cultures possessing metabolic capabilities to activated sludge does not guarantee enhanced degradative abilities. The ability to metabolise a chemical is a necessary but not a sufficient condition for the organism to effect the transformation in a mixed culture. [3]

6.     BIOAUGMENTATION OF SOIL

Terrestrial environments such as soils are typically complex microbial environments that contain large, diverse microbial populations. Of the many types of micro flora found within soils, bacteria are particularly critical for in situ bioremediation. Soil bacteria are simple prokaryotic organisms with diverse characteristics, including variable terminal electron acceptors that allow for aerobic or anaerobic modes of respiration, as well as heterotrophic and autotrophic modes of nutrition. Coupled with this is their capability of remaining dormant for long periods of time within soil, and yet they are biologically engineered for rapid growth and fluid genetic changes. Thus, they are perfectly designed for and adapted to soils, which consist of an inorganic and organic matrix with fluctuating abiotic conditions. Normally, soil bioavailable microbial substrate becomes self-limiting, and soil bacteria for the most part exist under starvation conditions. Overall, then, soils are a harsh environment for bacteria, and yet they normally support diverse culturable bacterial populations of 108 to 109 organisms per gram of soil.

The addition of metal or organic contaminants to soils can impose additional stress on microbial communities, resulting in decreased viable bacterial populations and/or activities. This situation can be exacerbated when pollution results in soils co-contaminated with both metals and organics. In this case the double stress imposed on soil bacterial communities means that for effective in situ bioremediation of the organic contaminant, there must be metal-resistant microbes with appropriate degradative genes, or a consortium of metal-resistant microbes with the appropriate catabolic capabilities. High soil metal concentrations can inhibit the microbial degradation of organics that are normally easily degraded within soils. Some of the available in situ soil remediation techniques, such as excavation, transport, landfilling of contaminated soils, acid leaching, chemical stabilization, and electro reclamation are associated with high cost, low efficiency, and are environmentally destructive.

In such cases bioaugmentation may enhance degradation and may even be a prerequisite foreffective bioremediation. Bioaugmentation has been defined as the introduction of specific microbes into a contaminated site for the purpose of enhancing the biological activity of the existing populations. The major problems associated with bioaugmentation are the rapid decline in numbers or death of the introduced microbe that can occur because of biotic or abiotic stress and the difficulty in getting the introduced microbes dispersed throughout the contaminated site.

These problems can occur when the expected enhanced degradation is caused by activity from the introduced whole cells. [7]

7.    PROBLEMS ASSOCIATED WITH BIOAUGMENTATION

A large number of exogenous microorganisms decrease shortly after addition to a site. There are abiotic and biotic stresses causing the death of introduced organisms. The abiotic stresses may include fluctuations or extremes in temperature, water content, pH, and nutrient availability, along with potentially toxic pollutant levels in contaminated soil. In addition, the added microorganisms almost always face competition from indigenous organisms for limited nutrients, along with antagonistic interactions including antibiotic production by competing organisms, and predation by protozoa and bacteriophages. It can also be difficult to deliver the inoculant to the desired location. This is not problematic for surface soils where the inoculant can be mechanically incorporated into the contaminated material, but in subsurface environments direct incorporation ranges from difficult to impossible. Technologies such as use of ultramicrobacteria, bacteria with altered cell surface properties, and/or addition of surfactants may facilitate greater transport through the soil matrix.The ability to distribute the inoculant also depends on what organism is being used. Fungi, which are larger than bacteria, are usually restricted to surface applications while bacteria are more adaptable to surface or subsurface applications. [6]

8.     BENEFITS OF BIOAUGMENTATION

Enhancement of biodegradation has several benefits:

·         conversion of toxic compounds to

·         nontoxic end products,

·         lower costs of disposal or no disposal at all

·         reduced health and ecological risks,

·     reduced long-term liabilities usually associated with non-destructive treatment methods

·   ability to perform the treatment in situ with a very low disturbance of native ecosystems. [1]

REFERENCES

1. G. Malina and I. Zawierucha,2007,Potential of bioaugmentation and biostimulation for enhancing intrinsic biodegradation in oil hydrocarbon– contaminated soil,Bioremediation Journal, vol. 11(3), 141–147

2. H. Van Limbergen á E. M. Top á W. Verstraete, 1998, Bioaugmentation in activated sludge: current features and future perspectives, Applied Microbiology and Biotechnology, vol. 50, 16-23

3. Henry McLaughlin,Alan Farrell, and BridQuilty, 2006, Bioaugmentation of Activated Sludge with Two Pseudomonas putidaStrains for the Degradation of 4-Chlorophenol, Journal of Environmental Science and Health Part A, vol. 41, 763–777

4. In-SooKim, KaluibeEkpeghere, et.al.,2013, An eco-friendly treatment of tannery wastewater using bioaugmentation with a novel microbial consortium,Journal of environmental science and health,vol. 48, 1732–1739

5. Regional municipality of Haltonbiosolids master plan, Report on effective microorganisms andbioaugmentation

6. Terry J. Gentry, Christopher Rensing, and Ian L. Pepper,2004, New approaches for bioaugmentation as a remediation technology, Environmental Science and Technology, vol. 34, 447–494

7.  Timothy M. Vogel, 1996, Bioaugmentation as a soil bioremediation approach, Current opinion in biotechnology, vol. 7, 311-316