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Prom ++ paasege oF an pron thee BLA undudrauw Sybtt < / CUT Udi aL » atawoe Le wt 329 | le 4 Les be Fi on ~Biovemediahiar, Introduction As is cle; ‘. compere ie 1. Sell “bioremediation* (bio + remediation) should involve two he teen atte live component and romedt ee sormaminant, The term denotes ihe comPOneM and remediation i.e. the treatment of the be remediated. Therefore, betare discuss what do we ney Present state of our environment Rapid industriatization and urbanization over the Suramination of all the components ot even our food. The process began it compounds for use as solvents, pesieit contamination of the environ like fugitive emissions waste, Over the years in organic contaminants hazardous or toxic, By and the food chain th Should be worried about such poltutame Past many decades has resulted in he environment that isthe air, the water, the soils and the generation of a plethora of synthetic organic s, refrigerants and chemical intermediates ete. The “dena 80 Secured through a varity of industrial operstions gc Gettal spills and leaks, discharge of effluents or dumping of discriminate use of the s has released several such ‘0 natural degradation and may also tum sag leased in the sewers, the compounds come back we so system Ad sludge, Nevertheless, the next question is why we Potential hazards sed | 'upon the type of contaminant, chemical species, spatial distribut route of exposure etc. Apart from this, the nature of matrix (for example, hydrogeological characteristics in case of soil) also affect ‘he potential hazards. Broadly the effect could be sung ized in two categories Short-term hazard: Direct contacts, inhalation of toxic dust/fumes or immediate risk of fire/explosion. Long-term hazard: Resulting by movement to other com generation of secondary toxic products, For example, rain water followed by percolation to ground water | i ponents of the environment or transportation of contaminants through and contamination of the drinking water fee develop a wide variety of physico-chemical and biological techniques that can remediate the hazardous contaminants fiom the environment without causing furtha, damage. The contatinal techniques of icineratonland-fils et, basically transfer pollutanis en ‘medium to another, are expensive and energy demanding, Further, these techniques are often inefficient for handling voluminous effluents containing complexing organic net and low Sereantaton of contaminants, Biotehnological approeches that are designed te cote son iches have, therefore, recived great deal of attention in the recent years Bioremediation is an attractive and potential altemative for treatment of contaminated sites. Let us now examine in detail what exactly the term bioremediation implies and the host oflong ter is pas Sane ‘must be conducted throughout the process to confirm that degradation 8 at rates consistent with ‘meeting cleanup objectives. eee srnetiation technologies, natural attenuation has several advantages structures are required) and Cuts OF remediation wastes; less intrusive (as few surface conditions and el and may be applied to all or Part of a given site, depending on site (active) roar ¥eS: Further it may be wed in conjunction with ora follow, up to, other (active) remedial meas, i v% a : eee es and the overall cost will likely be lower than active Biostimulation Bioventing Similar to biostimulation but it involves Venting of oxygen through soil to stimulate growth of natural or introduced microorganisms. Thus bioventing may complement biostimulation as Wel 3s bioaugmentation. It is a promising technology that stimulates the natural in situ Diodegradation of any acrobically degradable compounds in soil by providing oxygen to cxiStng soil microorganisms. Bioventing typically uses low ait flow tes to provide only Gnough oxygen to sustain microbial activity. Oxygen is most commonly supplied through direct air injection into resi ‘dual contamination in sol. In addition to degradation of adsorbed fuel residuals, volatile comy ating techniques have been successfully used to remediate soils contaminated by petroleum hydrocarbons, nonchlornated solvents, some pesticides, wood Peete a other organic chemicals. This technique shows considerable promise of Stabilizing or removing inorganics from soil as it can induce changes in the vale atte of tnorganies and cause adsorption, uptake, accumulation, and concentration of inorganis fa iro or macroorgenisms. However, several factors may limit the applicbilty and effectiveness of the process for example highly saturated soils, extremely low molstore onfent of low permeability soils negatively affect the bioventing performance. Build up of ‘apours needs to be avoided by extracting the air followed by monitoring of off-zases atthe Soil surface. The biggest limitation is that aerobic biodegradation of many chlorinated compounds may not be effective unless a eo-metaboliteor anaerobic cycle is present Bioaugmentation Addition of pollutant-degrading microorganisms engineered) to augment the biodegradative capacity termed as bioaugmentation. Sometimes microorga collected, separately cultured, and retuned to the sit microorganism population atthe site. Usually an attempt is made to isolate and accelerate the growth of the population of natural microorganisms that preferentially feed on the Contaminants at the site. In some situations diff ferent microorganisms may be added at Aiferent stages of the remediation process because the contaminants change in abundance se (caturalexotic/acclimatized/genetically of indigenous microbial populations is nisms from the remediation site are te as a means of rapidly increasing thethe degradation procee, ‘microorganisms is ben Als. However the ficial in the sit Te is no evidence to suggest thatthe use of non- ations tested, Biofilters Toa 6 Use of microbial stripping columns (Containing microorganism enriched compostsoil is to teat organic eases (volatile organi compounds), Bioreactors Biodegradation of contaminants in a liguid efftuents/stu large tank or reactor. Bioreactors can be used to treat ries or contaminat ted solid waste/soil Composting Composting is aerobic, thermophilic treatment Process in which contaminated material is ied with a bulking agent (compost rsh in bioremediation microorganisms). This is a centoled biological process by which orang ‘contaminants (¢.g,, PAHS) are converted by microorganisms to safe, ts. Typically, thermophilic conditions (54 to 65°C) must be maintain. 0st soil contaminated with hazardous organic contaminants and in mos fed by the use of indigenous microorganisms, Stabilized byproduct ied to properly comp cases, this is achieve Soils are excavated and mixed with bulki chips, animal, and vegetative wa decomposed. Maximum degrada ‘moisture as necessary, and close ing agents and organic amendments, such as wood sles etc, to enhance the porosity of the mixture to be ion efficiency is achieved through maintaining aeration and ly monitoring moisture content, and temperature Basically thee different process designs are used in composting: * Acrated static pile composting where compost is forme into piles and aerated with blowers or vacuum pumps * Mechanically agitated in-ves mixed and aerated Windrow composting where compost is periodically mixed with mobile equipment el composting where compos is placed in a reactor vessel, Placed in long piles known as windrows and Of explosives (TNT, RDX, and HMX), ammonium pirate (or yellow-D), and sercncg Teuly fo acceptable levels and is also applicable to PAH-contaminated soil. The substentil Ty ament of space and aeration coupled withthe need fr excavation of contaminate cal {imi the application of composting. If VOC (volatile organic compounds) or SOK (semi- ‘latte organic compounds) contaminants are present in sols, off-gas contol may be Gyuuted: Lastly, this method cannot treat metals and due to’ addition of amendwen, ultimately leads to volumetric inerease in the amount,Landfarming Itis a solid phase treatments "m for contaminated soil wh System for ¢ eae Ca ys contaminated soil : it souRe the growth of benef area, Different conditions that are contolig ed fa 2 Moisture content (usually by ization or spraying), + Acration (by tiling the ft Soil With a predet OH (buffered near neutral pH by adding * Other amendments (6.9. Soi bulking. here tilling and soil amendment microorganisms in contaminated ming are: quency) 18 crushed limestone or agricultural lime). Agents, nutrients, et) Temay be done in situ or ina treatment ell petroleum spills, Wood areserving waste Pesticides in the soil. The lan prevention of volatile gases summarizes the advantages and has been successfully used to remove large (PCP and creosote), coke wastes, and certain Se Tequirement of space, proper management of leachates and ard ato the limitations associated with landfarming, Table 1 and disadvantages of different types ofbioemediat i Techniques | Examples Advantages In situ ln situ |Most cost efficient Environmental constraints bioremediation | Noninvasive [Extended treatment times Bioventing Relatively passive Monitoring difficulties [Natural attenuation Biostimulation Bioaugmentation [Treats soil and water Ex situ Land farming | Costefcient [Extended treatment time [Composting Can be done on site [Need to control abiotic loss Mass transfer problem Bioavailability limitation ‘Requires excavation Relatively high capital ‘cost & operating cost 'Biopiles/Biocells Bioreactors |Slurry/aqueous | Rapid degradation kinctiow reactors [Optimized environmental parameters Enhances mass transfer Effective use of inoculants| and surfactants (Source: M, Vidal Bioremediation An overview Pare Appl. Chem, 73, 1163-1172, 200)) Tsita new concept? Around the world bioremediation technologies are categorized as the fechnologies". However, this does not mean that it is a novel phenomenon, bioremediation has been going on since the life began on this planet. Its a relatively slow rooess, but eventually nature has healed itself of all the disturbances. As a contaminant fy inirodued into the environment, the microbes ofthe surrounding area get pradally adapted Ho this changed environment. They begin to elaborate the process of degrading the mnovative Actually‘contaminant by evolvir r proces, the a Hae ably tose tas a exon or energy sues, Dig tis natu re prces shor ai a utonal and physiological needs ofthe bacteria and the contaminants at a staggering hi N ig. 1). With our fast development we have introduced the contains 8 Hagring high rte and ow we nol inst clan seltion at ot i aeiLRnERaN Hae ‘The natural process can however be speeded up by roan conden Ce: 18s disused inthe eater scion ie. cites by providing contaminated site How teehee the number of efficient microorganisms at the rie ee Vere S e e e eR in Fig. . After taking air ve view of the diferent bioremediation tsnolgs et ws ake an insight into what degraded and how does microbe metabolism handle them. ied s 2) Fig. 1: (a) Natural and (b) Man-Assisted bioremediation Bioreactor Years Mechanism of Bioremediation Broad categories of contaminants ‘As noticed above, if favourable nutritional and environmental conditions occur, the bacteria ane able to readily incorporate the simple organi substances into their cells and oxidize them. However, degradation of complex organic compounds with longer molecular structies is ‘lower, Some compounds are so complex that they cannot be degraded at all, which are termed ag recalitrant or refractory compounds, Still other may be toxic and thus inhibit the growth of microorganisms and their metabolic activity. Such compounds need special Techniques or integration of physico-chemical and biological techniques, for effective remediation (Fig. 2).ism by which microbes gain energy, they are broadly cap torized into three categories (Table 2). However, several xenobiotic contaminants might not be amenable to one of the at ibove described categories and other mechanisms are ‘employed by the microorganisms for degradation of such compounds. ‘Table 2: Mechanisms of energy generation by the microbes ‘Mechanism Electron donor |Electron acceptor [Product ‘Aerobie respiration | Organic Oxygen 00;, 10 compound [Anaerobie Organic INOs, 80s, Fe, Ma® Na, HS, CH respiration compound CO: Reduced metals [Fermentation | Organic [Organi compound | Organie acids, ‘compound alcohols, Hs & CO, Reductive dehalogenation: It plays very im portant role in the detoxification of halogenated organic contaminants. Microorganisms. cat talyze a reaction in which halogen atom of ontaminant is replaced by hydrogen stom. Thus the reaction adds? electrons t conearnacs and reduces it. It yields no energy but seems to be detonificn tion mechanism as etialogenated derivatives are less oxi and susceptible to further microbial devs,CCometabolism: In this ease also th z 5 on-enefcil transformation ig gc°™Net!on reaction yields no benefit othe cell. This eatuitous metabolism, Costeabol 8 secondary utilization, cometabolism or hich enzymes produced for ph ON form of secondary substrate transformation in reader ua foallioat mary substrate oxidation are capable of degrading the : nffcient energy to sustain the even though the secondary substrates do not_afford : ae Population, It is thus defined as degradation of a wiht OFBAnc compound that serves as a primary energy us aes degrade corsets tthaotopi activity, which has been demonstrated effet inated solvents, ; et jy oneal 3} S, such as vinyl chloride and Trichloroethane (TCE), Natrient and pit ‘Mathane and Adjustment Oxygen Addition To Further Treatment or Discharge Groundwater Extraction Walle t Vadose Zone itu ground water remediation via cometabolic activity (Source: EPA, 1993 n Situ Bioremediation: Biodegradation ofTricMloroethylene and Tetrachloroethylene by Injection of Air and Methane, Inovatve Remedial Technology Information Request Gude) Contaminant structure Vs biodegradability relation Biodegradability is essential for bioremediation of organic pollutants. Chemical structure of pollutant governs the ability of microorganisms to metabolize them, especially the rate and ‘extent of biodegradation. As we discussed above, some compounds are readily biodegradable ‘whereas others are not. Let us look at the general ules that determin this,+ Low to mid molecular wei ‘odewradable Charest ‘ight hydrocarbons and alcohols represent readily «Branched and polynuclear comps chain and Sipe nocroebe GABGURII eo oe ee ea Halocabons are resistant to bodoyradal Gonpounds: DRT an to biodegradation ‘and ate often termed as xenobiotic decreases the rule of te ifficult to degrade, Increasing degree of halogenation Mondehloro| CERES iodegradation, For example Dichloromethane as well as Ma Re degraded fast as these are used as carbon and energy lve end ESH # an the other hand Trichloroethane or TCE (widely used aes i lorinated biphenyls (widely used in transformers/pesticides) are vonv/energy source and are degraded slowly through cometabolic route. uch rules have been more clearly defi below: ed for broad structural categories of the pollutants as (A) Petroleum hydrocarbons «Short chain alkanes are more toxic and relatively difficult to biodegrade « Nealkanes of intermediate chain length (Cip-Czs) degraded most rapidly «Very long chain alkanes are increasingly resistant to degradation + When molecular weight is greater than 500, such long chain alkanes are no longer feasible as carbon source «Aromatic hydrocarbons (especially condensed polynuclear aromatic compounds) degrade more slowly than alkanes © Some aromatic compounds (benzene, toluene) metabolized under anaerobic conditions but the rates are slower than aerobic reactions «© Alicyclic compounds cannot serve as earbon source unless they have long aliphatic side chains, hence often degraded by cometabolism (B) Halocarbons «Most common mechanism is stepwise reductive dehalogenation under anerobic conditions e.g, cE ————————>* Viny| Chloride ——> Ethane Stepwise dechlorination ‘Cometabolic degradation by methanogenic consortium also reported ‘Aerobic degradation of TCE by methane utilizing consortium is also possible ‘Microcosm studies report both aerobic/anaerobic transformations In case of haloaromatics such as chlorobenzenes, aerobic biodegradability decreases with number of halosubstituents «Extensively chlorinated haloaromatcs degraded by stepwise anaerobic dechlorination ‘The consecutive dechlorination steps are more dificult, slow and often incomplete but the mono and dichloro substitutes can be aerobically degraded for example 10Hexachlorobenzene Anaerobi ic, fast Reductive dechlorination 1,3, 5 ~Trichtoro benzene Anaerobic, Slow Dichloro or Monchlorobenzene Fast ‘Aerobic biodegradation (Pseudomonas, Alcaligens) Chlorocatechols, (Q)Nitroaromatics ‘+ Nitrosubstitution makes the compounds less biodegradable + Excessively nitrosubstituted aromatics transformed under anaerobic conditions by stepwise conversion of nitro groups to amino groups Bioremediation Microorganisms/Agents Different microorganisms are able to degrade different contaminants depending upon the nature and coencentration of contaminant and the metabolic needs of the microorganisms. Scientists around the world are continuously working to find out novel and more efficient biodegraders. Let us discuss the important bioremediation microorganisms under the following heads Pure Cultures Since the conventional techniques of microbiology are designed for pure culture studies and also it is easy to work out the efficiency of microbes in such systems, most of the laboratory studies focus on biodegradative capacity of pure cultures. Table 3 shows the famous bioremediation microorganisms. Pseudomonas species are most widely detected microbes in the contaminated site due to their extensive biodegradation capacities (Table 4). Among the fugal groups, phanerochaete has been proposed for the biodegradation of various pollutants such as DDT, TNT, high molecular weight polynuclear aromatics like benzo (a) pyrene and plastics such as polyethylene. Acclimatized Microorganisms & Molecular Breeding ‘The metabolic range of naturally occurring microbiota may not be capable of degrading certain compounds or certain classes of compounds, There it may be necessary to supplement with the specialized microbes. One way of developing such specialized microbes is by repeatedly exposing them to higher concentration of contaminants, Often the microorganisms with specialized degradation ability can also be enriched from the contaminated sitesiya pall Table 3: Microorganisms (Pure cultures }) helpful in. bioremediation Pollutant : Microorganism(s) Reference in me Asheibarter Species [Singh eral, 2008 Feeudomonas sp. strain |Shapir and R-T, Mandelbaum, 5 1997 Ghlorpyrifos Aspergillus niger Mukherjee and Trichoderma viride Se cee Bacteri i [SINR @7a17 004 a ire rndreoni et al, 2003 Ciel ee eutropha (pJP4) |Daane and Higgblom, 1999 Reisen earopha [Roane et al, 2001 (Carbon tetrachloride [Pseudomonas stutzeri KC_|Dybas et al, 2002 [Methyl tert-butyl ether |Hydrogenophaga flava |Streger et al., 2002 ENV735, B-proteobacterium strain |Smith etal, 2005 PMI BTEX B. sp. Strain JS150 [Kahng et al. 2001) 1B. Cepacia G4 Shields etal. (1995) IR. Picketii PKOT [Byme etal, (1995) 'S. yanoikuyae BI [Kim and Zyistra, (1999) | [Orange 3, 4-(4- Pleurotus ostreatus /Xueheng Zhao et.al.,(2006) nitrophenylazo) aniline ‘Table 4: Pseudomonas strains with proven ability for bioremediation of BTEX P. putida MTS Keil etl., (1985) P. putida Fl Zylstra and Gibson, (1989) P. putida mt-2 (PaW1) Burlage et al., (1989) P_ aeruginosa J1L04 Kitayama et al., (1996) P. stutzeri OX1 Bertoni et al., (1998) P. putida MTS3 Kok et al. (1999) Pseudomonas putida, P, fluorescens |Hojae Shim (2002) To achieve a complete catabolic pathway for a xenobiotic compound is the ultimate objective for its biodegradative clean up. Biochemical pathways are under constant evolution and this, process can be accelerated by plasmid mediated genetic exchange, recombination. A. 12ly constructed Pseudom A speci }onas strain ‘effective for degradati it this way (as 1 wast tees OPES f clorobenoate and shen, SN ieee int yal of haloaromatic ical manufacturing plant where yes eres imroduced removal hydrocarbons, plant where it could bring about the small concentration of xenobiotics cher a related but utilizable Enrichment T=WeeksiMonths Substrate peers JM FH concentration ‘concentration ee decreased increased oA if rani eel Spontaneous mutant with increased abil nt nt with inereased ability to utilize Xenobllics (9. specially construct Pseudomonas stain 9F degradation of chlorobenzoate & chlorophenols Fig. 4: Molecular Breeding Genetically Engineered Microorganisms (GEMS) ‘The microorganisms can be genetically engineered to enhance its enzymatic capability to degrade wide range of compounds. GEMS have the advantage of possessing high growth affinity, rapid growth rate and resistance to toxicity. With such solutions several shortcomings of the bioremediation can be overcome, However, practically the potential Tesults of release of such GEMS into the environment cannot be predicted because ‘suboptimal conditions exist in the field and the GEMS might also face tough competition from the native communities. The artificially introduced genes can persist in environment for example phenol-degrading plasmid has been found in sol 6 years after addition of GEM. A Tot of debate exists concerning safety, persistence, containment, and potential ecological damage associated with the release of GEMS in the environment. Due to these constrains it is unlikely that GEMS could be utilized for large-scale bioaugmentaton in the coming days. To overcome the problem of persistence and long-term ecological damage suicidal ticroorganisms have been designed. These cells have suicide mechanisms (e.g, hok/sok system) so that the cells or the recipients of plasmid die in absence of pollutant. ‘Adhesion deficient microorganisms: Natural adhesive properties of native bacteria limit their Penetration through soil and rock matrix. A specialized adhesion deficient TCE-degrading Stain B. cepacia has been developed (which could rapidly disperse 25 feet depth) and used successfully for in-situ bioremediation of TCE-contaminated aquifer.Consortium Processes unch; : Even in the ividually cannot mineral f waste characterized consort co cmt mai a a el es Specific combination of rmiroorgne mand tat aa call We eae pollutant is a : . llutants of this type include pol rtium’) and pala ise nares ms (8 “conso pe lyaromatic. hy Prghatic and aromatic), polychlorinated hn a carbons, halogenated organics (both ITNT), , Multi-nitrated arenes (such as the explosive 2, 4, G-trinitrotoluene [ ed biphe: rbicides. In such cases, t Organophosphorous i the polluted biotope fe solution for sucessful Waktiehatin ia! te oeeT anisms — bioaugmentation. Complete mineralization can be ace ‘ complished thro) rtium of microor ied through @ sequential a ome nisms and involves syergsm and conti atoms (ig 8). Table 5: Consorti ; tium for degradation of recalcitrant compounds i Compound 5 [ aemeoas| jpowet = I Organism Reference Polychlorinate /Arthrobacter sp. BIB and R - s . jalstoni bipheny! (soil) /eutrophus 850 ond Ranronia. ‘Singer et al., 2000 /BTEX Methanogenic consortia [Da Silva and Alvarez, 2004 (Chloroethenes (Consortium that contains Dehalococcoides|Lendvay etal. 2003 (Consortium that contains Dehalococcoides [Adamson et al., 2003 Consortium that contains Dehalococcoides [Major etal, 2002 [Chiorobenzenes PP putida GI, P, aeruginosa RHOI and P. |Wenderoth eta. putida FLAC 2003 TjLi-Tvichloroethane |Butane-utlizing enrichment culture itmuyanont eta, 2001 ‘Atrazine Consortia degrading atrazine [Goux etal, 2003 Toluene ~atrate-reducing genera Azoarcus and |Harwood, CS "Tavera iron-reducing Geobacter _et.al(1997) metallireducens Tolune Feewiomonss pulida tran me-2 _ |Meckenstock etl, ‘Thavera aromatica strain K172, (1999) Geobacter metallireducens al i nated, cohesive microbial communties, composed of a consortium of rb mas are amine recy orf Oe or Mats generally include anoxygenic photoautotrophs (purple bacteria) ant ipa = co Mas pvc in ee anal Ste Pa ey sugue th e success of microbial mats and their ‘broad array of microbial activities suggest that t 2 microbial ecosystems might be useful to bioremediation of environmental pollutants ar 4metalloids, ‘contrasting mechanisms fo reaniee ais for removal which occur atthe emits, mats display er vel of ae cin Micctial Galea sonra, Meri ma ah Se apamte aS ns (chlordane), 2.4.6 rnieeolnen Coe a FEY 2 dnircluene (DNT), ind absorbable organ atl absorbable orgenoehlorine compounds 4 the community level of the ene ‘organic compounds also and orate ene ese include pateleum dil, the pesticides (carbofuran, paraquat (AOX) as effluents from he, ts Taipeete a soe [at intermediate oe 2 intemedite intermediate 0, HO. Fig. 5: Mineralization of contaminant by microbial consortia Enzymes jon of polluted sites could be that of utilizing cell ‘An appealing alternative to the bioremediati ad from their originating cells. Extra celular enzymes include @ Taree free enzymes isolat Frage of oxidoreductases and liydolases which may explicate « degrada function and rane one yer, suerte erin peta, Seema cede) FOOT! Eo be cecil upnken by eels, For instance, partial oration oF esata pollutants such as PAIL, By extn celular oxidative enzymes give rie 1 proses of increased polarity and eae rotary and thus with a higher biodeeredbiliy, Pestle of different chemical Pree ry recaicaae geome ike psalicig polychlorophenols, PAHS and others toxic pollutants were suecessfully transformed (in lab-scale studies) by thes oi otis ola oy upeak, atere! tt Fal col several advantages over the use of microbial cells. The most significant features of cell-free enzymes are eit AINE substrate-specificity and catalytic Foaros eis capability to act inthe presence of even recalcitrant, substances, Pe cP eweide, roage of ecsioumenia coma ia ‘often unfavourable to active Cellsfree enzymes can offer 15sion of ceases leapt ncea iminants); . creators, inhibitors of aoe thei ow sensitivity or gay ANB high and low centrations. However, inn sony Olona ty the eens of Sedo ural ey 7 changes in contaminant disadvantages may hinder or diminish the eaayi oe 8, 0 several drawbacks oF gjsadvantages of in situ application Inic Potential of these of enzymatic catalysts. The eyes, may depend on both the puters OR cella, celtacoted or oell-fe peseace of several polluting substances in a cece Foe instance, the simultanous retve, effects on the enzyme eff & contaminated site with synergistic, often en th syne wrfication of free enzymes, the low subhiy on osts associated withthe isolation and Y acu to restrict the wide us enzymes tothe harsh conditions of sol all © of enzymes as re : mediatin sation of cise ating agents of polluted soils. However, capi ght nts Ploitation of genome using advanced technologies, PH and salin Implementing Bioremediat : The Engineering Concepts Till now we have discussed the fundamentals on which the technology of bioremediation stands. It is very essential to realize and understand these principles before we think of the practical implementation where the main objectives are: + To enhance the rate and extent of biodegradation of the pollutant + Toutilize or develop microorganisms that can survive the toxic effect of pollutants. + Toutilize microorganisms in such a way that no toxic products are produced ‘As the following discussion on engineering aspects of bioremediation proceeds, we will realize that translating these fundamentals into field applications is not an easy task. It requires @ judicious exchange of skills among professionals from various disciplines as shown in Fig. 6. Imcrebicess) iplinary Activity 16ion can be performed pire ants objective oF he eae ate oee the local penis the remedial he decision fen dents upon the view? ation and. subseque tuthories, When oe ably of finds snd Piet os citrine SPLICE eT Polktion effect shifow sal ances er pile (contaminant material is pil (ote bioremediation) is and oie Mea MAS RM te cas ae must be fine the contaminant and leachates, rand ob 4 clean ie) both of in Boremeliation 153) rr shows the design of the inst fi" He faction of the unaltered ee (8B) system. At first the physically mont Recovery. The remaining fat inant (e.g, oil) is removed. Its called a8 Fe to bioremediation, Fi 1g fraction that cannot be easily removed in this way is yen ry effects of high Free product recovery prevents the microorganisms from pesly igh concentration of contaminant and makes the bioremediation more ne. After free product recovery, water is pumped from the recovery ‘well and passed ‘a filter and then above ground treatment system such as ‘ioreactoriair-stripper. The and gun supplemented wit ntiensllero eet and returned to the aquifer close pie source of contamination through injection wells. Oxygen is supplied by sPa ing, ait, gure oxygen and sometimes by addition of hydrogen peroxide. First inorganic nutrients are AJ wich soloed by oxygen ation erase cimalancost ditions may cause PIUB sien. Gravity and pumping actions pall reienvmiorobe rich watcr ino saturated 2006 Mpere it comes in contact with the ‘contaminant and eventually treats it Nutrients ‘contaminated 2078 siovertno @ Fig. 7 In-situ piremciation EH jr Eta awn from S. Saval, Bioremediation: clean-up Bi cad ‘Biotechnology and cleaner bioprocesses (is Es Olt: 7parameters affecting ISB aportant parameters that affect [ ‘The impo’ SB ae discuss, ed below: level te. yeaa: ve el ative electron acceptor o ing saturation ‘of the i ‘ic conditions. Ox; he cil with water, the mage leven the sil is increased xo 10 ley soir roiding compaction, voing igh lee pat et or ee ea materials, To ensure thet nb high redox potential, and low i an OSS fae pase because at Ligh concentrons i is eae Fie oF come aot reson tends to decompose into water and oxygen rapidly in te stituents. Anaerobic conditions may be used to degrade le chlorinated contaminants, alth fay although at a very slow rate. This can ‘et treatment to complete biodegradation of the parially dechlor eae ‘eit the other contaminants ce rater Content vote serves asthe Unspon metium trough which nin and orgie consents pss into the microbial cell and metabolic waste products pass out of the cell. However, too much water can be detrimental, however, because it may inhibit the en = wayfutess anaerobic conditions aredesind). [See Nutrients Nutrients required for cell growth are nitrogen, phosphorous, potassium, sulfur, magnesium, ‘calcium, manganese, iron, Zinc, copper, and trace elements. If nutrients are not available in suificient amounts, microbial activity will become limited. Nitrogen and phosphorous are the uttients most likely to be deficient in the contaminated environment. These are usually ‘edged to the bioremediation system in a useable form (e-g., as ammonium for nitrogen and as phosphate for phosphorous). Phosphates can cause soil plugging as a result oftheir reaction wth minerals, such as iron and calcium, to form stable precipitates that fill the pores in the soil and aquifer. pH pH affects the solubility, and consequently the availability, ‘of many constituents of soil, vihch ean affect biological activity. Many metals that are potentially toxic to microorganisms tre insoluble at elevated pH; therefore, elevating the pH of the treatment system can reduce the risk of poisoning the microorganisms. Temperature ‘Temperature affects microbial activity in the environment. The biodegradation rate will slow With decreasing temperature; thus, in cold climates bioremediation may be ineffective during, Fait ofthe year unless itis carried out in climate-controlied facility. The microorganisms remain viable at temperatures below freezing but will resume activity when the emperairs tises, Heating the bioremediation site, such as by use of warm alr injection, may speed up the remediation process, At Belson AFB, Alaska, pasive solar warming by incubation tanks (ex 18¢ application of heated water below th iu) investigated. On the 1¢ ground surface 6 es se oer and 9 see the contaminated vadose so oorganiams, eset TGnEey eae eee also be detrimental also affects nonbiological losses a t sses of contamin eo contains igh onperats The ase vo oaantaid oat ae oe ae eT ily of oman typically igs ‘than at high Leeper aA CCRT ycarbons are more soluble at low as temperature. ly, oxygen solubility decreases with ses to take precautions and it prevent the followit it 109% of pumped water nto See anne yo issive use of mutients ascert ‘of micrrorganisms in the unwanted area Tawanted interaction of soil/ground water ix ‘i matrix with added nutrients fo Gaya an Mn can et rested uon vation ile 3 ‘tis quite clear that a lot of investi pets i investigations and meticulous pl s suet : ti m planning is requires fp the full-scale ISB. TO begin with itis very essential to know whether the site is i ble fo ‘bioremediation or not supsin SB sion about the site characterises shoud te examined beforehand to evaluate Informa of pioremediation technology, which should be followed by confirmation of the viability of Dire mi gegrtave activity at microcosm evel and then at larger scale Site characterization site cbaracterizations must Be dor following major questions: Met chemical compounds are BEES 2 contaminants and whether the contamination is superficial or has reached the subsoil? «Wats the depth and extension of ‘contaminant plume and the water table? * Tethe contaminant biodegradable and whether the microorganisms present atthe site can degrade it? Are the mrotironmental conditions concuelve ‘to biodegradation? 4 litposible to build bioreactor a he site? by the following characterizations: we in a logical sequence to be able 19 respond to the The answers to these questions ate obtained (9olutant and geohydrochemical characterization Seana ane cation involves aecints ti composition, concentration, toxicity, The examination of biosvaabilit Tecpel itty, solubility, sorption and vole gation of all the pollutant Iyological characteristics of the Pestand the mobility of the site is essential 10 Un contaminant, The following 860 icted to determine: . stechnical tests ‘should be conduc dale conductivity seryor permeability of S0H 1° determine bow 1 ttents can move through the saturat tedivadose ZONE § Specific yield and storage coefficient of the aquifer ee of influence of recovery and injection adi and the ground water ov. direction ation exchange capacity of soils t© estimate the nutrient forption on the soil particles © Anionici feationis composition ofthe soils 24 9pe, favourable and unfavourable con sjementation of the bior tions have be is % nS a ce i bles, water emticity cabot pipes, sewers and. sr buildings, roads and parking ose eee ce SSE uct Table 6: Favorabl le and unfavorable conditions for implementation of the Bioremediation Fay worable [Unfavorable Chemi [Few organic contaminants Characteristics Numerous contaminants; complex mixture of inorganic and organic contaminants 2, [Non-toxic concentration Toxic concentrations 3, [Diverse microbial groups [Sparse microbial activity [Suitable electron acceptors [Absence of suitable electron acceptors a, [Pao8 pit extremes Hydrogeological characteristics (Granular porous media [Fractured rock High permeability [Low permeability [Uniform mineralogy (Complex mineralogy Hlomogenous saturated medium [Heterogeneous | medium, ‘unsaturated saturated conditions source: Trejo M and QuntroR (2000) Bioremediation of contin sols, In Olguin EJ, Sanchez G and sec (eds) Environmental Bechrology and Cleaner Biprocse Taylor and Francis, pp 179-188) (6) Microbiological characterization 1 hrc important prerequisites for successtl ISB sr «Appropriate microorganism should be press 8 the subsurface » These should be adapted to the contaminants » A neeessary nutrients should be present or mus added anid 1970's the general belief was that there 2° extremely low number sas all of them shall be retained by natural hown that naturally Interestingly even till af microorganisms in deep soil layers/around Woe ot era the upper sol columns. However, er it was sl treuring species of Pseudomonas and Artiraba” Se involved in the disappearance of cares pagotine, Since then several hydoeat degrading microorganisms have been reported. It is desirable to analyze the native vnierobial flora with respect to its degradative Capacity and sizeof the population with degradative potential Biotreatabilty studies ‘These test are normally, peeformed ata. mewenen level and efforts are made to simulatereproduce the environmental conditions that prevail in the field. For example if a 20‘ |. On shallow stratum is to be remediated, large trays or jars filled with pai sea ca eae the other hand if contamination is present at the ground water table, column pi ae contaminated soil can be used as for biotreatbility studies. The main purpose of biotreatability studies is to determine the nutritional requirement of microorganism: perform biodegradation for which following parameters are monitored Oxygen consumption Carbon dioxide generation Exhaustion of a Contaminant re , idded nutrients (Nitrogen and phosphorus sources) moval Biotic and abiotic ¢, move the behaviour of bior, ‘ontrols should be included to ensure that the contaminant is being arg) inicrobial activity. When the tests ae properly conducted its possible to predict remediation and the time for large scale applications. Advantages of ISB 1 creme sltioe 270) ania ocdRW GRA’ CHERLAEED ercRiVE BY in public as an able ble waste treatment process for contaminated material such as soil. Microbes able to degrade the contaminant increase in numbers when the contaminant is present; When the contaminant is degraded, the biodegradative population declines. The residues for the treatment are usually harmless products and include carbon dioxide, water, and cell biomass. Bioremediation is useful for the complete destruction of a wide variety of contaminants. Many hazardous com; spounds can be transformed to harmless products This climinates the chance of future liabilit 'y associated with treatment and disposal of contaminated material. 3. Instead of transferring contaminants from one environmental medium to another, for example, from land to water or air, the complete destruction of target pollutants is possible, 4. Bioremediation can often be carried out on site, often without causing a major disruption of normal activities. This also eliminates the need to transport quantities of waste off site and the potential threats to human health and the environment that ean arise during transportation, 5. ISB is almost always faster than baseline pump-and-treat remediation, 6. ISB may be used in both short and long term timeframes, either by itself or following a more aggressive source zone treatment technology. 1. Bioremediation can prove less expensive than other technologies that are used for clean-up of hazardous waste. Limitations to ISB Factors that may limit the applicability and effectiveness of the process include: Bioremediation is limited to those compounds that are biodegradable. There are some concerns that in some cases the products of biodegradation may be more persistent or toxic than the parent compound. baie ‘ Biological mn Seca ly speifc. Important ste factors required for nee include the presence of metabolically capable microbial populations, suitable environmental growth conditions, and appropriate levels of nutrients and contaminants. Cleanup goals may not be attained if the soil matrix prohibits contaminant-microorganism 2contact. A surface treatm: i lent system, required to treat such 2 a ction or disposal It is difficult to. extra lat operations. ipolate from bench and pilot- sets pilot-scale studies to full-scale field circulation of water-bas i -based solu mobility and necessitate tre sR SMOLIN tt : Se Te 1 ead lala deel eR and of nutrient and water injetion wells. Se ral eae ai cir Gea On, Be excavation and erase ation slows at low temperatures. oe RIS EER eRe chlorinated organics, long chain toxic to micro organisms. C of hydrogen peroxide oncentat greater th trations micro organisms. han 100 to 200 ppm in groundwater inhibit the activity of + Research is needed appropriate fr sit ra develop and engineer bioremediation technologies that are tes with complex mixtures of contaminants that are not in the environment. evenly dispersed + Regulato Ee ae Tes Sa regarding acceptable performance criteria for There 18 no accepted definition of “clean”, evaluating performance of bioremediation is diffi treatments, ficult, and there are no acceptable endpoints for ‘bioremediation Many of the above factors can be controlled with proper attention t good engineering practice. The length of time required for treatment can range f of ‘ean range from 6 months to 5 years and is ‘dependent on many site-specific factors. oe Microbes in Reclamation of Wastelands including OM Spills Petroleum hydrocarbons, although not xenobiotics, are one of the main potential sources of tavironmental contamination due to their large-scale 6 Soil and groundwater are offen contaminated with gasoline or diesel fuel from jeaking underground storage tanks and ecause of accidental spills and leakage from pipelines. Due to their mobility, these ompounds may cause considerable damage 1 nly in soils but also in water intakes or froundwater reservoirs. As @ consequent qoxteffective bioremediation techniques have teen developed during this period, copecially to clean up oil- and gas-polluted si been jum contaminated soils and antes Y sastitte about 60% of the sites where bioremediation is being used in filed demonstrations or fll-seale operations Steps in Bioremediation of 2 contaminated site “The basic steps involve inthe developmest of such bioremediation strategies ae (Fi. 8) (A) Microbe Isolation and jdentification () Selection of petroleum degrading bacteria {(C) Generation of biomass (D) Application of biomass the contaminated site (&) Maintenance ofthe sect bacterial population at the site (4) Mierobe Isolation and identifint* by inoculating trypticae 0/2 broth with the contaminated sol i js usually isolate f ne wy gga png oer of nminimal nitrogen medium (nj can be used, After 4! 8 hours ) covered with petroleum layer (carbon source) the peoleum degrading beter can be streaked CRBS Ct ae colonies are picked SIDNA anal an be done be ail the pure bacterial cultures are obtained. The lysis. Care she standard biochemical tests, fatty acid analysis or by 16 better to cons ould be take pee oasis! the screening and is that the selected organisms are non-pathogenic. It is ea is be best survive around 7 aan at around 20°C temperatures as most of the pathogenic natu lowever, further confirmation must be done to ensure re before procee Proceeding with the bioremediation experiments. Petroleum Oy ni WC) {(A) Microbe Isolation and identfication nts & a \2la.| Q | Bbq cetcoronat | = | 5 aaoea | {(€) Generation of biomass (©) Application ef biomass at ste Fig, 8: Steps in bioremediation of petroleum contaminated site (B) Selection of petroleum degrading bacteria ‘As shown in Fig, 9, further confirmation of the petroleum biodegradation ability of the A cated strains should be done by inoculating petroleum laden serie soil with the selected settare and incubating it for about 15 days, Total bacteria! com and total petroleum trydocarbon (TPH) ofthe samples can be monitored 10 select the most efficient strains. (C) Generation of biomass ‘Aner the microorganism has been identifi and it bits degrade petroleum oil has been ner te laboratory, he next step is 0 ineesse he number of microorganisms for field application, Biomass production is done Tange fermentors (Fig. 8) and the subsequent colture is concentrated (by high speed ‘rentrfugation) to produce mud like precipitated cells sroncentated biomass is packed in plastic tight sealing bags and transported 0 the contaminated site om ice B