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Bioaugmentation as a soil bioremediation approach Timothy M Vogel

The debate over the efficacy of bioaugmentation rages on, with research continuing to demonstrate that its advantages for soil bioremediation are difficult to predict; however, when it works, the results are often very encouraging. The difficulties arise from, among others, the diversity of the microorganisms used, environmental heterogeneity, and variations in the influence of critical parameters (e.g. humidity, microbial predation and 'bioavailability') which, unfortunately, are not even always identified.

Address Rh6ne-Poulenc Industrialisation, 24 avenue Jean-Jaur6s, 69153 D~cines-Charpieu, France; e-mail: TIMOTHY.VOGEL@univ-lyonl .fr Current Opinion in Biotechnology 1996, 7:311-316 Current Biology Ltd ISSN 0958-1669 Abbreviations PAH polycyclic aromatic hydrocarbon PCP pentachlorophenol

performed and the associated parameters yet to be discovered or understood. Among the parameters that appear to be important are the pollutant characteristics (e.g. bioavailability [19,20], concentration [21] and microbial toxicity [22]), the soil physico-chemical characteristics (e.g. humidity or water content [23], organic matter content [23], clay content [23] and pH), microbial ecology (e.g. presence of predators [24] and interspecies competition), microbiology (e.g. the presence of co-substrates [25], genetics of the relevant organisms, and enzyme stability and activity [26]), and methodology (e.g. inoculation concentration [27,28] method of inoculation [28,29], the presence/absence of indigenous activity [9,30], and inoculum heterogeneity [31]). Therefore, when individual attempts at bioaugmentation research are reported, the difficulty in characterizing, quantifying, and evaluating all of the potential parameters leads to general conclusions that might not be widely acceptable. This review discusses the above parameters; the lack of current understanding on the relative importance of these parameters demands an intuitive approach to a certain extent. The rationale for the use of bioaugmentation is the perceived inability of indigenous microorganisms to perform satisfactorily during the bioremediation of contaminated soil. This 'failure' is categorized and characterized differently depending on the chemicals to be degraded, time of treatment, etc. Thus, the opinion that bioaugmentation is better or worse than bioremediation using indigenous microorganisms is always relative! Nonetheless, a review of the literature concerning the factors responsible for the performance of indigenous microorganisms to bioremediate a contaminated soil (biostimulation) would find many issues in common with this one (e.g. potential additives for bioremediation enhancement [32]). Recent literature (1994-1995) contains a high percentage of articles concerning the injection, tracking and fate of added microorganisms during bioaugmentation, with a smaller number of articles concerned with their effectiveness once in the ground. This apparent emphasis is the result, at least in part, of the application of molecular biology techniques such as gene probes.
Indigenous microorganisms

Introduction Bioaugmentation, the addition of microorganisms to enhance a specific biological activity, has been practiced intentionally for years in a number of areas, including agriculture and forestry [1] and wastewater treatment [2]. Research attempting to evaluate the value of bioaugmentation for soil remediation is not new either (e.g. [3,4]). Bioaugmentation is not generally accepted as an efficient technique for soil bioremediation, although proponents continue to demonstrate possible advantages. It is often viewed negatively by those either who have had cautionary experiences or worse (e.g. the addition of bioaugmentation products either decreased biodegradation rates or clogged aquifers) or who do not believe that the advantages of increasing the biocatalyst activity offset the advantages of niche fitness demonstrated by indigenous microorganisms [5-8]. T h e utility of bioaugmentation is supported by studies showing the incompetence of indigenous microorganisms in some cases and the apparent enhanced bioremediation rate after the addition of competent microorganisms [9,10",11,12,13]. The reinoculation of soil with indigenous microorganisms directly isolated from the same soil is often included in the term bioaugmentation [14,15].
With the wealth of experiments that demonstrate the potency and problems of bioaugmentation, several critical factors have become evident [2,16,17]. Increased interest in this area is highlighted by the recent publication of a book entitled Bioaugmentation for Site Remediation based on conference presentations [18]. Certainly, today, researchers can design experiments to either succeed (or not); what is at issue is the way in which these experiments are

T h e adaptation capacity of indigenous microorganisms is currently under study by several groups, and suggestions are starting to be made that this capacity is tremendous and perhaps more rapid than previously thought. For example, environmental stress might enhance mutation rates (soil environments are comparatively more harsh than laboratory conditions). As these rapidly adapting microorganisms are going to be the reference for bioaugmentation,

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consideration should be given to their applicability to the bioremediation of contaminated soil. T h e y are often better distributed, in general, than added microorganisms, although not necessarily with regard to the target pollutant because both the pollutant and the added microorganisms often enter the soil by similar methods ('dumping'). One important concept is, thus, related to the distance between the target compound and microorganism. Perhaps added microorganisms are closer to compounds recently 'added' and indigenous microorganisms are closer to older 'historic' pollution, although concepts such as size exclusion [31] might be important determinants of microbe-compound proximity. Are indigenous microorganisms more likely to be found within soil aggregates than 'added' microorganisms? Are recently 'added' chemicals to be found outside aggregates near added microorganisms? Are soil surface characteristics going to influence temporal distributions of compounds and microorganisms? Answers to these questions depend in part on recent techniques such as the use of gene probes. Genetic techniques can be used not only to monitor the presence of specific microorganisms [33,34], but also to monitor the contact between compound and microorganism using luminescence genes [35,36",37].

contaminated with compounds of low bioavailability. Clearly, our understanding is limited of the mechanisms of adaptation regarding critical initial concentrations of pollutants.

Physico-chemical environmental characteristics

Environmental conditions play a pivotal role in determining biological activity, whether of indigenous microorganisms, added microorganisms, or cultured indigenous microorganisms returned to the soil. These conditions fall into two general categories: those that reduce the microbial activity, such as temperature, humidity and ionic strength (which, in one report, also increased cell attachment [43"]); and those that restrict the mass transfer (mainly by diffusion) of the compound to the microorganism, such as clay and organic-matter content [23]. In addition, several aspects of the bioaugmentation process are affected by advective transport, such as permeability. This affects the addition of microorganisms during in situ bioaugmentation as well as the addition of nutrient and electron acceptors during both bioaugmentation and biostimulation. As stated above, emphasis recently has been on the understanding of the movement and fate of added microorganisms during bioaugmentation. Under uncontrolled conditions, microorganisms added in situ via injection wells can clog well-heads and lead to overall failure of the bioremediation strategy [5]. Curiously, the relatively few microorganisms that traverse considerable distances in the subsurface are considered insufficient by those interested in bioaugmentation--column studies have demonstrated an average microbial concentration 10-fold or higher in the first 5cm than elsewhere in the soil column [44]. Accordingly, most research focuses on both controlling environmental conditions [43"] and controlling microbial development (e.g. resuscitation of starved non-sticky ultramicrobacteria [45]) to prevent microorganisms from clogging well-heads, but also to induce them to attach in sufficient numbers in the aquifer to improve contaminant degrad)tion. Even small concentrations of microorganisms are of concern to either those monitoring the spread of fecal contamination [46] or those who believe that microorganisms can enhance the movement of contaminants in a manner similar to colloids [47].

Compound characteristics
Pollutant toxicity is often used as justification for bioaugmentation because, conceptually, this toxicity could inhibit the degradative activity of indigenous microorganisms. Although few sites with obvious toxicity have been reported, the sites that have been described are of clear potential for bioaugmentation if the added microorganisms can also resist the toxicity. One site where exogenous microorganisms were employed required dilution by soil washing or bioslurry techniques to achieve pollutant degradation [22]. Compound 'biodegradability', which is associated with many factors, including those discussed below, is sometimes related to compound structure and its related physico-chemical characteristics such as solubility and bioavailability (which itself is not intrinsic to the compound, but related to the interactions between the compound, the microorganism(s) and the soil). Curiously, reports about the limited use of bioaugmentation relative to biostimulation often study compounds that are either known for their 'non-availability' (e.g. low concentrations of polycyclic aromatic hydrocarbons [PAHs] [20]) or relatively easy to degrade when other limiting conditions (e.g. nutrients) are provided such as petroleum hydrocarbons [6,7] or crude oil [38]. Even so, for compounds that are considered relatively recalcitrant but generally 'available', bioaugmentation has been demonstrated to be beneficial (e.g. 2,4,6-trinitrotoluene [9], carbon tetrachloride [39,40], and pentachlorophenol [PCP] [41,42]). Care must be taken not to conclude too much from such data as 'adapted' microorganisms are frequently found in soils

Niche adjustment
Although microbial ecology issues are among the most important in bioaugmentation approaches, unfortunately, they are rarely addressed. Parameters that could influence the performance of added (or indigenous) microorganisms include niche fitness (competition, synergy, etc.), steady-state microbial concentrations, and predators. One can demonstrate niche fitness in an inverse sense by instituting in soils changes that favor the development and performance of a particular added microorganism. This is exemplified by the enhanced growth and activity of both Pseudomonas sp. strain KC under slightly alka-

Bioaugmentation as a soil bioremediation approach Vogel 313

line conditions (pH8.2 [39]) and the white-rot fungus Phanerochaete chrysosporium under slightly acidic conditions (pH5 [6], pH4.5 [48] and p H 6 [491). Fitness for the soil environment can be defined by the quantity of microorganisms (i.e. development), but for t h e treatment goals of bioaugmentation, performance is usually the more important factor. An interesting example is where one nutrient in excess and another in limitation both lead to an improved performance [501. T h e 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 the treatment process (biostimulation [7,38]) or hinder the process by consuming the added nutrient or carbon source (e.g. starch [8]). In cases where co-metabolism is desired, the consumption of added substrate by indigenous microorganisms incapable of co-metabolizing the pollutant leaves little for the added microorganisms and, thus, results in poor performance [25]. T h e difficulties in adjusting the environment or in selecting microorganisms fit for their target environment have led to the development of techniques for protecting the added microorganisms such as encapsulation [51,52]. In general, these techniques improve the long-term viability of added cells [51]. Real engineering performance evaluation is hindered by the unrealistic experimental design (e.g. the use of freshly added pollutant, microorganisms and laboratory conditions), but the initial results are promising. T h e fate of added microorganisms is not unrelated to the ecologically stable microorganism concentration one observes in soils. Because the decrease (or increase) in microbial populations tends toward an asymptote, recent attempts to model this behavior follow a certain logic [53]. T h e appealing aspect of this model approach is the connection between growth/decay and a natural ecological population density/concentration. For example, the Eschetichia coli modeled had a final natural concentration 8-30 orders of magnitude less than the Pseudomonas species modeled [53]. T h e application of this approach to encapsulated microorganisms and to a range of pollutant concentrations would possibly provide important insights into added microorganism survival during bioaugmentation. In addition, an attempt to connect natural levels of genetic 'aptitude', either from indigenous or added microorganisms, to these models would be stimulating. Microbial ecology is very important in evaluating both the potential success of bioaugmentation and its possible advantages over biostimulation. Microorganisms are affected by maintenance energy, the production of, and resistance to, antibiotics and toxic metabolites, predation, etc. T h e necessity for using 'long-lasting' microorganisms in bioaugmentation, on the basis of the potentially slow reaction kinetics (which are controlled by slow compound desorption), is debatable. Improved understanding of the microbial ecology, microbiology and genetics of competent

pollutant-degrading microorganisms has increased our ability both to monitor the fate of added microorganisms and possibly to discover 'better-suited' species or strains for bioaugmentation.

Microbial ecology
The problems of monitoring microbial survival raised above are now somewhat routinely addressed through the use of gene probes [25], PCR technology [33,34] or immunoassay technology [54",55"]. These techniques provide a general absolute minimum detection limit of 100 microorganisms per gram of soil; thus, survival of an exogenous microorganism after inoculation at concentrations up to ten orders of magnitude greater (e.g. [27]), can easily be monitored. Even so, detection by gene probes and immunoassays does not provide evidence of microbial activity (with the possible exception of mRNA probes) and, therefore, our prediction of the performance of these added microorganisms still lacks easy assessment. Indeed, the fate of added microorganisms might not be related to their activity, but that of the indigenous microflora that have recovered relevant genetic material from the inoculated species. This potential natural exchange of genetic material is much easier to measure using recent applications of molecular biology [56,57]. The above genetic approaches have also stimulated research to improve the capacity of microorganisms to degrade xenobiotic compounds [58,59]. T h e related debate is whether these microorganisms are 'better' at degrading difficult compounds than naturally 'trained' microorganisms. 'Better' is defined not by microbial numbers, but by performance, given the possibility to biostimulate the indigenous population, as mentioned at the beginning of this review. Performance has an economic aspect, also. Some confusion is caused by the definition of the problem in terms of the long-term effects of bioremediation. Issues related to changes in the ecology, the geochemistry, and the hydrogeology resulting from either biostimulation or bioaugmentation are difficult to specify. For example, suggestions regarding the use of suicide genes to prevent long-term survival of added microorganisms returns us to the definition of the 'best' characteristics of added microorganisms. Microorganisms that are fast-acting (although slow with chemicals that are not rapidly available), short-lived (i.e. no long-term 'danger'), mobile (i.e. capable of penetrating into the system), adhesive (except near the injection point to avoid clogging), resilient (i.e. resistant to fluctuations in pH, ionic strength, heavy metal concentrations, etc.) and inexpensive with a wide range of degradative activity represent the ideal for bioaugmentation.

Engineering process design

Given the uncertainties regarding the environmental characteristics, microbial ecology and microbiology of bioaugmentation, the lack of comprehensive engineering guidelines is understandable. Yet, the 'brute force'

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approach has been shown to be relatively effective in some situations. For example, experiments where very high numbers of microorganisms--for example, from 107 bacteria (g soil)-I [10 ] to 10 lz bacteria (g soil)-! [ 2 7 ] - - h a v e been added to contaminated soils where 'bioavailability' is not particularly the limiting factor have been relatively successful. T h e disagreeable aspect of the 'brute force' approach is the lack of engineering design. Engineering design requires an understanding of the parameters that control the bioaugmentation process. Good engineering needs to be applied to bioaugmentation process design at the experimental level. Reports of the advantages/disadvantages of bioaugmentation from experiments where nutrients included in bioaugmentation products have not been tested separately from the added microorganisms [17], where the activity of indigenous microorganisms have not been compared [59], etc., do not help engineers determine the critical parameters involved. Engineering experiments cannot, however, cover all variables and, therefore, need to concentrate on the critical parameters defined in part by microbiologists: microbial inoculation quantity (with zero being an option), technique (what form of inoculum, e.g. freeze-dried [29], and with what nutrients) and then the parameters that are the same for biostimulation (e.g. humidity, pH and electron acceptor concentration).

Conclusions
Bioaugmentation clearly provides certain advantages over biostimulation in cases where pollutant toxicity or a lack of appropriate microorganisms (both quantity and quality) are important. Determination of the potential success of bioaugmentation requires an understanding of the bioavailability of the pollutant, the survival and activity of the added microorganism(s) or its genetic material, and the general environmental conditions that control soil bioremediation rates. T h e recent application of genetic techniques has aided the comprehension of the fate of added microorganisms, bringing us closer to identifying the critical parameters for the engineering design of bioaugmentation processes. Recent examples of both successes and failures associated with bioaugmentation provide a cautionary note to those who believe that the debate is over.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: * 1. of special interest of outstanding interest Jasper DA: BioremediaUon of agricultural and forestry soils with symbiotic micro-organisms. Aust J Soil Res 1994, 32:1301-1319. RittmannBE, Whiteman R: Bioaugmentation: a coming of age. Wat C)ual Int 1994, 1:12-16. Portier R, Bianchini M, Fujisaki K, Henry C, McMilin D: Comparison of effective toxicant biotransformation by autochthonous microorganisms and commercially available cultures in the in situ reclamation of abandoned industrial sites. Schriftenr Ver Wasser Boden Lufthyg 1988, 80:273-292. Pritchard PH: Use of inoculation in bioremediation. Curt Opin Biotechno11992, 3:232-243. Maxwell CR, Baqai HA: Remediation of petroleum hydrocarbons by inoculation with laboratory-cultured microorganisms. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:129-137. McGugan BR, Lees ZM, Senior E: Bioremediation of an oilcontaminated soil by fungal intervention. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:149-156. Neralla S, Wright AL, Weaver RW: Microbial inoculants and fertilization for bioremediation of oil in wetlands. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:31-38. M611er J, Gaarn H, Steckel T, Wedebye EB, Westermann P: Inhibitory effects on degradation of diesel oil in soilmicrocosms by a commercial bioaugmentation product. Buff Environ Contain Toxicol 1995, 54:913-918. Shin CY, Crawford DL: Biodegradation of trinitrotoluene (TNT) by a strain of Clostridium bifermentans. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:57-69.

Although geological characteristics, such as heterogeneity, can pose daunting engineering hurdles to the use of bioaugmentation for in situ bioremediation, the use of added microorganisms in ground treatment is easier to implement and justify. Bioreactors for the treatment of groundwater are an example where bioaugmentation is inapplicable, even if indigenous microorganisms selected on the target contaminants are used. On the other hand, soil treatment 'reactors' such as bioslurry systems [15] or biopiles [13"] are easier to control and tend to provide more convincing results when bioaugmentation is compared with biostimulation. This does not discount recent advances in in situ bioaugmentation, such as the use of plants, their root systems and rhizospheric bacteria [60], only that the heterogeneity is difficult to assess.

2. 3.

4. 5.

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Finally, all of the above discussion has been made independently of economic aspects other than those relating improved rates to implicit cost reduction. Yet, the decision of if and how to apply bioaugmentation really should be more objectively based. As for biostimulation, bioaugmentation has a calculable cost that will vary with compound type, soil type, reactor type, and inoculum type and quantity. As mentioned at the beginning of this article, generalizations about the advantages of bioaugmentation relative to other techniques can be easily disproven in a given example. Careful decision-making processes need to be employed to avoid misconceptions about fantastic or frightful results.

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Briglia M, Middeldorp PJM, Salkinoja-Salonen MS: Mineralization performance of Rhodococcus chlorophenolicus strain PEP-1 in contaminated soil simulating site conditions Soil Biol Biochem 1994, 26:377-385. Demonstrates the influence of several operating parameters on the success of PCP degradation by Ft. chloropheno/icus. Degradation rates increased with increasing POP concentrations and microbial numbers; bioavailability

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and soil humidity also played a role. The bacterium performed poorly in saturated soils, although little difference in degradation rate was observed between 50% and 80% of field capacity. A high level of organic matter (peat) apparently reduced the PCP bioavailability. 11. 12. Riggle D: Successful bioremediation with compost. Biocycle 1995, 36:57-59. Dave H, Ramakrishna C, Bhatt BD, Desai JD: Biodegradation of slop oil from a petrochemical industry and bioreclamation of slop oil contaminated soil. World J Microbiol Biotechno/1995, 10:653-656.

soil by bioaugmentaUon. Appl Microbiol Biotechnol 1993, 38:681-687. 29. RomichMS, Cameron DC, Etzel MR: Three methods for large-scale preservation of a microbial inoculum for bioremedistion. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:229-235. Liang R, McFadand MJ: Biodegradation of pentachlorophenol in soil amended with the white rot fungus Phanerochaete chrysosporium. Hazard Waste Hazard Mater 1994, 11:411-421. Petrich CR, Stormo KE, Knaebel DB, Ralston DR, Crawford RL: A preliminary assessment of field transport experiments using encapsulated cells. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:237-244. Bajpai RK, Zappi ME, Gunnison D: Additives for establishment of biologically active zones during in situ bioremedlation. Ann NY Acad Sci 1994, 721:450-465. Selenska-Pobell S: How to monitor released rhizobia. Plant Soft 1994, 166:187-191. Burlage RS, Palumbo AV, McCarthy J: Signal quantification of bacteria for a groundwater transport experiment. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:139-148. Masson L, Comeau Y, Brousseau R, Samson R, Greet C: Construction and application of chromosomally integrated lac-lux gene markers to monitor the fate of a 2,4dichlorophenoxyacetlc acid-degrading bacterium in contaminated soils. Microb Releases 1993, 1:209-216.

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LamarRT, Davis MW, Dietrich DM, Glaser JA: Treatment of a pentachlorophenol- and creosote-contaminated soil using the lignin-degrading fungus Phanerochaete sordida: a field demonstration. Soil Biol Biochem 1g94, 26:1603-1611. A good example of small-scale field application of bioaugmentation, with controls to establish the real benefits of the addition of microorganisms. Application of the fungus Phanerochaete sordida to PCP-contaminated and PAH-contaminated soil resulted in a relative increase in compound biodegradation relative to controls. Three-ring PAHs were degraded further in nontreated soil, whereas four-ring PAHs were degraded further in fungus-treated soil. Five-ring and six-ring PAHs were not significantly reduced. 14. Phelps TJ, Siegrist RL, Korte NE: Bioremediation of petroleum hydrocarbons in soil column lysimeters from Kwajalein Island. Appl Biochem Biotechnol 1994, 45/46:835-845. Otte M-P, Gagnon J, Comeau Y, Matte N, Greet CW, Samson R: Activation of an indigenous microbial consortium for bioaugmentaUon of pentachlorophenol/creosote contaminated soils. Appl Microbiol Biotechnol 1994, 40:926-932. Atlas RM: Bioaugmentation to enhance microbial bioremediation. In Biotreatment of Industrial and Hazardous Waste. Edited by Levin MA, Gealt MA. New York: McGraw-Hill; 1993:19-37. ForsythJV, Tsao YM, Bleam RD: Bioremediation: when is augmentation needed? In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:1-14. Hinchee RE, Frederickson J, Alleman BC (Eds): Bioaugmentation for Site Remediation. Columbus: Battelle Press; 1995. Harms H, Zehnder AJB: Bioavailabllity of sorbed 3chlorodibenzofuran. Appl Environ Microbio/1995, 61:27-33. Rothmel RK, Gaudet JL, Schul WH, Shannon MJR, Kishnamoorthy R, Smith JR, Unterman R: Biostimulation versus bioaugmentation: two strategies for treating PCB-contaminated soils and sediments. In Abstracts of the g4th General Meeting of the American Society for Microbiology. Lss Vegas: ASM; 1gg4:#Q-153. Aamand J, Bruntse G, Jepsen M, Jqrgensen C, Jensen BK: Degradation of PAHs in soil by indigenous end inoculated bacteria. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:121-12?. Baud-Grasset F, Vogel TM: Bioaugmentation: biotreatment of contaminated soil by adding adapted bacteria. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:39-48. Godbout JG, Comeau Y, Greet CW: Soil characteristics effects on introduced bacterial survival and activity. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:115-120. RamadanMA, EI-Tayeb OM, Alexander M: Inoculum size as a factor limiting success of inoculation for biodegradaUon. Appl Environ Microbiol 1990, 56:1392-1396. Barriault D, Sylvestre M: Factors affecting PCB degradaton by an implanted bacterial strain in soil microcosms. Can J Microbio11993, 39:594-602. Trombly J: Engineering enzymes for better bioremediation. Environ Sci Technol 1995, 29:550A-564A. Pearce K, Snyman HG, Oellermann RA, Gerber A: Bioremediation of petroleum-contaminated soil. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:71-76. Comeau Y, Greet CW, Samson R: Role of inoculum preparation and density on the bioremediation of 2,4-D-contaminated

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Blackburn NT, Seech AG, Trevors JT: Survival and transport of lac-lux marked Pseudomonas fluorescens strain in uncontaminated and chemically contaminated soils. Syst Appl Microbiol 1994, 17:574-580. Reports an interesting use of luminescent bacteria for monitoring bacterial transport and survival. Bacteria are shown to survive better at lower temperatures (10"C) and in non-contaminated soils. The influence of contaminated soils on the survival of bacteria capable of growing on the contaminant (PAHs) is not shown. 3?. King JMH, Digrazia PM, Applegate B, Burlage R, Sanseverino J, Dunbar P, Larimer F, Sayler GS: Rapid, sensitive bioluminescent reporter technology for naphthalene exposure and biodegrsdation. Science 1990, 249:778-781. Venosa AD, Suidan MT, Haines JR, Wrenn BA, Strohmeier KL, Eberhart BL, Kadkhodayan M, Holder E, King D, Anderson B: Field bioremediation study: spilled crude oil on Fowler Beach, Delaware. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:49-56. Dybas MJ, Tatara GM, Knoll WH, Mayotte TJ, Criddle CS: Niche adjustment for bioaugmentation with Pseudomonas sp. strain KC. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:77-84. Witt ME, Dybas MJ, Heine RL, Nair S, Criddle CS, Wigged DC: Bioaugmentation and transformation of carbon tetrachloride in a model aquifer. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:221-227. Holroyd ML, Caunt P: Large-scale soil bioremediation using white-rot fungi. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:181-187. Edgehill RU: Removal of pentachlorophenol from soil by Arthrobacter strain ATCC 33790. In Bioaugmentation/or Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:85-90.

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Shonnard DR, Taylor RT, Hanna ML, Boro CO, Duba AG: Injection-attachment of Methylosinus trichosporium OB3b in a two-dimensional minature sand-filled aquifer simulator. Water Resource Res 1g94, 30:25-35. Description of the possible factors influencing the attachment of injected bacteria in sandy aquifers. The experiments did not study cell straining, but did investigate colloid-like filtration, where solution ionic strength plays an important role. 44. JenningsDA, Petersen JN, Skeen RS, Peyton BM, Hooker BS, Johnstone BL, Yonge DR: An experimental study of

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microbial transport in porous media. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:97-103. 45. Bryers JD, Sanin S: Resuscitation of starved ultramicrobacteria to improve in situ bloremediation. Ann NY Acad Sci 1994, 30:61-76. Ramos-Cormenzana A, Castillo A, Incerti C, Gomez-Palma LF: Bacteriological indicators of faecal contamination: result of a loading experiment with untreated urban wastwater. J Appl Bacteriol 1994, 76:95-99. Kim SH, Corapcioglu MY: Effects of mobile bacteria in bloremediaUon operations. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Allman BC. Columbus: Battelle Press; 1995:91-96. Venttea RT, Hicks RJ, Lewis RF: Comparison of three lignindegrading fungi for degrading cyclodiene insecticides. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Allman BC. Columbus: Battelle Press; 1995:157-164. Field JA, Feiken H, Hag A, Kotterman MJJ: Application of a white-rot fungus to biodograde benzo(a)pyrene in soil. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Allman BC. Columbus: Battelle Press; 1995:165-171, Kotterman MJJ, Wasseveld R, Field JA: Influence of nitrogen sufficiency and manganese deficiency on PAH degradation by Bjerkandera sp. In Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Allman BC. Columbus: Battelle Press; 1995:189-194. Lin J-E, Lantz S, Schultz WW, Mueller JG, Pritchard PH: Use of microbial encapsulation/immobllizaton for biodegradaUon of PAHs. In: Bioaugmentation for Site Remediation. Edited by Hinchee RE, Fredrickson J, Alleman BC. Columbus: Battelle Press; 1995:211-220. Leung K, Cassidy MB, Holmes SB, Lee H, Trevors JT: Survival of k-carrageenan-encapsulated and unencapsulatad Pseudomones aeruginosa UG2Lr cells In forest soil monitored by polymerese chain reaction and spread plating. FEMS Microbio/Ecol 1995, 16:71-82.

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Vandepitte V, Quataert P, De Rore H, Verstraete W: Evaluation of the Gompertz function to model survival of bacteria Introduced into soils. Soil Biol Biochem 1995, 27:365-3'72. Describes the best function, to date, for describing the asymptotic changes in added microbial populations, application of which could aid in accurately determining the effective concentrations of microorganisms for bioaugmenration. Significantly, the fitting of this function often provided results that were logical (e.g.E. coil had final populations considerably smaller than Pseudomonas sp.) Schloter M, Abmus B, Hartmann A: The use of immunological methods to detect and identify bacteria in the environment. Biotechnol Adv 1995, 13:75-90. A general review of the potential uses and problems of immunological methods for monitoring bacteria in environmental samples. Winkler J, Timmis KN, Snyder RA: Tracking the response of Burkholderia cepacia G4 5223-PR1 in aquifer microcosms. Appl Environ Microbiol 1995, 61:448-455. Describes an interesting application of monoclonal antibody immunofluorescence for counting added microorganisms under different experimental conditions. The technique was able to compare the effect of sterile nonsterile conditions on bacterial survival with microorganisms that lacked niche adaptation. 56. Barkay T, Kroer N, Rasmussen LD, S6rensen SJ: Conjugal transfer at natural population densities in a microcosm simulating an estuarlne environment. FEMS Microbiol Ecol 1995, 16:43-54.

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