a uthor : Jez Supreme (30098333) contact : jezsupreme@gmail.com

1. author: Jez Supreme (30098333) contact: jezsupreme@gmail.com Author’s Profile There is a great deal we do not understand in this world, and it can be difficult to eliminate any given area in the quest for knowledge of everything; my name is Jez Supreme and I am a biological studyholic. I have Majors in Biotechnology, Molecular Biology and Biomedical Science, with a Minor in Applied Statistics, a strong interest in environmental technologies and a love of the physics underlying our existence. Using biological processes to reconstruct suitable ecological conditions for life which benefits our species is one of the great challenges we face as a result of the path we have walked since the industrial revolution. The harnessing of scientific knowledge for the mitigation of technologically driven damage represents a laudable use of our collective prefrontal cortex. The use of biodegradation of toxic substances, especially in the forms of pesticides and herbicides, is a potentially game changing approach to environmental remediation that might help our species to sustainably improve the carrying capacity of the rock we live on. As a representative chemical contaminant, Atrazine is a good choice to illustrate methodology, in that it has been widely used for 40 years for herbicidal qualities, is relatively persistent in contaminated soils, and is toxic. The exploration of effective microbial communities for biodegradation is examined here using published research in the field.

2. Papers of Comparison Biodegradation of atrazine by Agrobacterium radiobacter J14a and use of this strain in bioremediation of contaminated soil Struthers, J. K., Jayachandran, K. & Moorman, T. B. (1998). Biodegradation of atrazine by Agrobacterium radiobacter J14a and use of this strain in bioremediation of contaminated soil. Applied and Environmental Microbiology64, 3368-3375. Bioremediation of atrazine-contaminated soil by repeated applications of atrazine-degrading bacteria Newcombe, D. A. & Crowley, D. E. (1999). Bioremediation of atrazine-contaminated soil by repeated applications of atrazine-degrading bacteria. Appl Microbiol Biotechnol51, 877-882.

3. Executive Summary A comparison of two experiments in biodegradation of the toxic herbicide atrazine, this study shows that the bioremediation of polluted sites is best approached by use of multiple inoculations of pure culture using a fermenter-distributor apparatus. This bioaugmentation is feasible in cases where selective enrichment of soils and/or native microbial degraders are not available as mechanisms towards biodegradation. Note: image from microblogs.org

4. Background 1 A commonly used herbicide over the last 40 years, atrazine has been known to be fairly persistent in soils after regular use (Seller et al., 1992). Over 1 billion pounds of atrazine have been applied to soils worldwide (Gianessi, 1987) and measured levels of atrazine in soils and surface waters have often been detected in excess of the environmental protection agency’s (EPA) regulations in areas of high use (Buhler et al., 1993; Kello, 1989). Presence of this compound in the environment has the potential to impact local ecologies, having been indicated as an endocrine disruptor and with the ability to disrupt sexual development in amphibians at even levels in the environment considered safe by the EPA (Hayes et al., 2002), and with the potential to lead to carcinogenic effects in humans (Biradar & Rayburn, 1995). atzA, atzB and atzC genes are linked to catabolism of atrazine by bacteria and resulting biodegradation (Abdelhafid et al., 2000; Boundy-Millset al., 1997; Sadowsky et al., 1998). Note: X (2), R-1(4) and R-2 (6) are the substituted groups in s-triazine ring in 2, 4 and 6 positions respectively.

5. Background 2 Studies have shown that some microorganisms are able to utilize atrazine as an N source (Alvey & Crowley, 1995; Cook & Hutter, 1981) and that high N presence in soils inhibit atrazine catabolism (Abdelhafid et al., 2000). The number of microorganisms able to catalyse a compound has been observed to increase with repeated exposure to herbicides such as 2,4-D (Ka et al., 1994), MCPA (Smith et al., 1986). Atrazine degradation rate has been shown to be Faster when higher concentrations of atrazine are present (Ghosh & Philip, 2004). 

6. Biodegradation of atrazine by Agrobacterium radiobacter J14a and use of this strain in bioremediation of contaminated soil Struthers, J. K., Jayachandran, K. & Moorman, T. B. (1998). Biodegradation of atrazine by Agrobacterium radiobacter J14a and use of this strain in bioremediation of contaminated soil. Applied and Environmental Microbiology64, 3368-3375. Note: where information is derived directly from the paper under consideration, no referencing will be provided within this summary. Overview The researchers examined the use of Agrobacterium radiobacter J14a (J14a) to increase the rates of biodegradation of atrazine, in a variety of culture conditions and soils. In order to do this they isolated and characterized J14a, followed by conducting tests on the effects of changing culture conditions, such as differences in secondary carbon and nitrogen sources in culture and soils. Differential media (i) Minimal salts + carbon, trace elements & vitamins (ii) basal minimal salts + carbon, trace elements and atrazine as sole N source (iii) C- and N-limited medium, with only basal minimal salts, trace elements, vitamins & atrazine (iv) complete BMA medium with 5g NH4NO3/L (v) basal minimal salts medium with atrazine, vitamins & trace elements. Methods Culture levels determinations Atrazine incorporation into J14a cells measured via ethyl-14C atrazine and radioactivity measurement in a scintillation vial, using a 1mL medium aliquot and filtration prior to measurement. Metabolites of atrazine degradation assessed by high performance liquid chromatography (HPLC) analysis after incubation. Atrazine-degradative ability of isolate was assessed by mineralisation of the 14C-U-ring. Soil determinations Soil collected from two chemical dealerships in Iowa by removing the top 5cm of soil from likely affected locations. Soil from first dealership had 75% sand, 17% silt, 8% clay, 3.2% organic C and 0.007% total N, pH 7.9. Soil from second dealership had 78% sand, 18% silt, 4% clay, 2.4% organic C and 0.005% total N, pH 6.5. Both soils were sieved and adjusted to 10% moisture. HPLC used to determine atrazine, DEA, DIA and DEDIA concentrations. Residual ethyl-14C atrazine in soil was determined through combustion was an OX500 biological oxidiser.

7. Results & Discussion Addition of J14a to atrazine-contaminated soils resulted in 2-5 fold increase in mineralisation than by the indigenous microbial cultures in the soils from the first site. Sucrose addition had no effect. Addition of J14a to atrazine-contaminated soils resulted in an increase in mineralisation over the indigenous microbial cultures in the soils from the second site, though only those with added sucrose showed significance in difference. umax for this species is unlikely to be met by atrazine concentrations in contaminated soils. In organic C limitation, mineralisation of atrazine is limited, in conditions of N-limitation J14a is able to utilise atrazine as the sole N-source, and mineralisation is increased as compared to degradation in conditions of organic N availability. In addition, in conditions of N-limitation, J14a demonstrated wide substrate utilisation ability. Degradative enzymes are constitutively expressed. Biodegradation was incomplete at 63 days, and populations of J14a had declined to low levels 60 days after inoculation. Final Conclusions J14a is an effective bioaugmentation to increase the degradation of atrazine in contaminated soils, however availability of chemicals and cell survival are important factors for success of this strategy. • Fig. 3. Degradation of 50 mg of [14C-U-ring]atrazine ml21 in N-limited medium by strain J14a. Atrazine remaining in the medium (A) is shown in relation to cell density (B) and 14CO2 production (C). Fig. 4. (left) Mineralization of 50 (A) or 200 (B) mg of [14C-U-ring]atrazine ml21 added to soil from the Alpha site. Soils were amended with strain J14a (105 cells g of soil21), sucrose, or both J14a and sucrose 3 days after atrazine treatment (right) Mineralization of 50 (A) or 200 (B) mg of [14C-U-ring]atrazine ml21 added to soil from the Bravo site. Soils were amended with strain J14a (105 cells g of soil21), sucrose, or both J14a and sucrose 3 days after atrazine treatment.

8. Bioremediation of atrazine-contaminated soil by repeated applications of atrazine-degrading bacteria Newcombe, D. A. & Crowley, D. E. (1999). Bioremediation of atrazine-contaminated soil by repeated applications of atrazine-degrading bacteria. Appl Microbiol Biotechnol51, 877-882. Note: where information is derived directly from the paper under consideration, no referencing will be provided within this summary. Overview The researchers examined the effectiveness of biodegradation of atrazine by single inoculation of an atrazine degrader, Pseudomonas sp. Strain ADP (ADP) or bacterial consortium in atrazine contaminated soil against a system of multiple inoculations at regular intervals. Species and Consortia Atrazine-degrading consortium (Alvey & Crowley, 1996) and the atrazine-degrading isolate ADP. Methods Soil microcosms were established in the laboratory and also in the field. Moisture content for all soil microcosms was maintained at 15% (w/w). Mesocosms received 1, 4 or 8 applications of either a pure culture or consortium. Atrazine and metabolite extraction from soil was conducted with three consecutive treatments with 90% acetonitrile and 10% 0.1M HCl solution. Analysis of atrazine mineralisation was performed using HPLC and results subject to statistical analysis. Results All inoculated soils saw significantly increased mineralisation rates as compared to the uninoculated controls.Single inoculation of pure ADP saw significantly higher mineralisation of atrazine than the consortium inoculated soils. Multiple inoculation of pure ADP cultures saw significantly higher mineralisation of atrazine than the consortium inoculated soils.

9. Discussion & Final conclusions Where there are not indigenous degraders of atrazine present, bioaugmentation to mineralise and degrade atrazine to safe levels can provide a sufficient reduction in atrazine concentration in polluted soils. The levels of degrader population necessary in the field to achieve appropriate degradation of atrazine are higher than can be sustained by a single application, and may require as many as 8 applications of a pure culture of degraders in an environment in which the culture is not favoured for growth. For this purpose an automated fermenter and delivery system may be a feasible means to promote biodegradation in affected soils. A pure culture is concluded to be more effective at such degradation, possibly due to more efficient energy kinetics in not requiring metabolite exchange between multiple members of the consortium. Fig. 6. Mineralization of ring-labelled [14C]atrazine in soil microcosms. Each point represents the mean of four replicates. Error bars represent one standard deviation from the mean

10. Comparison of Papers While Struthers et al. Made the comparison of degrader inoculation with an existing atrazine degrader, Newcombe & Crowley did not do so. In addition, Struthers et al. controlled for several variations in conditions which Newcombe & Crowley did not. The use of both repeated and single inoculations by Newcombe & Crowley, as well as use of consortium inoculation in addition to a single isolate showed greater strength of design in these respects over Struthers et al. While extraction and analysis were similar between the experiments, the delivery methods used were the main differential besides specific inoculums; these led to the differing conclusions in these articles, and the Newcombe & Crowley paper thus addresses the main concern raised in summation of Struthers et al. Table 1. Comparison of reported experiments

11. Critical Comments The work performed by Struthers et al. Provided an insight into the effectiveness of Agrobacterium radiobacter j14a in biodegradation of s-triazines like atrazine. More importantly, provides elucidation of the hurdles encountered in biodegradation in an environment where a native degrader is not already present. The use of a wide range of culture conditions enables examination of the effects of differing environments on the use of pure cultures to carry out bioremediation, and demonstrates the importance of understanding environmental conditions when biodegradation of pollutants is attempted. The claims made by the authors are fairly well supported by the evidence, and in line with similar other examinations (Abdelhafid et al., 2000; Ghosh & Philip, 2004; Yassir et al., 1999). In not exploring contaminated soil treatment in the field, the results are unfortunately not able to be best extrapolated to large-scale use. In addition, the lack of other strains and/or consortiums for analysis in biodegradation limits the ability to infer comparative effectiveness of this strain’s atrazine degradation over other possible cultures. Newcombe & Crowley’s work, taking place both in the laboratory and the field, provides a better ability to extrapolate results beyond test conditions. The comparison of multiple delivery cycles in regard to degradative cultures as well as concurrent testing of pure cultures and consortium enable a good ability to explore potentially effective approaches to limiting dangerous levels of atrazine in soils. A weakness of their experimental design was the lack of adequate culture condition differences, thus failing to enable extrapolation of results to widely different conditions. The claims made by the authors are well supported, and their suggested delivery mechanism for atrazine-degradative cultures addresses a major problem in bioremediation (Alvey & Crowley, 1995; Satsuma, 2009; Schaffer et al., 2008) .

12. Personal Comments The work presented in both of these papers was relatively rigorous and sufficiently investigated their questions of interest. Biodegradation is a potentially effective and cost-effective means of preserving water supply from contamination by herbicides like atrazine in the case of polluted soils; the work of Newcombe & Crowley provides a good example of how this might be applied in an actual scenario. The field is one of great difficulties, especially in cases where there are not an intrinsic population of degradative microbes, and the conditions are not conducive to bacterial atrazine degraders. The work of finding further isolates and consortiums which are capable of surviving in a range of conditions is being pursued by many (Cook & Hutter, 1981; De Souza et al., 1998a; De Souza et al., 1998b; Ghosh & Philip, 2004; Rhine et al., 2003; Piutti et al., 2002) is of great importance to the applicability of this approach to remediation. There are further approaches to pursue, and molecular biological means may also prove of some use to the expansion of this field as well (De Souza et al., 1998c).

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