Microbial Degradation of 2,4,6-Trinitrotoluene (TNT) and Bioremediation of Contaminated Areas with TNT

Yıl 2016, Cilt: 4 Sayı: 7, 38 - 50, 03.06.2016

Zehra Gün Gök Murat İnal Mustafa Yiğitoğlu

Öz

2,4,6-trinitrotoluene (TNT) is a widely used explosive chemical. TNT was used as the main explosive in both two World wars due to characteristics such as safe manufacture and storage of TNT, its low melting point and its chemical and thermal stability. The manufacturing, use and disposal of TNT for military activities have caused contamination of both soil and groundwater. Although TNT is widely used as an explosive material, there are toxic effects of TNT on many organisms including humans and because of the symmetrical arrangement of the nitro groups in the structure of TNT, it can be stay in nature without degradation for a long time. Remediation of soil and groundwater contamination caused by TNT with biological approaches is very important in terms of human health and ecosystems. The availability of biological treatment of a toxic compound depends on the possibility of biodegradation of the compound. Though TNT is persistent in nature for a long time, it is sensitive to microbial attacks. A variety of aerobic and anaerobic bacteria and fungi can degrade TNT and TNT’s transformation metabolites with nitroreductase enzymes which they synthesize. For the remediation of contaminated areas with TNT, various biological-based technologies employing microorganisms that are capable of degrading TNT have been developed. In this study, by examining relevant literature, microbial degradation mechanisms of TNT and microorganism species that have TNT degradation capacity have been designated. Furthermore, bioremediation methods used for the treatment of contaminated sites with TNT were investigated, studies on the implementation of the methods and advantages and disadvantages of these methods in practice were compiled from literature sources. 

Anahtar Kelimeler

Nitroaromatic compounds, 2 4 6-trinitrotoluene, microbial degradation, bioremediation

2,4,6-Trinitrotoluen (TNT)’in Mikrobiyal Degradasyonu ve TNT ile Kirlenmiş Bölgelerin Biyoremediasyonu

Öz

2,4,6-trinitrotoluen (TNT) yaygın olarak kullanılan patlayıcı bir kimyasaldır. TNT güvenli üretimi ve depolanması, düşük erime noktası, kimyasal ve termal kararlılığı gibi özelliklerinden dolayı her iki dünya savaşında ana patlayıcı madde olarak kullanılmıştır. TNT’nin askeri faaliyetler için imalatı, kullanımı ve imha edilmesi hem toprak hem de yeraltı sularının kirlenmesine neden olmaktadır. TNT’nin yaygın olarak patlayıcı madde olarak kullanılmasına karşın insan dahil birçok organizma üzerinde toksik etkileri vardır ve TNT yapısında bulunan nitro gruplarının simetrik düzeninden dolayı doğada uzun süre bozunmadan kalabilmektedir. TNT’nin hem yer altı sularında hem de toprakta neden olduğu kirliliğin biyolojik yöntemlerle remediasyonu insan sağlığı ve ekosistem açısından oldukça önemlidir. Bir toksik bileşiğin biyolojik olarak muamele edilebilirliği bu bileşiğin biyodegradasyonunun mümkün olmasına bağlıdır. TNT doğada uzun süre bozunmadan kalabilmesine karşın mikrobiyal ataklar karşısında hassastır. Çeşitli aerobik ve anaerobik bakteriler ve mantarlar sentezledikleri nitroredüktaz enzimleri ile TNT ve TNT’nin transformasyon metabolitlerini parçalayabilmektedir. TNT ile kirlenmiş bölgelerin ıslahı için, TNT’yi parçalayabilme yeteneğine sahip mikroorganizmaların kullanıldığı çeşitli biyolojik temelli teknolojiler geliştirilmiştir. Bu çalışmada konuyla ilgili literatür incelenerek TNT’nin mikrobiyal degradasyon mekanizmaları ve TNT degradasyon kapasitesine sahip mikroorganizma türleri belirlenmiştir. Ayrıca, TNT ile kirlenmiş bölgelerin ıslahında kullanılan biyoremediasyon yöntemleri araştırılmış, yöntemlerin uygulanmasına ait çalışmalar ve yöntemlerin uygulamadaki avantaj ve dezavantajları literatür kaynaklarından derlenmiştir. 

Anahtar Kelimeler

Nitroaromatik bileşikler, mikrobiyal degradasyon, biyoremediasyon, 2 4 6-trinitrotoluen

Kaynakça

• Anonim, Trinitrotoluene, University of Torino, http://lem.ch.unito.it/didattica/infochimica/2008_Esplosivi/TNT.html (Erişim Tarihi: 12.10.2015).
• ATSDR. 1995. U.S. Agency for Toxic Substances and Disease Registry. Toxicological Profile for 2,4,6-trinitrotoluene. Georgia, 1-208.
• Ayoub, K., Hullebusch, E.D., Cassir, M., Bermond, A. 2010. Application of advanced oxidation processes for TNT removal: A review. Journal of Hazardous Materials 178, 10-28. doi: 10.1016/j.jhazmat.2010.02.042.
• Bae, B., Autenrieth, R.L., Bonner, J.S. 1995. Aerobic biotransformation and mineralization of 2,4,6-trinitrotoluene. 231-238. In R. E. Hinchee, R. E. Hoeppel, and D. B. Anderson (ed.), Bioremediation of recalcitrant organics. Battelle Press, Columbus, Ohio.
• Bayman, P., Radkar G.V. 1997. Transformation and Tolerance of TNT (2,4,6-trinitrotoluene) by Fungi. International Biodeterioration & Biodegradation 39, 45-53.
• Boopathy, R., Kulpa, C.F. 1992. Trinitrotoluene as a sole nitrogen source for a sulfate-reducing bacterium Desulfovibrio sp. (B strain) isolated from an anaerobic digester. Current Microbiology 25, 235-241.
• Boopathy, R., Kulpa, C.F. 1994. Biotransformation of 2,4,6-trinitrotoluene (TNT) by a Methanococcus sp. (strain B) isolated from a lake sediment. Canadian Journal of Microbiology 40, 273-278.
• Bruns-Nagel, D., Drzyzga, O., Steinbach, K., Schmidt, C., von Löw, E., Gorontzy, T., Blotevogel, K.H., Gemsa, D. 1998. Anaerobic/aerobic composting of 2,4,6-trinitrotoluene-contaminated soil in a reactor system. Environmental Science & Technology 32, 1676-1679.
• Bruns-Nagel, D., Scheffer, S., Casper, B., Garn, H., Drzyzga, O., von Löw, E., Gemsa, D. 1999. Effect of 2,4,6-trinitrotoluene and its metabolites on human monocytes. Environmental Science & Technology 33, 2566-2570.
• Bruns-Nagel, D., Steinbach, K., Gemsa, D., von Löw, E. 2000. Composting (humification) of nitroaromatic compounds. 357-393.
• In Biodegradation of nitroaromatic compounds and explosives. Ed: by J. Spain, J.B. Hughes, and H.-J. Knackmuss. Publishers, Boca Raton, Fla
• Bumpus, J.A., Tatarko, M. 1994. Biodegradation of 2,4,6-trinitrotoluene by Phanerochaete chrysosporium: identification of initial degradation products and the discovery of a TNT metabolite that inhibits lignin peroxidase. Current Microbiology 28, 185-190.
• Chien, C-C., Kao, C-M., Chen, D-Y., Chen, S.C., Chen, C-C. 2014. Biotransformation of Trinitrotoluene (TNT) by Pseudomonas spp. Isolated from a TNT-contaminated Environment. Environmental Toxicology and Chemistry 33 (5), 1059-1063.
• Clark, B., Boopathy, R. 2007. Evaluation of bioremediation methods for the treatment of soil contaminated with explosives in Louisiana Army Ammunition Plant, Minden, Louisiana. Journal of Hazardous Materials 143, 643-648.
• Claus, H. 2014. Microbial Degradation of 2,4,6- Trinitrotoluene In Vitro and in Natural Environments. 15-38. In the Biological remediation of explosive residues. Ed: S.N. Singh. Environmental Science and Engineering, Springer International Publishing Switzerland, doi: 10.1007/978-3-319-01083-0_2.
• Claus, H., Bausinger, T., Lehmler, I., Perret, N., Fels, G., Dehner, U., König, H. 2007. Transformation of 2,4,6-trinitrotoluene (TNT) by Raoultella terrigena. Biodegradation 18, 27-35.
• Das, P., Datta, R., Makris, K.C., Sarkar, D. 2010. Vetiver grass is capable of removing TNT from soil in the presence of urea. Environmental Pollution 158, 1980-1983.
• Dindar, E., Topaç Şağban, F.O., Başkaya, H. 2010. Kirlenmiş Toprakların Biyoremediasyon İle Islahı. Uludağ Üniversitesi Mühendislik-Mimarlık Fakültesi Dergisi 15(2), 123-137.
• Drzyzga, O., Bruns-Nagel, D., Gorontzy, T., Blotevogel, K.H., Gemsa, D. 1998. Mass balance studies with 14C-labeled 2,4,6-trinitrotoluene (TNT) mediated by an anerobic Desulfovibrio species and an aerobic Serratia species. Current Microbiology 37, 380-386.
• Duque, E., Haidour, A., Godoy, F., Ramos, J.L. 1993. Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene. Journal of Bacteriology 175, 2278-2283.
• Ederer, M.M., Lewis, T.A., Crawford, R.L. 1997. 2,4,6-Trinitrotoluene (TNT) transformation by Clostridia isolated from a munition-fed bioreactor: comparison with non-adapted bacteria. Journal of Industrial Microbiology & Biotechnology 18, 82-88.
• Eilers, A., Rüngeling, E., Stündl, U.M., Gottschalk, G. 1999. Metabolism of 2,4,6-trinitrotoluene by the white rot fungus Bjerkandera adusta DSM 3375 depends on cytochrome P450. Applied Microbiology Biotechnology 53, 75-80.
• Esteve-Nuñez, A., Caballero, A., Ramos, J.L. 2001. Biological Degradation of 2,4,6-Trinitrotoluene. Microbiology and Molecular Biology Reviews 65, 335-352. doi: 10.1128/MMBR.65.3.335–352.
• Esteve-Nuñez, A., Lucchesi, G., Philipp, B., Schink, B., Ramos, J.L. 2000. Respiration of 2,4,6-Trinitrotoluene by Pseudomonas sp. Strain JLR11. Journal of Bacteriology 182, 1352-1355.
• Esteve-Nuñez, A., Ramos, J.L. 1998. Metabolism of 2,4,6-Trinitroroluene by Pseudomonas sp. JLR11. Environmental Science & Technology 32, 3802-3808.
• Fernando, T., Bumpus, J.A., Aust, S.D. 1990. Biodegradation of TNT (2,4,6 trinitrotoluene) by Phanerochaete chrysosporium. Applied Environmental Microbiology 56, 1666-1671.
• Fiorella, P.D., Spain, J.C. 1997. Transformation of 2,4,6-trinitrotoluene by Pseudomonas pseudoalcaligenes JS52. Applied Environmental Microbiology 63, 2007-2015.
• French, C.E., Nicklin, S., Bruce, N.C. 1998. Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase. Applied Environmental Microbiology 64, 2864-2868.
• Gallagher, E.M. 2010. Anaerobic degradation of 2,4,6-trinitritoluene (TNT): molecular analysis of active degraders and metabolic pathway. The State University of New Jersey, PhD Thesis, 115s, New Jersey.
• Gao, D., Du, L., Yang, J., Wu, W.M., Liang, H. 2010. A critical review of the application of white rot fungus to environmental pollution control. Critical Reviews in Biotechnology 30, 70-77.
• Gumuscu, B., Tekinay, T. 2013. Effective biodegradation of 2,4,6-trinitrotoluene using a novel bacterial strain isolated from TNT-contaminated soil. International Biodeterioration&Biodegradation 85, 35-41.
• Haselhorst, L. 1999. Bioremediation of 2,4,6-Trinitrotoluene (TNT) at munitions sites. Restoration And Reclamination Rewiew 4 (7), 1-9.
• Hughes, J.B., Wang, C., Yesland, K., Bhadra, R., Richardson, A., Bennet, G., Rudolph, F. 1998. Reduction of 2,4,6-trinitrotoluene by Clostridium acetobutylicum through hydroxylamino intermediates. Environmental Toxicology and Chemistry 17, 343-348.
• Jarvis, A.S., McFarland, V.A., Honeycutt, M.E. 1998. Assesment of the effectiviness of composting for the reduction of toxicity and mutagenicity of explosives-contaminated soil. Ecotoxicology and Environmental Safety 39, 131-135.
• Ju, K.S., Parales, R.E. 2010. Nitroaromatic Compounds, from Synthesis to Biodegradation. Microbiology and Molecular Biology Reviews 74 (2), 250-272.
• Kalafut, T., Wales, M.E, Rastogi, V.K., Naumova, R.P., Zaripova, S.K., Wild, J.R. 1998. Biotransformation patterns of 2,4,6-trinitrotoluene by aerobic bacteria. Current Microbiology. 36, 45-54.
• Kalderis, D., Juhasz, A.L., Boopathy, R., Comfort, S. 2011. Soils contaminated with explosives: Environmental fate and evaluation of state-of the-art remediation processes (IUPAC Technical Report)*. Pure and Applied Chemistry 83 (7), 1407-1484.
• Khan, M.I., Lee, J., Park, J. 2013. A Toxicological Review on Potential Microbial Degradation Intermediates of 2,4,6-Trinitrotoluene, and Its Implications in Bioremediation. KSCE Journal of Civil Engineering 17 (6), 1223-1231.
• Kim, H.Y., Bennett, G., Song, H.G. 2002. Degradation of 2,4,6- trinitrotoluene by Klebsiella sp. isolated from activated sludge. Biotechnology Letter 24, 2023-2028.
• Lewis, T.A., Ederer, M.M., Crawford, R.L., Crawford, D.L. 1997. Microbial transformation of 2,4,6-trinitrotoluene. Journal of Industrial Microbiology and Biotechnology 18, 89-96.
• Lewis, T.A., Newcombe, D.A., Crawford, R.L. 2004. Bioremediation of soils contaminated with explosives. Journal of Environmental Management 70, 291-307.
• Lin, H., Yu, C., Chen, Z. 2013. Aerobic and anaerobic biodegradation of TNT by newly isolated Bacillus mycoide. Ecological Engineering 52, 270-277.
• Maeda, T., Kadokami, K., Ogawa, H.I. 2006. Characterization of 2,4,6-Trinitrotoluene (TNT)-Metabolizing Bacteria Isolated from TNT-Polluted Soils in the Yamada Green Zone, Kitakyushu, Japan. Journal of Environmental Biotechnology 6, 33-39.
• Maeda, T., Nakamura, R., Kadokami, K., Ogawa, H.I. 2007. Relationship between mutagenicity and reactivity or biodegradability for nitroaromatic compounds. Environmental Toxicology and Chemistry 26, 237-241.
• Martin, J.L., Comfort, S.D., Shea, P.J., Kokjohn, T. A., Drijber, R.A. 1997. Denitration of 2,4,6-trinitrotoluene by Pseudomonas savastanoi. Canadian Journal of Microbiology 43, 447-455.
• McCormick, N.G., Feeherry, F.E., Levinson, H.S. 1976. Microbial transformation of 2,4,6-TNT and other nitroaromatic compounds. Applied Environmental Microbiology 31, 949-958.
• Mercimek, H.A. 2011. 2,4,6-trinitrotoluen’in Mikrobiyal Biyodegradasyonu. Çukurova Üniversitesi, Doktora Tezi, 158s, Adana.
• Mercimek, H.A., Dincer, S., Guzeldag, G., Ozsavli, A., Matyar, F. 2013. Aerobic biodegradation of 2,4,6-trinitrotoluene (TNT) by Bacillus cereus isolated from contaminated soil. Microbial Ecology 66, 512-521.
• Mercimek, H.A., Dincer, S., Guzeldag, G., Ozsavli, A., Matyar, F., Arkut, A., Kayis, F., Sumengen Ozdefne, M. 2015. Degradation of 2,4,6-trinitrotoluene by P. aeruginosa and characterization of some metabolites. Brazilian Journal of Microbiology 46, 104-111.
• Montpas, S., Samson, J., Langlois, E., Lei, J., Piche, Y., Chevenert, R. 1997. Degradation of 2,4,6-trinitrotoluene by Serratia marcescens. Biotechnology Letters 19, 291-294.
• Mulla, S.I., Talwar, M.P., Ninnekar, H.Z. 2014. Bioremediation of 2,4,6-Trinitrotoluene Explosive Residues. 201-233. In the Biological remediation of explosive residues. Ed: S.N. Singh. Environmental Science and Engineering, Springer International Publishing Switzerland, doi: 10.1007/978-3-319-01083-0_2.
• Muter, O., Potapova, K., Limane, B., Sproge, K., Jakobsone, I., Cepurnieks Bartkevics, V. 2012. The role of nutrients in the biodegradation of 2,4,6-trinitrotoluene in liquid and soil. Journal of Environmental Management 98, 51-55.
• Naumova, R.P., Selivanovskaya, S.L.U., Mingatina, F.A. 1988. Possibilities for the deep bacterial destruction of 2,4,6-trinitrotoluene. Mikrobiologia 57, 218-222.
• Nyanhongoa, G.S., Aicherniga, N., Ortnera, M., Steinerb, W., Guebitza, G.M. 2009. Incorporation of 2,4,6-trinitrotoluene (TNT) transforming bacteria into explosive formulations. Journal of Hazardous Materials 165, 285-290.
• Oh, B.T., Sarah, G., Shea, P.J., Drijber, R.A., Comfort, S.D. 2000. Rapid spectrophotometric determination of 2,4,6-trinitrotoluene in a Pseudomonas enzyme assay. Journal of Microbiological Methods 42, 149-158.
• Oh, K.H., Kim, Y.J. 1998. Degradation of Explosive 2,4,6-Trinitroroluene by s-Triazine Degrading Bacterium Isolated from Contaminated Soil. Bulletin of Environmental Contamination and Toxicology 61, 702-708.
• Özcan, G., Türkdoğan, F.İ. 2014. Askeri Alanlardaki Kirliliklerin Gideriminde Biyoremediasyon Teknikleri. KSU Mühendislik Bilimleri Dergisi, 17, 31-36.
• Pak, J.W., Knoke, K.L., Noguera, D.R., Fox, B.G., Chambliss, G.H. 2000. Transformation of 2,4,6-trinitrotoluene by purified xenobiotic reductase B from Pseudomonas fluorescens I-C. Appl. Environmental Microbiology 66, 4742-4750.
• Panz, K., Miksch, K. 2012. Phytoremediation of explosives (TNT, RDX, HMX) by wild-type and transgenic plants. Journal of Environmental Management 113, 85-92.
• Park, C., Kim, T.H., Kim, S. 2003b. Optimization of biodegradation of 2,4,6-trinitrotoluene (TNT) by Pseudomonas putida. Journal of Bioscience and Biengineering 95, 567-571.
• Park, C., Kim, T-H., Kim, S., Lee, J., Kim, S-W. 2002. Biokinetic Parameter Estimation for Degradation of 2,4,6-Trinitrotoluene (TNT) with Pseudomonas putida KP-T201. Journal of Bioscience and Biengineering 94, 57-61.
• Preuss, A., Fimpel, J., Dickert, G. 1993. Anaerobic transformation of 2,4,6-trinitrotoluene (TNT). Archives of Microbiology 159, 345-353.
• Qasim, M.M., Moore, B., Taylor, L., Honea, P., Gorb, L., Leszczynski, J. 2007. Structural Characteristics and Reactivity Relationships of Nitroaromatic and Nitramine Explosives – A Review of Our Computational Chemistry and Spectroscopic Research, International Journal of Molecular Sciences 8, 1234-1264.
• Rahal, A.Gh., Moussa, L.A. 2011. Degradation of 2,4,6-Trinitrotoluene (TNT) by Soil Bacteria Isolated From TNT Contaminated Soil. Australian Journal of Basic and Applied Sciences 5(2), 8-17.
• Rezaei, M.R., Abdolı, M.A., Karbassı, A., Baghvand, A., Khalilzadeh, R. 2010. Bioremediation of TNT Contaminated Soil by Composting with Municipal Solid Wastes Soil and Sediment Contamination 19, 504–514.
• Rodgers, J.D., Bunce, N.J. 2001. Treatment methods for the remediation of nitroaromatic explosives. Water Research 35, 2101-11.
• Rylott, E.L., Bruce, N.C. 2009. Plants disarm soil: engineering plants for the phytoremediation of explosives. Trends in Biotechnology 27, 73-81.
• Sangwan, P, Mary Celin, S., Hooda, L. 2015. Response Surface Methodological Approach for Optimizing Process Variables for Biodegradation of 2,4,6-Trinitrotoluene using Acinetobacter Noscomialis. European Journal of Advances in Engineering and Technology 2(4), 51-56.
• Schackmann, A., Müller, R. 1991. Reduction of nitroaromatic compounds by different Pseudomonas species under aerobic conditions. Applied Microbiology and Biotechnology 34, 809-813.
• Scheibner, K., Hofrichter, M., Fritsche, W. 1997a. Mineralization of 2-amino-4,6-dinitrotoluene by manganese peroxidase of the white-rot fungus Nematoloma frowardii. Biotechnology Letters 19, 835-839.
• Scheibner, K, Hofrichter, M., Herre, A., Michels, J., Fritsche, W. 1997b. Screening for fungi intensevely mineralizing 2,4,6-trinitrotoluene. Applied Microbiology and Biotechnology 47, 452-457.
• Shim, C.Y., Crawford, D.L. 1995. Biodegradation of trinitrotoluene (TNT) by a strain of Clostridium bifermentan. 57-69. In R. E. Hinchee, J. Fredrickson, and B. C. Alleman (ed.), Bioaugmentation for site remediation. Battelle Press, Columbus, Ohio.
• Spain, J. 1995. Biodegradation of nitroaromatic compounds. Annual Review of Microbiology 49, 523-555.
• Tan, E.L., Ho, C.H., Griest, W.H., Tyndall, R.L. 1992. Mutagenicity of trinitrotoluene and its metabolites formed during composting. Journal of Toxicology and Environmental Health 36, 165-175.
• Üzer, A. 2004. Bazı Nitrofenollerin Temel ve Türev Spektrofotometrik Analizi. İstanbul Üniversitesi, Yüksek Lisans Tezi, 77s, İstanbul.
• Van Aken, B., Skubusz, K., Naveau, H., Agathos, S.N. 1997. Biodegradation of 2,4,6-trinitrotoluene by the white-rot basidiomycete Phlebia radiata. Biotechnology Letters 19, 813-817.
• Vanderberg, L.A., Perry, J.J., Unkefer, P.J. 1995. Catabolism of 2,4,6-trinitrotoluene by Mycobacterium vaccae. Applied Microbiology and Biotechnology 43, 937-945.
• Vorbeck, C., Lenke, H., Fischer, P., Knackmuss, H.J. 1994. Identification of a hydride-Meisenheimer complex as a metabolite of 2,4,6-trinitrotoluene by a Mycobacterium strain. Journal of Bacteriology 176, 932-934.
• Vorbeck, C., Lenke, H., Fischer, P., Spain, J.C., Knackmuss, H.J. 1998. Initial reductive reactions in aerobic microbial metabolism of 2,4,6-trinitrotoluene. Applied and Environmental Microbiology 64, 246-252.
• Widrig, D.L., Boopathy, R., Manning, J.F. 1997. Bioremediation of TNT contaminated soil: A laboratory study. Environmental Toxicology and Chemistry 16, 1141-1148.
• Williams, M., Reddy, G., Quinn, M., Johnson, M. 2015. Wildlife Toxicity Assessments for Chemicals of Military Concern. Elsevier Inc., Oxford, UK.
• Williams, R.T., Ziegenfuss, P.S., Sisk, W.E. 1992. Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions. Journal of Industrial Microbiology and Biotechnology 9, 137-144.
• Yan, C. , Wang, Y., Xia, B., Li, L., Zhang, Y., Liu, Y. 2002. The retrospective survey of malignant tumor in weapon workers exposed to 2,4,6-trinitrotoluene. Chinese Journal of Industrial Hygiene and Occupational Diseases 20(3), 184-188.
• Yinon, J., 1990. Toxicity and Metabolism of Explosives. CRC Press, Florida.
• You, S-H., Zhu, B., Han, H-J., Wang, B., Peng, R-H., Yao, Q-H. 2015. Phytoremediation of 2,4,6-trinitrotoluene by Arabidopsis plants expressing a NAD(P)H-flavin nitroreductase from Enterobacter cloacae. Plant Biotechnology Reports 9, 417-430.