Biotechnology and Molecular Biology Reviews
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Article Number - 07DB69840318

Vol.8(2), pp. 35-42 , October 2013
DOI: 10.5897/BMBR10.037
ISSN: 1538-2273


Molecular and biological techniques used in landfill investigations: A mini-review

umphrey O. Zebulun*, Hilary I. Inyang and Helene Hilger

Department of Civil and Environmental Engineering, University of North Carolina, Charlotte, NC, USA.


 Accepted: 20 September 2013  Published: 31 October 2013

Copyright © 2013 Author(s) retain the copyright of this article.
This article is published under the terms of the Creative Commons Attribution License 4.0

The purpose of this research paper was to review the different molecular biology techniques that are used in landfill investigations. The methods discussed include polymerase chain reaction (PCR), fluorescent in situ hybridization (FISH) and phospholipid fatty acid analysis (PLFA). Operation of landfills as bioreactors is now becoming a common practice, which involves the identification of different microbiological activities that facilitate the eventual breakdown of landfill wastes into useful and innocuous materials. In this review, the two important microbial activities that are discussed include methanotrophic process, carried out by methanotrophic bacteria, and methanogenic processes, carried out by methanogenic bacteria. Other bacteria encountered in landfills such as Nitrosospira and Nitrosomonas are also briefly discussed. As the name of these processes imply, methane oxidation and methane production by these microbial activities in landfills constitute another main focus of this paper. The application of these molecular biological techniques in real-time has also been demonstrated in studies involving the investigation of methanogenic diversity and activity in municipal solid waste landfill leachates and this is also discussed further. The results and conclusions of different research studies that focused on these techniques are hereby identified, discussed and summarized.


Key words: Bioreactors, polymerase chain reaction (PCR), fluorescent in situ hybridization (FISH), phospholipid fatty acid analysis (PLFA), methanogens, methanotrophs, landfilling.

Abichou T, Powelson D, Chanton J, Escoriaza S, Stern J (2006). Characteristics of methane flux and oxidation at a solid waste landfill. J. Environ. Eng. 132:220-228.
Barlaz MA, Bareither CA, Hossain A, Saquing J, Mezzari I, Benson CH, Yazdani R (2010). Performance of North American Bioreactor Landfills. II: Chemical and Biological Characteristics. J. Environ. Eng. 136(8):839-853.
Bodelier PLE, Meima-Franke M, Zwart G, Laanbroek HJ (2005). New DGGE strategies for the analyses of methanotrophic microbial communities using different combinations of existing 16S rRNA-based primers. FEMS Microbiol. Ecol. 52:163-174.
Borjesson G, Chanton J, Svensson BH (2001). Atmospheric pollutants and trace gases: methane oxidation in tow Swedish landfill covers measured with carbon-13 to carbon-12 isotope ratios. J. Environ. Qual. 30:369-376.
Borjesson G, Sundh I, Svensson B (2004). Microbial oxidation of CH4 at different temperatures in landfill cover soils. FEMS Microbiol. Rev. 48:305-312.
Borjesson G, Sundh I, Tunlid A, Svensson BH (1998). Methane oxidation in landfill cover soils, as revealed by potential oxidation measurement and phospholipid fatty acid analyses. Soil Biol. Biochem. 30:1423-1433.
Charlier RH, Finkl CW, Krystosyk-Gromadzinska A (2012). Throw it overboard: A commentary on coastal pollution and bioremediation. J. Coast. Res. 28(4):881-890.
Chen AC, Imachi H, Sekiguchi Y, Ohashi A, Harada H (2003). Archaeal community compositions at different depths (up to 30 m) of a municipal solid waste landfill in Taiwan as revealed by 16S rDNA cloning analyses. Biotechnol. Lett. 25:719-724.
Chen AC, Ueda K, Sekiguchi Y, Ohashi A, Harada H (2003). Molecular detection and direct enumeration of methanogenic archaea and methanotrophic bacteria in domestic solid waste landfill soils. Biotechnol. Lett. 25:1563-1569.
Chen Y, Dumont MG, Cébron A, Murrell JC (2007). Identification of active methanotrophs in a landfill cover soil through detection of expression of 16S rRNA and functional genes. Environ. Microbiol. 9(11):2855–2869
Copty NK, Ergene D, Onay TT (2004). Stochastic model for landfill gas transport and energy recovery. J. Environ. Eng. 130:1042-1049.
Domingo JWS, Berry CJ, Hazen TC (1997). Use of conventional methods and whole cell hybridization to monitor the microbial response to triethylphosphate. J. Microbiol. Methods 29:145-151.
Dorigo U, Volatier L, Humbert J-F (2005). Molecular approaches to the assessment of biodiversity in aquatic microbial communities. Water Res. 39:2207-2218.
Einola J-KM, Kettunen RH, Rintala JA (2007). Responses of methane oxidation to temperature and water content in cover soil of a boreal landfill. Soil Biol. Biochem. 39:1156-1164.
Frantz JH, Wood RM, Sawyer WK, Delozier DL (2000). Recovery and utilization of methane from solid waste landfills. Proceedings; SPE/CERI Gas Technology Symposium, 3-5 April 2000, Calgary, Alberta, Canada. pp. 9-15.
Garcia L-L, Patel BKC, Ollivier B (2000). Taxonomic, phylogenetic, and ecological diversity of methanogenic archaea. Anaerobe 6:205-226.
Gupta R, Morris JW (2013). Expanding the options. Civil Engineering, Retrieved September 16, 2013 from
Han JS, Kim CG (2010). Characterization of molecular biological indicators to define stabilization of landfills. Korean J. Chem. Eng. 27: 868-873.
Han JS, Kim CG (2009). Comparative assessment of gene quanti-fication using real-time PCR and water quality parameters in unsanitary landfill. Water Sci. Technol. 59(2):331-338.
Hanson RS, Hanson TE (1996). Methanotrophic bacteria. Microbiol. Rev. 60:439-471.
PMid:8801441 PMCid:PMC239451
Hartlieb N, Ertunc T, Andreas S, Klein W (2003). Mineralization, metabolism and formation of non-extractable residues of 14C-labelled organic contaminants during pilot-scale composting of municipal biowaste. Environ. Pollut. 126:83-91.
He, R., Ruan, A., Jiang, C., and Shen, D. (2008). Responses of oxidation rate and microbial communities to methane in simulated landfill cover soil microcosms. Bioresource Technology, 99(15), 7192-7199.
He R, Wooller MJ, Pohlman JW, Catranis C, Quensen J, Tiedje JM, Leigh MB (2012). Identification of functionally active aerobic methanotrophs in sediments from an arctic lake using stable isotope probing. Environ. Microbiol. 14(6):1403–1419.
Henneberger R, Chiri E, Blees J, Niemann H, Lehmann MF, Schroth MH (2013). Field-scale labelling and activity quantification of methane-oxidizing bacteria in a landfill-cover soil. FEMS Microbiol. Ecol. 83(2):392-401.
Hilger HA, Wollum AG, Barlaz MA (2000). Landfill methane oxidation response to vegetation, fertilization, and liming. J. Environ. Qual. 29: 324-334.
Hunt M (2006). Real time PCR. Retrieved September 20, 2006 from edu/pcr/realtime-home.htm.
Jung Y, Imhoff PT, Augenstein DC, Yazdani R (2009). Influence of High-Permeability Layers for Enhancing Landfill Gas Capture and Reducing Fugitive Methane Emissions from Landfills. J. Environ. Eng. 135(3):138-146.
Kallistova AU, Kevbrina MV, Nekrasova VK, Shnyrev NA, Einola JM, Kulomaa MS, Nozhevnikova AN (2007). Enumeration of Methanotrophic Bacteria in the Cover Soil of an Aged Municipal Landfill. Microb. Ecol. 54(4):637-645.
Kim D-J, Dong L, Keller J (2006). Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH. Bioresour. Technol. 97:459-468.
Kong JY, Su YY, Zhang QQ, Bai YY, Xia FF, Fang CR, He RR (2013). Vertical profiles of community and activity of methanotrophs in landfill cover soils of different age. J. Appl. Microbiol.115(3):756-765.
Laloui-Carpentier W, Li T, Vigneron V, Mazeas L, Bouchez T (2006). Methanogenic diversity and activity in municipal solid waste landfill leachates. Antonie van Leeuwenhoek 89:423-434.
Latorre I, Hwang S, Montalvo-Rodriguez R (2012). Isolation and molecular identification of landfill bacteria capable of growing on di-(2-ethylhexyl) phthalate and deteriorating PVC materials. J. Environ. Sci. Health A Tox. Hazard. Subst. Environ. Eng. 47(14):2254-2262.
PMid:22934997 PMCid:PMC3432978
Li Y, Cleall PJ (2010). Analytical solutions for contaminant diffusion in double-layered porous media. J. Geotechn. Geoenviron. Eng. 136(11):1542-1554.
Limam RD, Bouchez T, Chouari R, Li T, Barkallah I, Landoulsi A, Sghir A (2010). Detection of WWE2-related Lentisphaerae by 16S rRNA gene sequencing and fluorescence in situ hybridization in landfill leachate. Can. J. Microbiol. 56:846–852.
Lockhart RJ, Dyke MIV, Beadle IR, Humphreys P, McCarthy AJ (2006). Molecular biological detection of anaerobic gut fungi (Neocallimastigales) from landfill sites. Appl. Environ. Microbiol. 72: 5659-5661.
PMid:16885325 PMCid:PMC1538735
Lowry MI, Bartelt-Hunt SL, Beaulieu SM, Barlaz MA (2008). Development of a coupled reactor model for prediction of organic contaminant fate in landfills.Environ. Sci. Technol. 42(19):7444-7451.
Ludvigsen L, Albrechtsen H-J. Holst H, Christensen TH (1997). Correlating phospholipid fatty acids (PFLA) in a landfill leachate polluted aquifer with biogeochemical factors by multivariate statistical methods. FEMS Microbiol. Rev. 20:447-460.
Luesken FA, Wu ML, Op den Camp HJ, Keltjens JT, Stunnenberg H, Francoijs K, Strous M, Jetten MS (2012). Effect of oxygen on the anaerobic methanotroph 'Candidatus Methylomirabilis oxyfera': kinetic and transcriptional analysis. Environ. Microbiol. 14(4):1024–1034. emi_2682 1024..
Maxfield PJ, Brennand EL, Powlson DS, Evershed RP (2011). Impact of land management practices on high-affinity methanotrophic bacterial populations: evidence from long-term sites at Rothamsted. Europ. J. Soil Sci. 62:56-68.
McDonald JE, Houghton JN, Rooks DJ, Allison HE, McCarthy AJ (2012). The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters. Environ. Microbiol. 14(4):1077–1087.
McDonald JE, Lockhart RJ, Cox MJ, Allison HE, McCarthy AJ (2008). Detection of novel Fibrobacter populations in landfill sites and determination of their relative abundance via quantitative PCR. Environ. Microbiol. 10(5):1310-1319.
Mehta R, Barlaz MA, Yazdani R, Augenstein D, Bryars M, Sinderson L (2002). Refuse decomposition in the presence and absence of leachate recirculation. J. Environ. Eng. 128:228-236.
Mellendorf M, Huber-Humer M, Gamperling O, Huber P, Gerzabek MH, Watzinger A (2010). Characterisation of microbial communities in relation to physical–chemical parameters during in situ aeration of waste material. Waste Manage. 30(11):2177-2184.
Mer JL, Roger P (2001). Production, oxidation, emission and consumption of methane by soils. a review. Europ. J. Soil Biol. 37: 25-50.
Mnif S, Zayen A, Karray F, Bru-Adan V, Loukil S, Godon J, Sayadi S (2012). Microbial population changes in anaerobic membrane bioreactor treating landfill leachate monitored by single-strand conformation polymorphism analysis of 16S rDNA gene fragments. Int. Biodeterior. Biodegradation 73:50-59.
Mohammadi T, Pietersz RN, Vandenbroucke-Grauls CM, Savelkoul PH, Reesink HW (2005). Detection of bacteria in platelet concentrates: comparison of broad-range real-time 16S rDNA polymerase chain reaction and automated culturing. Transfusion 45:731-736.
Mojiri A, Aziz AH, Aziz SQ (2013). Trends in Physical-Chemical Methods for Landfill Leachate Treatment. Int. J. Sci. Res. Environ. Sci. 1(2):16-25.
Mor S, Ravindra K, Visscher AD, Dahiya RP, Chandra A (2006). Municipal solid waste characterization and its assessment for potential methane generation: a case study. Sci. Total Environ. 371:1-10.
Nakamura K, Terada T, Seiguchi Y, Shinzato N, Meng X-Y, Enoki M, Kamagata Y (2006). Application of pseudomurein endoisopeptidase to fluorescence in situ hybridization of methanogens within the family Methanobacteriaceae. Appl. Environ. Microbiol. 72:6907-6913.
PMid:16950902 PMCid:PMC1636154
National Human Genome Institute (2007). Florescence in situ hybridization (FISH). Fact Sheet. Retrieved May 31, 2007 from nhgri_ PDFs/ fish_TXT.pdf.
Nozhevnikova AA, Glagolev MM, Nekrasova VV, Einola JJ, Sormunen KK, Rintala JJ (2003). The analysis of methods for measurement of methane oxidation in landfills. Water Sci. Technol. 48(4):45-52.
Pombo SA, Kleikemper J, Schroth MH, Zeyer J (2005). Field-scale isotopic labeling of phospholipid fatty acids from acetate-degrading sulfate-reducing bacteria. FEMS Microbiol. Ecol. 51(2):197-207.
Powelson DK, Chanton J, Abichou T, Morales J (2006). Methane oxidation in water-spreading and compost biofilters. Waste Manage. Res. 24:528-536.
Providenti MA, Mautner SI, Chaudhry O, Bombardier M, Scroggins R, Gregorich E, Smith ML (2004). Determining the environmental fate of a filamentous fungus, Trichoderma reesei, in laboratory-contained intact soil-core microcosms using competitive PCR and viable plating. Can. J. Microbiol. 50:623-631.
Rachor IM, Gebert JJ, Gröngröft AA, Pfeiffer EM (2013). Variability of methane emissions from an old landfill over different time-scales. Europ. J. Soil Sci. 64(1):16-26.
Ran Y, Ping G, MacKay AA, Shouliang Z, Smets BF (2010). Presence, distribution, and diversity of iron-oxidizing bacteria at a landfill leachate-impacted groundwater surface water interface. FEMS Microbiol. Ecol. 71(2):260-271.
Reinhart DR (1993). A review of recent studies on the sources of hazardous compounds emitted from solid waste landfills: a U.S. experience. Waste Manage. Res. 11:257-268.
Ren L, Li H, Lu W (2012). Response of community structure and activity of methanotroph to different CH4/O2 ratios. Procedia Environ. Sci. 16:697 – 704.
Riesenfeld CS, Schloss PD, Handelsman J (2004). Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet. 38: 525-552.
Sanchez R, Hashemi M, Tsotsis TT, Sahimi M (2006). Computer simulation of gas generation and transport in landfills II: dynamic conditions. Chem. Eng. Sci. 61:4750-4761.
Sanz JL, Kochling T (2007). Molecular biology techniques used in wastewater treatment: an overview. Process Biochem. 42:119-133.
Srivastava AK, Nema AK (2011). Fuzzy Parametric Programming Model for Integrated Solid Waste Management under Uncertainty. ASCE J. Environ. Eng. 137(1):69-83.
Staley BF, de los Reyes FL, Barlaz MA (2012). Comparison of B acteria and A rchaea communities in municipal solid waste, individual refuse components, and leachate. FEMS Microbiol. Ecol. 79(2):465-473.
Stralis-Pavese N, Bodrossy L, Rechenauer TG, Weilharter A, Sessitsch A (2006). 16S rRNA based T-RFLP analysis of methane oxidizing bacteria – assessment, critical evaluation of methodology performance and application for landfill site cover soils. Appl. Soil Ecol. 31:251-266.
Stralis-Pavese N, Sessitsch A, Weilharter A, Rechenauer T, Riesing J, Csontos J, Murrel JC, Bodrossy L (2004). Optimization of diagnostic microarray for application in analyzing landfill methanotroph communities under different plant covers. Environ. Microbiol. 6:347-363.
Sundberg C, Stendahl JS, Tonderski K, Lindgren P-E (2007). Overland flow systems for treatment of landfill leachates – potential nitrification and structure of the ammonia-oxidizing bacterial community during a growing season. Soil Biol. Biochem. 39:127-138.
Themelis NJ, Ulloa PA (2007). Methane generation in landfills. Renew. Energy 32:1243-1257.
Tolaymat TM, Green RB, Hater GR, Barlaz MA, Black P, Bronson D, Powell J (2010). Evaluation of landfill gas decay constant for municipal solid waste landfills operated as bioreactors. J. Air Waste Manage. Assoc. 60:91-97.
Urmann K, Lazzaro A, Gandolfi I, Schroth MH, Zeyer J (2009). Response of methanotrophic activity and community structure to temperature changes in a diffusive CH4/O2 counter gradient in an unsaturated porous medium. FEMS Microbiol. Ecol. 69(2):202-212.
Van Dyke MI, McCarthy AJ (2002). Molecular biological detection and characterization of Clostridium populations in municipal landfill sites. Appl. Environ. Microbiol. 68:2049-2053.
PMid:11916731 PMCid:PMC123838
Vishwakarma P, Dubey SK (2010). DNA microarray analysis targeting pmoA gene reveals diverse community of methanotrophs in the rhizosphere of tropical rice soils. Curr. Sci. 99(8):1090-1095.
Visvanathan C, Pokhrel D, Cheimchaisri W, Hettiaratchi JPA, Wu JS (1999). Methanotrophic activities in tropical landfill cover soils: effects of temperature, moisture content and methane concentration. Waste Manage. Res. 17:313-323.
Wang C, Lee P, Kumar M, Huang Y, Sung S, Lin J (2010). Simulta-neous partial nitrification, anaerobic ammonium oxidation and denitrification (SNAD) in a full-scale landfill-leachate treatment plant. J. Hazard. Mater. 175(1-3):622-628.
Watzinger A, Stemmer M, Pfeffer M, Rasche F, Reichenauer TG (2008). Methanotrophic communities in a landfill cover soil as revealed by [13C] PLFAs and respiratory quinones: Impact of high methane addition and landfill leachate irrigation. Soil Biol. Biochem. 40(3):751-762.
Yang N, Lu F, He P, Shao L (2011). Response of methanotrophs and methane oxidation on ammonium application in landfill soils. Appl. Microbiol. Bitechnol. 92:1073-1082.
Yu Y, Lee C, Kim J, Hwang S (2005). Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol. Bioeng. 89:670-679.
Zhou M, Chung YH, Beauchemin KA, Holtshausen L, Oba M, McAllister TA, Guan LL (2011). Relationship between rumen methanogens and methane production in dairy cows fed diets supplemented with a feed enzyme additive. J. Appl. Microbiol. 111:1148-1158.


APA (2013). Molecular and biological techniques used in landfill investigations: A mini-review. Biotechnology and Molecular Biology Reviews, 8(2), 35-42.
Chicago umphrey O. Zebulun, Hilary I. Inyang and Helene Hilger. "Molecular and biological techniques used in landfill investigations: A mini-review." Biotechnology and Molecular Biology Reviews 8, no. 2 (2013): 35-42.
MLA umphrey O. Zebulun, Hilary I. Inyang and Helene Hilger. "Molecular and biological techniques used in landfill investigations: A mini-review." Biotechnology and Molecular Biology Reviews 8.2 (2013): 35-42.
DOI 10.5897/BMBR10.037

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