Improved method for the extraction of high-quality DNA from lignocellulosic compost samples for metagenomic studies

Costa, Ângela M. A.; Santos, Andréia O.; Sousa, Joana; Rodrigues, Joana L.; Gudiña, Eduardo J.; Silvério, Sara C.; Rodrigues, Ligia R.

doi:10.1007/s00253-021-11647-7

Methods and Protocols
Published: 01 November 2021

Improved method for the extraction of high-quality DNA from lignocellulosic compost samples for metagenomic studies

Applied Microbiology and Biotechnology volume 105, pages 8881–8893 (2021)Cite this article

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Abstract

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality (> 99% of humic acids effectively removed) and sufficient quantity (10.5–13.8 µg g⁻¹ of compost) for downstream applications, namely functional metagenomic studies. The protocol takes about 4 h of bench work, and it can be performed using standard molecular biology equipment and reagents available in the laboratory.

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Key points/Highlights

• Metagenomic DNA was successfully extracted from compost samples rich in humic acids

• The improved protocol was established by optimizing the cell lysis method and buffer

• Complete removal of humic acids was achieved through the use of activated charcoal

• The suitability of the DNA was proven by the construction of a metagenomic library

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References

Ali N, Zhang Q, Liu Z-Y, Li F-L, Lu M, Fang X-C (2020) Emerging technologies for the pretreatment of lignocellulosic materials for bio-based products. Appl Microbiol Biotechnol 104:455–473. https://doi.org/10.1007/s00253-019-10158-w
Article PubMed CAS Google Scholar
Arevalo-Gallegos A, Ahmad Z, Asgher M, Parra-Saldivar R, Iqbal HMN (2017) Lignocellulose: a sustainable material to produce value-added products with a zero waste approach—a review. Int J Biol Macromol 99:308–318. https://doi.org/10.1016/j.ijbiomac.2017.02.097
Article PubMed CAS Google Scholar
Baker FS, Miller CE, Repik AJ, Tolles ED (1992) Activated carbon. Kirk-Othmer Encycl Chem Technol 4:1015–1037. https://doi.org/10.1002/0471238961.0103200902011105.a01
Article Google Scholar
Batista-García RA, del Rayo Sánchez-Carbente M, Talia P, Jackson SA, O`Leary ND, Dobson ADW, Folch-Mallol JL (2016) From lignocellulosic metagenomes to lignocellulolytic genes: trends, challenges and future prospects. Biofuels, Bioprod Biorefining 10:864–882. https://doi.org/10.1002/bbb.1709
Bergmann JC, Costa OYA, Gladden JM, Singer S, Heins R, D’haeseleer P, Simmons BA, Quirino BF (2014) Discovery of two novel β-glucosidases from an Amazon soil metagenomic library. FEMS Microbiol Lett 351:147–155. https://doi.org/10.1111/1574-6968.12332
Berini F, Casciello C, Marcone GL, Marinelli F (2017) Metagenomics: novel enzymes from non-culturable microbes. FEMS Microbiol Lett 364:fnx211. https://doi.org/10.1093/femsle/fnx211
Bernal MP, Sánchez-Monedero MA, Paredes C, Roig A (1998) Carbon mineralization from organic wastes at different stages during their incubation with soil. Agric Ecosyst Environ 69:175–189. https://doi.org/10.1016/S0167-8809(98)00106-6
Article CAS Google Scholar
Buchfink B, Xie C, Huson DH (2015) Fast and sensitive protein alignment using DIAMOND. Nat Methods 12:59–60. https://doi.org/10.1038/nmeth.3176
Article PubMed CAS Google Scholar
Devi SG, Fathima AA, Radha S, Arunraj R, Curtis WR, Ramya M (2015) A rapid and economical method for efficient DNA extraction from diverse soils suitable for metagenomic applications. PLoS ONE 10:e0132441. https://doi.org/10.1371/journal.pone.0132441
Article PubMed PubMed Central CAS Google Scholar
Dong D, Yan A, Liu H, Zhang X, Xu Y (2006) Removal of humic substances from soil DNA using aluminium sulfate. J Microbiol Methods 66:217–222. https://doi.org/10.1016/j.mimet.2005.11.010
Article PubMed CAS Google Scholar
Gabor EM, De Vries EJ, Janssen DB (2003) Efficient recovery of environmental DNA for expression cloning by indirect extraction methods. FEMS Microbiol Ecol 44:153–163. https://doi.org/10.1016/S0168-6496(02)00462-2
Article PubMed CAS Google Scholar
Goyal S, Dhull SK, Kapoor KK (2005) Chemical and biological changes during composting of different organic wastes and assessment of compost maturity. Bioresour Technol 96:1584–1591. https://doi.org/10.1016/j.biortech.2004.12.012
Article PubMed CAS Google Scholar
Gudiña EJ, Amorim C, Braga A, Costa Â, Rodrigues JL, Silvério S, Rodrigues LR (2020) Biotech green approaches to unravel the potential of residues into valuable products. In: Inamuddin M, Asiri AM (eds) Sustainable green chemical processes and their allied applications, 1st edn. Springer, Cham, pp 97–150
Chapter Google Scholar
Guerriero G, Hausman J-F, Strauss J, Ertan H, Siddiqui KS (2016) Lignocellulosic biomass: biosynthesis, degradation, and industrial utilization. Eng Life Sci 16:1–16. https://doi.org/10.1002/elsc.201400196
Article CAS Google Scholar
Hahnke RL, Meier-Kolthoff JP, García-López M, Mukherjee S, Huntemann M, Ivanova NN, Woyke T, Kyrpides NC, Klenk HP, Göker M (2016) Genome-based taxonomic classification of Bacteroidetes. Front Microbiol 7:2003. https://doi.org/10.3389/fmicb.2016.02003
Article PubMed PubMed Central Google Scholar
Haldar D, Purkait MK (2020) Lignocellulosic conversion into value-added products: a review. Process Biochem 89:110–133. https://doi.org/10.1016/j.procbio.2019.10.001
Article CAS Google Scholar
Harry M, Gambier B, Bourezgui Y, Garnier-Sillam E (1999) Evaluation of purification procedures for DNA extracted from organic rich samples: interference with humic substances. Analusis 27:439–442. https://doi.org/10.1051/analusis:1999270439
Article CAS Google Scholar
Isikgor FH, Becer CR (2015) Lignocellulosic biomass: a sustainable platform for the production of bio-based chemicals and polymers. Polym Chem 6:4497–4559. https://doi.org/10.1039/c5py00263j
Article CAS Google Scholar
Kalra YP (1995) Determination of pH of soils by different methods: collaborative study. J AOAC Int 78:310–324. https://doi.org/10.1093/jaoac/78.2.310
Article CAS Google Scholar
Lakay FM, Botha A, Prior BA (2007) Comparative analysis of environmental DNA extraction and purification methods from different humic acid-rich soils. J Appl Microbiol 102:265–273. https://doi.org/10.1111/j.1365-2672.2006.03052.x
Article PubMed CAS Google Scholar
LaMontagne MG, Michel FC Jr, Holden PA, Reddy CA (2002) Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis. J Microbiol Methods 49:255–264. https://doi.org/10.1016/S0167-7012(01)00377-3
Article PubMed CAS Google Scholar
Lear G, Dickie I, Banks J, Boyer S, Buckley HL, Buckley TR, Cruickshank R, Dopheide A, Handley KM, Hermans S, Kamke J, Lee CK, MacDiarmid R, Morales SE, Orlovich DA, Smissen R, Wood J, Holdaway R (2018) Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples. N Z J Ecol 42:10–50A. https://doi.org/10.20417/nzjecol.42.9
Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, Li Y, Li S, Shan G, Kristiansen K, Li S, Yang H, Wang J, Wang J (2010) De novo assembly of human genomes with massively parallel short read sequencing. Genome Res 20:265–272. https://doi.org/10.1101/gr.097261.109
Article PubMed PubMed Central CAS Google Scholar
Li W, Godzik A (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22:1658–1659. https://doi.org/10.1093/bioinformatics/btl158
Article CAS PubMed Google Scholar
Li Y, Tan W, Koopal LK, Wang M, Liu F, Norde W (2013) Influence of soil humic and fulvic acid on the activity and stability of lysozyme and urease. Environ Sci Technol 47:5050–5056. https://doi.org/10.1021/es3053027
Article PubMed CAS Google Scholar
Liesack W, Weyland H, Stackebrandt E (1991) Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed-culture of strict barophilic bacteria. Microb Ecol 21:191–198. https://doi.org/10.1007/BF02539153
Article PubMed CAS Google Scholar
Madhavan A, Sindhu R, Parameswaran B, Sukumaran RK, Pandey A (2017) Metagenome analysis : a powerful tool for enzyme bioprospecting. Appl Biochem Biotechnol 183:636–651. https://doi.org/10.1007/s12010-017-2568-3
Article PubMed CAS Google Scholar
Marsh H, Rodríguez-Reinoso F (2006) Activated carbons (origins). Activated carbon, 1st edn. Elsevier Science Ltd, Oxford, pp 13–86
Chapter Google Scholar
Miller DN, Bryant JE, Madsen EL, Ghiorse WC (1999) Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl Environ Microbiol 65:4715–4724. https://doi.org/10.1128/aem.65.11.4715-4724.1999
Article PubMed PubMed Central CAS Google Scholar
Mirete S, Morgante V, González-Pastor JE (2016) Functional metagenomics of extreme environments. Curr Opin Biotechnol 38:143–149. https://doi.org/10.1016/j.copbio.2016.01.017
Article PubMed CAS Google Scholar
Montella S, Amore A, Faraco V (2016) Metagenomics for the development of new biocatalysts to advance lignocellulose saccharification for bioeconomic development. Crit Rev Biotechnol 36:998–1009. https://doi.org/10.3109/07388551.2015.1083939
Article PubMed CAS Google Scholar
Montella S, Ventorino V, Lombard V, Henrissat B, Pepe O, Faraco V (2017) Discovery of genes coding for carbohydrate-active enzyme by metagenomic analysis of lignocellulosic biomasses. Sci Rep 7:42623. https://doi.org/10.1038/srep42623
Article PubMed PubMed Central CAS Google Scholar
Ning P, Yang G, Hu L, Sun J, Shi L, Zhou Y, Wang Z, Yang J (2021) Recent advances in the valorization of plant biomass. Biotechnol Biofuels 14:102. https://doi.org/10.1186/s13068-021-01949-3
Article PubMed PubMed Central Google Scholar
Oh HN, Lee TK, Park JW, No JH, Kim D, Sul WJ (2017) Metagenomic SMRT sequencing-based exploration of novel lignocellulose-degrading capability in wood detritus from Torreya nucifera in Bija Forest on Jeju Island. J Microbiol Biotechnol 27:1670–1680. https://doi.org/10.4014/jmb.1705.05008
Article PubMed CAS Google Scholar
Pang M-F, Abdullah N, Lee C-W, Ng C-C (2008) Isolation of high molecular weight DNA from forest topsoil for metagenomic analysis. Asia Pacific J Mol Biol Biotechnol 16:35–41
Google Scholar
Patel A, Shah AR (2021) Integrated lignocellulosic biorefinery: gateway for production of second generation ethanol and value added products. J Bioresour Bioprod 6:108–128. https://doi.org/10.1016/j.jobab.2021.02.001
Article CAS Google Scholar
Petric I, Philippot L, Abbate C, Bispo A, Chesnot T, Hallin S, Laval K, Lebeau T, Lemanceau P, Leyval C, Lindström K, Pandard P, Romero E, Sarr A, Schloter M, Simonet P, Smalla K, Wilke B-M, Martin-Laurent F (2011) Inter-laboratory evaluation of the ISO standard 11063 “soil quality - method to directly extract DNA from soil samples.” J Microbiol Methods 84:454–460. https://doi.org/10.1016/j.mimet.2011.01.016
Article PubMed CAS Google Scholar
Philippot L, Abbate C, Bispo A, Chesnot T, Hallin S, Lemanceau P, Lindström K, Pandard P, Romero E, Schloter M, Simonet P, Smalla K, Wilke B-M, Petric I, Martin-Laurent F (2010) Soil microbial diversity: an ISO standard for soil DNA extraction. J Soils Sediments 10:1344–1345. https://doi.org/10.1007/s11368-010-0265-8
Article CAS Google Scholar
Rajendhran J, Gunasekaran P (2008) Strategies for accessing soil metagenome for desired applications. Biotechnol Adv 26:576–590. https://doi.org/10.1016/j.biotechadv.2008.08.002
Article PubMed CAS Google Scholar
Saeki K, Ihyo Y, Sakai M, Kunito T (2011) Strong adsorption of DNA molecules on humic acids. Environ Chem Lett 9:505–509. https://doi.org/10.1007/s10311-011-0310-x
Article CAS Google Scholar
Sar A, Pal S, Dam B (2018) Isolation of high molecular weight and humic acid-free metagenomic DNA from lignocellulose-rich samples compatible for direct fosmid cloning. Appl Microbiol Biotechnol 102:6207–6219. https://doi.org/10.1007/s00253-018-9102-6
Article PubMed CAS Google Scholar
Schmeisser C, Steele H, Streit WR (2007) Metagenomics, biotechnology with non-culturable microbes. Appl Microbiol Biotechnol 75:955–962. https://doi.org/10.1007/s00253-007-0945-5
Article PubMed CAS Google Scholar
Sharma S, Sharma KK, Kuhad RC (2014) An efficient and economical method for extraction of DNA amenable to biotechnological manipulations, from diverse soils and sediments. J Appl Microbiol 116:923–933. https://doi.org/10.1111/jam.12420
Article PubMed CAS Google Scholar
Sheldon RA, Woodley JM (2018) Role of biocatalysis in sustainable chemistry. Chem Rev 118:801–838. https://doi.org/10.1021/acs.chemrev.7b00203
Article PubMed CAS Google Scholar
Soares LA, Borges Silva Rabelo CA, Sakamoto IK, Delforno TP, Silva EL, Varesche MBA (2018) Metagenomic analysis and optimization of hydrogen production from sugarcane bagasse. Biomass Bioenerg 117:78–85. https://doi.org/10.1016/j.biombioe.2018.07.018
Article CAS Google Scholar
Sorokin DY, Muntyan MS, Toshchakov SV, Korzhenkov A, Kublanov IV (2018) Phenotypic and genomic properties of a novel deep-lineage haloalkaliphilic member of the phylum Balneolaeota from soda lakes possessing Na⁺-translocating proteorhodopsin. Front Microbiol 9:2672. https://doi.org/10.3389/fmicb.2018.02672
Article PubMed PubMed Central Google Scholar
Tanase A-M, Mereuta I, Chiciudean I, Ionescu R, Milea L, Cornea CP, Vassu T, Stoica I (2015) Comparison of total DNA extraction methods for microbial community form polluted soil. Agric Agric Sci Procedia 6:616–622. https://doi.org/10.1016/j.aaspro.2015.08.102
Article Google Scholar
Tirado-Acevedo O, Chinn MS, Grunden AM (2010) Production of biofuels from synthesis gas using microbial catalysts. Adv Appl Microbiol 70:57–92. https://doi.org/10.1016/S0065-2164(10)70002-2
Article PubMed CAS Google Scholar
Tsai Y, Olson BH (1992) Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl Environ Microbiol 58:2292–2295. https://doi.org/10.1128/aem.58.7.2292-2295.1992
Article PubMed PubMed Central CAS Google Scholar
Verma SK, Singh H, Sharma PC (2017) An improved method suitable for isolation of high-quality metagenomic DNA from diverse soils. 3 Biotech 7:171. https://doi.org/10.1007/s13205-017-0847-x
Wang C, Dong D, Wang H, Müller K, Qin Y, Wang H, Wu W (2016) Metagenomic analysis of microbial consortia enriched from compost: new insights into the role of Actinobacteria in lignocellulose decomposition. Biotechnol Biofuels 9:22. https://doi.org/10.1186/s13068-016-0440-2
Article PubMed PubMed Central CAS Google Scholar
Wang H, Hart DJ, An Y (2019) Functional metagenomic technologies for the discovery of novel enzymes for biomass degradation and biofuel production. BioEnergy Res 12:457–470. https://doi.org/10.1007/s12155-019-10005-w
Article Google Scholar
Wnuk E, Waśko A, Walkiewicz A, Bartmiński P, Bejger R, Mielnik L, Bieganowski A (2020) The effects of humic substances on DNA isolation from soils. PeerJ 8:e9378. https://doi.org/10.7717/peerj.9378
Article PubMed PubMed Central CAS Google Scholar
Wu L, Li F, Deng C, Xu D, Jiang S, Xiong Y (2009) A method for obtaining DNA from compost. Appl Microbiol Biotechnol 84:389–395. https://doi.org/10.1007/s00253-009-2103-8
Article PubMed CAS Google Scholar
Yeates C, Gillings MR, Davison AD, Altavilla N, Veal DA (1998) Methods for microbial DNA extraction from soil for PCR amplification. Biol Proced Online 1:40–47. https://doi.org/10.1251/bpo6
Article PubMed PubMed Central CAS Google Scholar
Young JM, Rawlence NJ, Weyrich LS, Cooper A (2014) Limitations and recommendations for successful DNA extraction from forensic soil samples: a review. Sci Justice 54:238–244. https://doi.org/10.1016/j.scijus.2014.02.006
Article PubMed Google Scholar
Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322. https://doi.org/10.1128/aem.62.2.316-322.1996
Article PubMed PubMed Central CAS Google Scholar
Zhou M, Guo P, Wang T, Gao L, Yin H, Cai C, Gu J, Lü X (2017) Metagenomic mining pectinolytic microbes and enzymes from an apple pomace-adapted compost microbial community. Biotechnol Biofuels 10:198. https://doi.org/10.1186/s13068-017-0885-y
Article PubMed PubMed Central CAS Google Scholar
Zhu N, Yang J, Ji L, Liu J, Yang Y, Yuan H (2016) Metagenomic and metaproteomic analyses of a corn stover-adapted microbial consortium EMSD5 reveal its taxonomic and enzymatic basis for degrading lignocellulose. Biotechnol Biofuels 9:243. https://doi.org/10.1186/s13068-016-0658-z
Article PubMed PubMed Central CAS Google Scholar

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Acknowledgements

AC and JS acknowledge her research grants UMINHO/BPD/37/2018 and UMINHOBIM/2020/28, respectively, under the scope of the project LIGNOZYMES (POCI-01-0145-FEDER-029773). The authors also acknowledge the Portuguese composting units Terra Fértil and Lipor for kindly supplying the compost samples.

Funding

This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the Project LIGNOZYMES—Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-01–0145-FEDER-029773).

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CEB - Centre of Biological Engineering, Universidade Do Minho, 4710-057, Braga, Portugal
Ângela M. A. Costa, Andréia O. Santos, Joana Sousa, Joana L. Rodrigues, Eduardo J. Gudiña, Sara C. Silvério & Ligia R. Rodrigues

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Ângela M. A. Costa
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Andréia O. Santos
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Joana Sousa
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Joana L. Rodrigues
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Eduardo J. Gudiña
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Sara C. Silvério
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Ligia R. Rodrigues
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AC, AS and JS conducted the experiments. SS and EG conceived and designed research. JR, SS and LR analysed data. SS and LR obtained financial support and coordinated the research. AC wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Sara C. Silvério.

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Costa, Â.M.A., Santos, A.O., Sousa, J. et al. Improved method for the extraction of high-quality DNA from lignocellulosic compost samples for metagenomic studies. Appl Microbiol Biotechnol 105, 8881–8893 (2021). https://doi.org/10.1007/s00253-021-11647-7

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Received: 02 July 2021
Revised: 07 October 2021
Accepted: 13 October 2021
Published: 01 November 2021
Issue Date: December 2021
DOI: https://doi.org/10.1007/s00253-021-11647-7

Keywords

Lignocellulosic
Compost
Metagenomic DNA
Humic acids
Activated charcoal

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