Abstract
Cell growth needs to be monitored in biological studies and bioprocess optimization. In special circumstances, such as microbial fermentations in media containing insoluble particles, accurate cell growth quantification is a challenge with current methods. Only the Burton method is applicable in such circumstances. The original Burton method was previously simplified by adopting a two-step sample pretreatment in perchloric acid procedure to eliminate the need for DNA extraction. Here, we further simplified the Burton method by replacing the previous two-step perchloric acid pretreatment with a new and one-step diphenylamine reagent pretreatment. The reliability and accuracy of this simplified method were assessed by measuring the biomass of four model microorganisms: Escherichia coli, Streptomyces clavuligerus, Saccharomyces cerevisiae, and Trichoderma reesei grown in normal media or those containing solid particles. The results demonstrate that this new simplified method performs comparably to the conventional methods, such as OD600 or the previously modified Burton method, and is much more sensitive than the dry weight method. Overall, the new method is simple, reliable, easy to perform, and generally applicable in most circumstances, and it reduces the operation time from more than 12 h (for the previously simplified Burton method) to about 2 h.
Similar content being viewed by others
References
Aidoo KE, Hendry R, Wood BJB (1981) Estimation of fungal growth in a solid state fermentation system. Appl Microbiol Biotechnol 12(1):6–9
Bittner C, Wehnert G, Scheper T (1998) In situ microscopy for on-line determination of biomass. Biotechnol Bioeng 60(1):24–35
Burke D, Dawson D, Stearns T (2000) Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, Plainview
Burton K (1956) A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 62(2):315–323
Burton K (1968) Determination of DNA concentration with diphenylamine. Methods Enzymol 12:163–166
Damiano D, Wang SS (1985) Improvements in ethanol concentration and fermentor ethanol productivity in yeast fermentations using whole soy flour in batch, and continuous recycle systems. Biotechnol Lett 7(2):135–140
Daniels L, Hanson RS, Phillips JA (1994) Chemical analysis. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC, pp 534–535
de Bekker C, van Veluw GJ, Vinck A, Wiebenga LA, Wosten HA (2011) Heterogeneity of Aspergillus niger microcolonies in liquid shaken cultures. Appl Environ Microbiol 77(4):1263–1267
Deriaz RE, Stacey M, Teece EG, Wiggins LF (1949) Deoxy-sugars. Part I. The dische reaction for 2-deoxypentoses. J Chem Soc 265:1222–1232
Findlay RH, King GM, Watling L (1989) Efficacy of phospholipid analysis in determining microbial biomass in sediments. Appl Environ Microbiol 55(11):2888–2893
Gendimenico GJ, Bouquin PL, Tramposch KM (1988) Diphenylamine-colorimetric method for DNA assay: a shortened procedure by incubating samples at 50 degrees C. Anal Biochem 173(1):45–48
Gomez EJP, Martin JF, Hesketh A, Bibb MJ, Liras P (2008) Streptomyces clavuligerus relA-null mutants overproduce clavulanic acid and cephamycin C: negative regulation of secondary metabolism by (p)ppGpp. Microbiology 154(3):744–755
Ito K, Kimizuka A, Okazaki N, Kobayashi S (1989) Mycelial distribution in rice Koji. J Ferment Bioeng 68(1):7–13
Ju NH, Damiano D, Shin CS, Kim NK, Wang SS (1983) Continuous ethanol fermentation of Zymomonas mobilis using soy flour as a protective agent. Biotechnol Lett 5(12):837–842
Kissane JM, Robins E (1958) The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system. J Biol Chem 233(1):184–188
Kleyn JG, Vacano NL (1966) Some effects of soybeans on yeast growth and beer flavor. Brew Dig 41(11):95–99
Kniest FM, Bronswijk JEMH, Schober G, Bouma C (1990) Phosphatases as an index of biotic contamination of dust. Biotechnol Tech 4(4):269–274
Lancker MV, Gheyssens LC (1986) A comparison of four frequently used assays for quantitative determination of DNA. Anal Lett 19(5–6):615–623
Lecault V, Patel N, Thibault J (2009) An image analysis technique to estimate the cell density and biomass concentration of Trichoderma reesei. Lett Appl Microbiol 48(4):402–407
Lehninger AL (1975) Biochemistry. Worth, New York
Lejeune R, Nielsen J, Baron GV (1995) Morphology of Trichoderma reesei QM 9414 in submerged cultures. Biotechnol Bioeng 47(5):609–615
May BA, Vandergheynst JS, Rumsey T (2006) The kinetics of Lagenidium giganteum growth in liquid and solid cultures. J Appl Microbiol 101(4):807–814
Overend WG, Shafizadeh F, Stacey M (1950) Deoxy-sugars. Part IX. Some properties and reactions of 2-deoxy-d-galactose. J Chem Soc 131:671–677
Panda T, Bisaria VS, Ghose TK (1989) Method to estimate growth of Trichoderma reesei and Aspergillus wentii in mixed culture on cellulosic substrates. Appl Environ Microbiol 55(4):1044–1046
Penttila M, Nevalainen H, Ratto M, Salminen E, Knowles J (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61(2):155–164
Perez RR, Rodriguez GA, Martin JF, Liras P (1999) Deletion of the pyc gene blocks clavulanic acid biosynthesis except in glycerol-containing medium: evidence for two different genes in formation of the C3 unit. J Bacteriol 181(22):6922–6928
R Development Core Team (2012) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. Available at http://www.R-project.org
Rahumathulla R, Patel N, Thibault J (2009) Estimation of biomass concentration of Trichoderma reesei in insoluble medium. 8th World Congress of Chemical Engineering Montreal, Canada, pp 23–27
Sambrook J, Russell D (2002) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Santamarta I, Lopez GMT, Perez RR, Koekman B, Martin JF, Liras P (2007) Connecting primary and secondary metabolism: AreB, an IclR-like protein, binds the ARE(ccaR) sequence of S. clavuligerus and modulates leucine biosynthesis and cephamycin C and clavulanic acid production. Mol Microbiol 66(2):511–524
Schaffeld G, Illanes A (1982) Determination of cell mass in an insoluble lignocellulosic substrate in submerged fermentation. Biotechnol Lett 4(10):667–672
Schmidt G, Thannhauser SJ (1945) A method for the determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J Biol Chem 161:83–89
Schneider WC (1945) Phosphorus compounds in animal tissues: extraction and estimation of desoxypentose nucleic acid and of pentose nucleic acid. J Biol Chem 161:293–303
Schneider WC (1946) Phosphorus compounds in animal tissues: a comparison of methods for the estimation of nucleic acids. J Biol Chem 164(2):747–751
Slatkin DN, Lovtrup S (1983) DNA concentrations in the human cerebellum. Computation from kinetics of deoxyribose extraction in hot acid. Acta Chem Scand B 37(4):281–287
Sun Y, Zhou X, Liu J, Bao K, Zhang G, Tu G, Kieser T, Deng Z (2002) ‘Streptomyces nanchangensis’, a producer of the insecticidal polyether antibiotic nanchangmycin and the antiparasitic macrolide meilingmycin, contains multiple polyketide gene clusters. Microbiology 148(2):361–371
Swenson GJ, Hagenbuch IM, Pinckney JL, Long RA (2012) Fluorometric estimation of surface associated microbial abundance. J Microbiol Methods 88(2):297–303
van Veluw GJ, Petrus ML, Gubbens J, de Graaf R, de Jong IP, van Wezel GP, Wosten HA, Claessen D (2012) Analysis of two distinct mycelial populations in liquid-grown Streptomyces cultures using a flow cytometry-based proteomics approach. Appl Microbiol Biotechnol 96(5):1301–1312
Xiang SH, Li J, Yin H, Zheng JT, Yang X, Wang HB, Luo JL, Bai H, Yang KQ (2009) Application of a double-reporter-guided mutant selection method to improve clavulanic acid production in Streptomyces clavuligerus. Metab Eng 11(4–5):310–318
Acknowledgments
This work is supported by the Young Scientists Fund (grant no. 31000025) from the National Natural Science Foundation of China and by the Ministry of Science and Technology of China (grant no. 2009CB118905). We thank Dr. Zhiyang Dong and Yongzhi He (Institute of Microbiology, Chinese Academy of Sciences) for S. cerevisiae INVSc1and T. reesei QM8414.
Author information
Authors and Affiliations
Corresponding author
Additional information
Youbao Zhao and Sihai Xiang contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 34 kb)
Rights and permissions
About this article
Cite this article
Zhao, Y., Xiang, S., Dai, X. et al. A simplified diphenylamine colorimetric method for growth quantification. Appl Microbiol Biotechnol 97, 5069–5077 (2013). https://doi.org/10.1007/s00253-013-4893-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-013-4893-y