1887

Isolation and Characterization of Bacteria that Produce Polyhydroxybutyrate Depolymerases

    Authors: Emily A. Egusa1, Daniel J. Edwards2, MyLo L. Thao3, Larry L. Kirk2, Larry F. Hanne1,*
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    Affiliations: 1: Department of Biological Sciences, California State University, Chico, Chico, CA 95929; 2: Department of Chemistry and Biochemistry, California State University, Chico, Chico, CA 95929; 3: Department of Biological Sciences, California State University, Stanislaus, Turlock, CA 95382
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    Source: J. Microbiol. Biol. Educ. December 2018 vol. 19 no. 3 doi:10.1128/jmbe.v19i3.1682
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    Abstract:

    Isolation of environmental bacterial strains with diverse metabolic properties such as antibiotic production, sulfide oxidation, and the ability to use different nutrient sources is a common undergraduate lab experience. This article describes a useful protocol for the detection and isolation of bacteria that can degrade the common carbon storage biopolymer poly (3-hydroxybutyrate) (PHB). The procedure utilizes a low nutrient media supplemented with insoluble PHB that allows for the easy, visible detection of strains that produce extracellular PHB degrading enzymes. This method can be used to determine the percent of culturable bacteria from various environments that can degrade PHB. Further, PHB degrading exoenzyme activity can be monitored from pure cultures by: (1) Growing cells in broth, (2) inducing with PHB, and (3) measuring activity of supernatant with a UV/Vis spectrophotometer. The enzymatic breakdown of PHB presents an opportunity to expose students to the concept of using biodegradable plastics as a solution to the global plastic waste problem.

References & Citations

1. Anjum A, Zuber M, Zia KM, Noreen A, Anjum MN, Tabasum S 2016 Microbial production of polyhydroxyalkanoates (PHAs) and its copolymers: a review of recent advancements Int J Biol Macromol 89 161 174 10.1016/j.ijbiomac.2016.04.069 27126172 http://dx.doi.org/10.1016/j.ijbiomac.2016.04.069
2. Varsha YM, Savitha R 2011 Overview of polyhydroxyalkanoates: a promising biopol J Microb Biochem Technol 3 5 99 105
3. Shively JM 1974 Inclusion bodies of prokaryotes Annu Rev Microbiol 28 167 188 10.1146/annurev.mi.28.100174.001123 4372937 http://dx.doi.org/10.1146/annurev.mi.28.100174.001123
4. Muhammadi S, Afzal M, Hameed S 2015 Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: production, biocompatibility, biodegradation, physical properties and applications Green Chem Lett Rev 8 56 77 10.1080/17518253.2015.1109715 http://dx.doi.org/10.1080/17518253.2015.1109715
5. Jendrossek D, Knoke I, Habibian RB, Steinbuchel A, Schlegel HG 1993 Degradation of poly(3-hydroxybutyrate), PHB, by bacteria and purification of a novel PHB depolymerase from Comamonas sp J Environ Polym Degr 1 53 63 10.1007/BF01457653 http://dx.doi.org/10.1007/BF01457653
6. Nigam PS 2013 Microbial enzymes with special characteristics for biotechnological applications Biomolecules 3 597 611 10.3390/biom3030597 24970183 4030947 http://dx.doi.org/10.3390/biom3030597
7. Alariya SS, Sethi S, Gupta S, Gupta BL 2013 Amylase activity of a starch degrading bacteria isolated from soil Arch Appl Sci Res 5 1 15 24
8. Sirisha E, Rajasekar N, Narasu ML 2010 Isolation and optimization of lipase producing bacteria from oil contaminated soils Adv Biol Res 4 5 249 252
9. Brown MRW, Scott Foster JH 1970 A simple diagnostic milk medium for Pseudomonas aeruginosa J Clin Pathol 23 172 177 10.1136/jcp.23.2.172 4987392 474488 http://dx.doi.org/10.1136/jcp.23.2.172
10. Rosa DS, Lotto NT, Lopes DR, Guedes CGF 2004 The use of roughness for evaluating the biodegradation of poly-beta-(hydroxybutyrate) and poly-beta-(hydroxybutyrate-co-betavalerate) Polym Test 23 3 8 10.1016/S0142-9418(03)00042-4 http://dx.doi.org/10.1016/S0142-9418(03)00042-4
11. Engohand-Ndong J, Gerbig DG Jr 2013 Making the basic microbiology laboratory an exciting and engaging experience J Microbiol Biol Educ 14 1 125 126 10.1128/jmbe.v14i1.532 http://dx.doi.org/10.1128/jmbe.v14i1.532
12. Maniatis T, Fritsch EF, Sambrook J 1982 Molecular cloning, a laboratory manual 68 Media and Antibiotics Cold Spring Harbor Laboratory Cold Spring Harbor, NY

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2018-12-14
2019-08-19

Abstract:

Isolation of environmental bacterial strains with diverse metabolic properties such as antibiotic production, sulfide oxidation, and the ability to use different nutrient sources is a common undergraduate lab experience. This article describes a useful protocol for the detection and isolation of bacteria that can degrade the common carbon storage biopolymer poly (3-hydroxybutyrate) (PHB). The procedure utilizes a low nutrient media supplemented with insoluble PHB that allows for the easy, visible detection of strains that produce extracellular PHB degrading enzymes. This method can be used to determine the percent of culturable bacteria from various environments that can degrade PHB. Further, PHB degrading exoenzyme activity can be monitored from pure cultures by: (1) Growing cells in broth, (2) inducing with PHB, and (3) measuring activity of supernatant with a UV/Vis spectrophotometer. The enzymatic breakdown of PHB presents an opportunity to expose students to the concept of using biodegradable plastics as a solution to the global plastic waste problem.

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Figures

Image of FIGURE 1

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FIGURE 1

A) Structure of PHB polymer (average = 400). B) 3-hydroxybutyrate monomer. PHB = poly(3-hydroxybutyrate).

Source: J. Microbiol. Biol. Educ. December 2018 vol. 19 no. 3 doi:10.1128/jmbe.v19i3.1682
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Image of FIGURE 2

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FIGURE 2

Clearing around several colonies on PHB/nutrient agar plates. Polymeric PHB is insoluble (cloudy), but clears upon hydrolysis by PHB depolymerase exoenzyme (example colonies at tips of arrows). PHB = poly(3-hydroxybutyrate).

Source: J. Microbiol. Biol. Educ. December 2018 vol. 19 no. 3 doi:10.1128/jmbe.v19i3.1682
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Image of FIGURE 3

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FIGURE 3

Percentage of PHB degraders detected in each of three stages of sewage treatment. Ten-fold dilutions of sample were plated on PHB plates and incubated 4 days at 28ºC, and the percentage of colonies with clear zones of PHB degradation was determined. PHB = poly(3-hydroxybutyrate).

Source: J. Microbiol. Biol. Educ. December 2018 vol. 19 no. 3 doi:10.1128/jmbe.v19i3.1682
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Image of FIGURE 4

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FIGURE 4

A) Photograph of assay cuvettes from supernatants without (left) and with (right) enzyme activity. B) Enzyme activity (decrease in A) of test supernatant following 8-hour incubation with PHB or no PHB (control) for PHB-degrading isolate . PHB = poly(3-hydroxybutyrate).

Source: J. Microbiol. Biol. Educ. December 2018 vol. 19 no. 3 doi:10.1128/jmbe.v19i3.1682
Download as Powerpoint

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