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Group Members:
Lim Zhong Hui
Maung Thet Naing Win
Ong Tiet Ho

Abstract

Alcaligenes eutrophus is a bacterium that accumulates the polymer polyhydroxybutyrate (PHB) as reserve materials under nutritionally imbalanced conditions. Stored PHB acts as a carbon and energy source during starvation. This project examines the biodegradation of PHB using the bacteria Pseudomonas fluorescens and Pseudomonas putida. Both bacteria showed a higher growth rate in the presence of PHB, suggesting that they were able to degrade PHB and utilise it as a carbon source for growth. The piece of plastic buried in soil also showed progressive loss in mass, indicating its degradation by soil microorganisms. Use of biodegradable plastics will reduce the need for landfills and incineration. Used plastics can also be recycled by using them to support growth of beneficial bacteria such as P. fluorescens and P. putida.

Literature Review

In the US, up to 1.3 billion dry tons of cellulosic biomass could be produced per year, which has the energy content of four billion barrels of crude oil. This translates to 65% of American oil consumption. This biomass is made of cellulose which is usually found in newspapers, disposable diapers, shopping bags, fast food containers and what ultimately make up a large amount of wastes in landfills. (Dees, 1999)

To date, efforts directed toward developing means for degrading cellulosic materials using microorganisms have focused on cellulose degrading fungi (e.g., Trichoderma reesei) . However, there are disadvantages to working with fungi. Relative to bacteria, fungi are generally difficult to grow in a fermentor, relatively difficult to manipulate by means of genetic engineering and requires the addition of enzymes to prevent product inhibition of cellulase production.

A diverse spectrum of cellulolytic fungi and bacteria are the major mineralisers in soil and aquatic systems, including landfills. However, many of such microorganisms only release a scant amount of cellulase. Hence, prolific cellulase producers including bacteria of the Pseudomonas genus have received far more attention and research. (Eveleigh, 1987)

The present invention is a method for degrading cellulosic material comprising contacting the cellulosic material with an effective amount of a cellulase produced by a bacterial strain having the characteristics of Pseudomonas cellulosa mutant strain ATCC 202032 comprising within its genome a recombinant vector having a heterologous cellulase gene. Most preferably, the mutant strain produces 7 times, or even as much as 10 times more cellulase than ATCC 55702. Thus, it is crucial that we find out the optimal conditions in which these bacteria survive in order to produce the maximum amount of extracellular cellulase, which is a field that is currently not worked extensively on.

Collaborating with another biological project involving bacterial plastics, there is the possibility that the glucose obtained from the catalysis of cellulose could be used to supply the bacterium Alcaligenes eutrophus which can be grown in several carbon rich but nitrogen deficient sources, thus breaking down otherwise space-occupying cellulose and at the same time providing energy for the synthesis of biodegradable plastics.

Therefore, we would approach this problem by examining 3 life conditions which would affect their production of cellulase. They are the pH, temperature and osmotic pressure of the surroundings. Current studies have shown that with respect to the cellulase produced by ATCC 55703 and ATCC 55702, the operating temperature range is 5-70° C. while it has an operating pH range of 5-11. The preferred temperature and pH is therefore 55° C at pH 7.5. ATCC 55703 and ATCC 55702 have a relatively high salt tolerance, with an operating salt range of 8.5-32 mM sodium, with a preference of 8.5 mM sodium. (Dees, 1999) As we know the proposed optimal values, we would have to find out whether they are by varying the concentrations.

Results would be obtained by observing the area of clear zones around the bacteria grown on 0.1% CMC (carboxyl-methyl cellulose) which is a substitute for cellulose in the laboratory with the concentration of a variable changed. The cellulose in the media was visualized by first staining with Congo Red dye and then destaining with 1M NaCl. The area of clear zone around the bacteria is positively correlated to the amount of cellulase produced, therefore obtaining our desirable results.

Introduction

Common synthetic plastics today are highly non-biodegradable, coupled with reckless dumping and improper waste management, plastic wastes now form up to 80% of all marine debris worldwide. 1 million tons of plastic wastes have been discarded since the 1950s, and only 5.7% of all plastic trash is recycled. These plastic wastes can either be subjected to landfill treatment or incineration, both of which are problematic; the former requires space and global landfills will reach maximum capacity at one point, while the latter produces toxic fumes that may pose an environmental and health issue.

A. eutrophus, a gram-negative rod-shaped eubacterium species, naturally produce PHB, a polymer belonging to a larger group of linear polyesters termed as polyhydroxyalkanoates (PHAs), which are completely biodegradable. It uses PHB for storing carbon in environments with abundant carbon but comparatively limited in other nutrients like phosphorus and nitrogen, with the PHB existing as highly refractive granules (Madison & Huisman, 1999). Some bacteria can produce the extracellular PHB depolymerase enzyme that degrades PHB. Kita et al. (1995) studied the properties of a PHB depolymerase from a marine bacterium, Alcaligenes faecalis. The growth of the bacterium was dependent on PHB degradation. Choi et al. (2004) also studied the enzymatic and non-enzymatic degradation of poly(3HB-co-3HV), a copolymer produced by Alcaligenes sp. MT-16 grown in glucose. If biodegradable bacterial plastics take over the role of non-biodegradable synthetic polymers today, bacteria with PHB- degrading abilities will definitely be heavily relied on. This investigation targets exactly that - the ability of P. fluorescens, P. putida and other soil bacteria to degrade polymers produced by A. eutrophus.

For apparatus and materials, only key items are stated.

Apparatus

Centrifuge Machine
Glass Flasks
Incubator
Incubator Shaker
Laminar Hood
Micropipette
Pipette
Rotary Evaporator
Spectrophotometer
Waring Blender
Weighing Balance

Materials - Bacteria

Alcaligenes eutrophus bacterial strain (ATCC 17699)
Escherichia coli bacterial strain (MM294)
Pseudomonas fluorescens bacterial strain (ATCC 948)
Pseudomonas putida bacterial strain (ATCC 31800)

Materials - Others

Centrifuge Tubes
Chloroform
Cuvettes
Fermentation Medium*
Fresh Soil
Lysogeny Broth (LB)
Methanol
Micropipette Tips
Palm Olein
Peptone
Petri Dishes
Pipette Tips
Soybean Oil
Trypticase Soy Broth (TSB)

* The fermentation medium is made up of:
0.03g of MgSO₄
0.30g of NH₄Cl
0.41g of KH₂PO₄
0.57g of Na₂HPO₄
0.15ml trace minerals
150ml of deionised water

* The trace minerals, on the other hand, consist of:
0.00062g of CrCl₃
0.00118g of NiCl₂
0.00119g of CoCl₂
0.00156g of CuSO₄
0.078g of CaCl₂
0.097g of FeCl₃
10ml of deionised water

Obtaining Plastics from A. eutrophus

Before the other experimental procedures can begin, the PHB polymers have to be acquired from A. eutrophus beforehand. In this process, bacterium A. eutrophus is induced to store readily available carbon from carbon sources, in this case either palm olein or soybean oil, in the form of PHB polymers and it is then lysed before the plastics are precipitated. Yields of plastics from differing carbon sources can later be compared and discussed.

Before the actual fermenting process to induce PHB formation, A. eutrophus is firstly grown in of LB for a day, TSB for 2 days, and blended fermentation medium for 4 days, incubated at 30^oC in an incubator shaker. 2% palm olein or 2% soybean oil is blended using a sterile Waring blender into 150ml of fermentation medium contained in a flask, which is a nutritionally imbalanced medium providing a rich carbon supply but a relatively minimal nitrogen reserve, so that A. eutrophus is stimulated to stockpile this abundant carbon in the form of PHB.

These accumulated plastics are then obtained in the following process. PHB-containing A. eutrophus cell pellets are first centrifuged using a centrifuge machine and then refluxed in chloroform for 3 to 4 hours at 60^oC, before being filtered. The filtrate is then concentrated using a rotary evaporator and the PHB is subsequently precipitated using stirred ice-cold methanol.

Degradation and Utilisation of Plastics in Bacterial Growth

For the investigation of degradation and utilisation of plastic in bacterial growth, the correlation between the presence of PHB polymers in bacterial cultures, which act as carbon sources that can potentially sustain a higher rate of bacterial multiplication, and the bacterial growth rate is examined. For such a purpose, known PHB-degraders P. fluorescens and P. putida, together with experimental control non-PHB-degrader E. coli, are grown in peptone mediums, half of which include PHB pieces, so as to investigate if such a presence of PHB pieces affects bacterial growth and population. Different types of plastics, those produced from fermentation medium with 2% palm olein or that with 2% soybean oil, are also utilised in different tests. Other than having triplicates per experiment, the entire procedure is also carried out thrice for the two varying types of plastics.

Bacterial strains P. fluorescens, P. putida and control E. coli are first cultured in LB for a day. They are then transferred into 18 different 30ml tubes (6 tubes of each bacteria) of nutritionally imbalanced 0.5% peptone medium, which is high in nitrogen but low in carbon, contrary to the fermentation medium used to induce PHB synthesis, with half of the tubes (3 tubes of each bacteria) containing a pre-cut and sterilised PHB piece. With the medium highly lacking in carbon, bacterial cells are forced into degrading any PHB present so as to utilise it as a carbon source for higher growth rate. The cultures are then incubated in an incubator shaker for a week at 30^oC. The absorbance of the tubes, which is proportional to bacterial growth and density, are subsequently taken with a spectrophotometer on days 1 and 7, at 600nm using 0.5% peptone as blank. Absorbance values over the days are then compared, so as to check for any change in bacterial population and difference in bacterial multiplication rate between the cultures with PHB and those without.

Biodegradation of Plastic Pieces in Fresh Garden Soil

Lastly, the biodegradation of plastic pieces in fresh garden soil is examined. By completely covering PHB polymer pieces in fresh garden soil and thus exposing them to common soil microorganisms, and periodically taking their masses, a conclusion of whether the presence of such microorganisms will lead to biodegradation these polymers can be drawn. Similarly, different plastic types are used in this investigation, with duplicates being conducted in each of the three experiments carried out.

Pre-cut, pre-weighed and sterilised pieces of PHB are buried in 30g of either fresh garden soil. These plastic pieces are later removed from the soil after 7 days, cleaned, weighed, sterilised and re-buried into 30g of either fresh garden soil or autoclaved soil respectively. The cycle then goes on for four weeks, and the trends of the mass of separate PHB pieces are observed so as to investigate if the presence of soil microorganisms will lead to any biodegradation and hence decrease in mass of PHB fragments.

Obtaining Plastics from A. eutrophus

PHB was successfully yielded from the above experimental protocol using a nutritionally imbalanced fermentation medium blended with either 2% palm olein or 2% soybean oil. For A. eutrophus cultured in 2% palm olein, the average plastic mass isolated was 0.140g, accounting for an average of 7.592% of the cell mass which weighed 1.924g. Correspondingly for that in fermentation medium blended with 2% soybean oil, 0.070g of PHB was obtained from 1.087g of cells on the average, with the plastic making up a smaller 6.234% of the total cell mass. Hence on the average, higher yields of plastic can be obtained when the cells were cultured in fermentation medium with 2% palm olein, as compared to that grown in FM with 2% soybean oil.

Degradation and Utilisation of Plastics in Bacterial Growth

For the investigation of degradation and utilisation of plastic in bacterial growth, the average absorbance values taken after a week of incubation is as follows. The average absorbance of P. fluorescens grown in 0.5% peptone medium with polymers from 2% palm olein was 0.490, whereas its 2% soybean oil counterpart gave a reading of 0.642, both of which are higher than when no PHB was included, which gave averages of 0.305 and 0.307 in the two different control sets.

Next for P. putida, cells cultured in peptone medium with polymers from 2% palm olein gave an average absorbance value of 0.570, those with plastics from 2% soybean oil showed an absorbance of 0.453, with that grown in 0.5% peptone without any PHB showing a smaller 0.272 and 0.279 in the two different control sets. These can be compared to the values obtained from the E. coli cultures which were the controls, giving averages of 0.534 for PHB from palm olein, 0.734 for PHB from soybean oil, and 0.548 and 0.635 for that without any PHB, apparently displaying no clear trend.

There are apparent differences and trends in absorbance values obtained from the P. fluorescens and P. putida cultures, with those with PHB pieces included, be it from 2% palm olein or 2% soybean oil, giving higher absorbance values. It is thus evident that PHB acted as a carbon source that sustained a higher growth rate for these bacteria that secrete PHB-degrading enzymes. This can be compared to the E. coli values which are erratic and possess no general trend, despite there being a difference in averages. A T-test p value of 0.026 indicates that P. fluorescens displayed a significantly higher growth rate when cultured with PHB from soybean oil than without any PHB.

Biodegradation of Plastic Pieces in Fresh Garden Soil

The mass of buried plastic pieces in fresh garden soil are as follows.

From an average of 0.0650g, the PHB pieces from palm olein decreased in mass to 0.0640g in the first week, and then to average values of 0.0563g, 0.0539g, 0.0519g and 0.0375g over the following three weeks.

PHB polymers obtained from 2% soybean oil also displayed an apparent decrease in mass, from an average of 0.019g initially to 0.0170g, 0.0130g and then to 0.0070 over the subsequent weeks.

Despite significantly large error bars caused by fluctuating values, partially caused by the tearing and ripping of delicate plastic pieces in the cleaning process which inevitably led to misplacement of some small plastic portions, there is an obvious trend of plummeting PHB mass, be it those harnessed from FM with palm olein or those garnered from FM with soybean oil. Degradation of PHB by soil microorganisms is thus apparent.

Conclusions

As discussed above, there is sufficient evidence that the presence of PHB promoted higher growth rates of PHB-degrading bacteria P. fluorescens and P. putida, hence signifying that there indeed has been utilisation of PHB as a carbon source for bacterial growth. There is also apparent gradual decrease of PHB mass over a few weeks in the presence of soil microorganisms, showing that there has been degradation of PHB by certain soil microorganisms. However, there are existing limitations, firstly being that the decrease in PHB mass may not be significant enough, and secondly, that the biodegradation of PHB pieces require long periods of time.

Applications

With proof that PHB is indeed biodegradable, one can safely say that they provide a superior environmentally-friendly alternative to synthetic plastics today, which are highly non- biodegradable. If PHB is one day integrated into present day plastic products, it will indeed be advantageous as subjecting PHB wastes to P. fluorescens and P. putida treatment can potentially have multiple benefits.

Firstly, this will alleviate stress on landfill demand as plastic wastes that once occupy much space can now be biodegraded, and secondly, there will not be that much a need for plastic incineration.

Secondly, since the introduction of PHB into peptone medium can promote higher growth rates of P. fluorescens and P. putida, such PHB waste treatment can also be a means of culturing these useful bacteria, with PHB acting as a carbon source which these bacteria can utilise for growth. The uses of P. fluorescens include the production of the antibiotic muprocin for the treatment of skin, ear and eye disorders, while that of P. putida include bioremediation, or the ability to clean up oil spills.

Future Work

The degradation of PHB polymers using a mixture of Pseudomonas genus bacterial species can be further investigated, and these effects can then be compared to when only a single bacteria of the genus, for example purely P. fluorescens or purely P. putida, is utilised for the degradation of these plastics. The most effective and time-efficient mixture can thus be derived at, so that such a plastic degradation method can be optimised and made more productive.

References

Choi, G.G., Kim, H.W. and Rhee, Y.H. (2004). Enzymatic and non-enzymatic degradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolyesters produced by Alcaligenes sp. MT-16. The Journal of Microbiology, 42(4), 346-352.

Eismann, K., Mlejnek, K., Zipprich, D., Hoppert, M., Gerberding, H. and Mayer, F. (1995). Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm. Journal of Bacteriology, 177, 6309-6312.
Retrieved April 1, 2010 from http://jb.asm.org/cgi/reprint/177/21/6309.pdf

Kim, H.-Y., Park, J.-S., Shin, H.-D. and Lee, Y.-H. (1995). Isolation of a glucose utilizing mutant of Alcaligenes eutrophus, its substrate selectivity, and accumulation of poly-b- hydoxybutyrate. Journal of Microbiology, 33, 51 – 58.

Kita, K., Ishimaru, K., Teraoka, M., Yanase, H. and Kato, N. (1995) Properties of poly(3-hydroxybutyrate) depolymerise form a marine bacterium, Alcaligenes faecalis AE122. Applied and Environmental Microbiology, 61, 1727 – 1730.

Ojumu, T. V., Yu, J. and Solomon, B. O. (2004). Production of polyhydroxyalkanoates, a bacterial biodegradable polymer. African Journal of Biotechnology, 3, 18-24.
Retrieved April 1, 2010 from http://www.academicjournals.org/AJB/PDF/Pdf2004/JanPDFs2004/Ojumu%20et%20al.pdf

Paustian, T. (1998) Bacterial plastics.
Retrieved April 1, 2010 from http://www.bact.wisc.edu/Microtextbook/index.php?name=Sections&req=viewarticle&artid=155&page=1

Dees, C. H. (1999). Genetically Enhanced Cellulase Production in Pseudomonas Cellulosa Using Recombinant DNA Technology.
Available online at: http://www.freepatentsonline.com/5958740.html
[Last accessed 30.01.10]

Eveleigh, D. E. (1987). Philosophical Transactions of the Royal Society of London. Series A, Mathematical and Physical Sciences. Cellulase: A Perspective, 321, 435-447.
Available online at: http://www.jstor.org.libproxy.nlb.gov.sg/stable/37790
[Last accessed 30.01.10]

http://en.wikipedia.org/wiki/Alcaligenes_eutrophus
http://en.wikipedia.org/wiki/Polyhydroxyalkanoates
http://en.wikipedia.org/wiki/Polyhydroxybutyrate
http://web.mst.edu/~microbio/BIO221_2005/A_eutrophus.htm

Pictures

http://www.topnews.in/files/bacteria-456.jpg
http://www.racc.edu/Faculty/mkelly/PSCS2/Unit3Basic_TurnUpHeat/Open%20book.jpg http://www.freelancelife.co.za/uploads/blog/brown%20pen%20paper.jpg
http://www.gettyimages.com/detail/83911228/Photographers-Choice-RF
http://www.hku.hk/biochem/undergraduate/mbbs/images/Practical_pipette.jpg
http://www.eaas.co.uk/images/logbook/Jan17thLog%20Book.JPG
http://www.notaries-r-us.com/000801_0357_0197_tsls_pen_paper_184x125.jpg
http://churchwhisperer.files.wordpress.com/2008/09/after-much-discussion.jpg
http://www.wired.com/images_blogs/photos/uncategorized/2008/10/28/081028_library_books.jpg
http://media.bigoo.ws/content/background/color_multicolor/color_multicolor_67.jpg

Last but not least, we would like to thank our mentor for her continuous support throughout the course of the project.