Bacterial Cellulose

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Biodegradability of poly-3-hydroxybutyrate/bacterial cellulose composites under aerobic conditions - measured via evolution of carbon dioxide, spectroscopic and diffraction methods Dianne R Ruka, Parveen Sangwan, Christopher James Garvey, George P. Simon, and Katherine M Dean Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5044485 • Publication Date (Web): 12 Mar 2015 Downloaded from http://pubs.acs.org on May 3, 2015

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Environmental Science & Technology

PHB/bacterial cellulose (BC)

Poly(hydroxybutyrate) (PHB)

Fungal growth

Early stages of aerobic biodegradation of bacterial cellulose ( )

ACS Paragon Plus Environment


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Biodegradability of poly-3-

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hydroxybutyrate/bacterial cellulose composites

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under aerobic conditions - measured via evolution

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of carbon dioxide, spectroscopic and diffraction

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methods

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Dianne R. Ruka1, 2, Parveen Sangwan1, Christopher J. Garvey3, George P. Simon2, Katherine

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M. Dean 2,4.

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1

CSIRO Materials Science & Engineering, Gate 5 Normanby Road, Clayton 3168 Australia, 2

9 10 11

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Department of Materials Engineering, Monash University, Clayton 3800 Australia,

Australian Nuclear Science and Technology Organisation, Kirrawee DC 2232, Australia, 4

D3 Consulting Group Pty Ltd, Fitzroy North, 3068 Australia.

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[email protected], [email protected], [email protected],

13

[email protected], [email protected]

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*CORRESPONDING AUTHOR: Katherine Dean: Email [email protected], Tel:

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(+61) 437 254 462

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ABSTRACT Poly-3-hydroxybutyrate (PHB) and bacterial cellulose (BC) are both natural

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polymeric materials that have the potential to replace traditional, non-renewable polymers. In


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particular, the nanofibrillar form of bacterial cellulose makes it an effective reinforcement for

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PHB. Neat PHB, bacterial cellulose and a composite of PHB/BC produced with 10 wt%

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cellulose were composted under accelerated aerobic test conditions, with biodegradability

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measured by the carbon dioxide evolution method, in conjunction with spectroscopic and

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diffraction methods to assess crystallinity changes during the biodegradation process. The

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PHB/BC composite biodegraded at a greater rate and extent than that of PHB alone, reaching

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80% degradation after 30 days, whereas PHB did not reach this level of degradation until

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close to 50 days of composting. The relative crystallinity of PHB and PHB in the PHB/BC

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composite was found to increase in the initial weeks of degradation, with degradation

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occurring primarily in the amorphous region of the material and some recrystallisation of the

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amorphous PHB. Small angle X-ray scattering indicates that the change in PHB crystallinity

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is accompanied by a change in morphology of semi-crystalline lamellae. The increased rate

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of biodegradability suggests that these materials could be applicable to single-use

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applications, and could rapidly biodegrade in compost on disposal.

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Keywords Bacterial cellulose, poly-3-hydroxybutyrate, biodegradability, solution blending

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INTRODUCTION

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An increasing awareness of environmental issues relating to the production and disposal of

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traditional polymers has brought natural materials into the spotlight. Ideally, natural

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biodegradable materials may be developed for packaging material, as this comprises a

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significant proportion of the plastics market and degradability would be desirable as a waste-

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reduction option, as well as being advantageous if the packing becomes litter and is carried

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out to sea. These types of materials would be quite stable on the shelf, but could be degraded

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in compost or other environments after disposal. For these applications it is critical to



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investigate how long degradation would take in conditions that are relevant to real world

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situations.

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Poly-3-hydroxybutyrate (PHB) is a material that could replace traditional plastics, as it has 1, 2

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similar key properties to polypropylene

. Typically the morphology PHB is organised

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hierarchically, with two important length-scales: alternating layers of crystalline and

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amorphous polymer organised in partially ordered lamellae (of the order nm), for the lamellar

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are then organised into much larger spherulites. The size of both structures are highly

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dependent on the processing conditions3, 4. By comparison, bacterial cellulose possesses a

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high crystallinity and modulus5, and it is composed of nanosized fibrils. Given this

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morphology, it has potential to be the reinforcing phase in composites, including those where

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the matrix is itself biodegradable. Since both these materials are produced by soil bacteria,

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they can be used as nutrients by microbes present in the natural environment and completely

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broken down into carbon dioxide and water. Blends of these materials are expected to be

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completely mineralised when exposed to natural microbial flora, but this needs to be

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demonstrated experimentally.

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There are a number of different methods used to determine the degree and rate at which

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materials biodegrade6. Samples can be exposed to degrading enzymes or microbes in

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laboratory culture, or biodegraded by being submerged in soil, activated sludge or compost.

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Numerous metrics can be obtained from such experiments to quantify and assess the rate and

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extent of biodegradation. The loss of weight of materials is commonly used, however this

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method can be problematic, as degrading materials can absorb moisture which alters their

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weight, or the materials could disintegrate into smaller fragments, causing difficulty in

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recovering the materials to determine the weight loss. The materials can also be examined for

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mechanical properties before and after degradation, but this too does not give a direct

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measure of the biodegradation or the rate of biodegradation, but rather simply provides


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evidence that degradation has taken place. The measurement of carbon dioxide production or

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oxygen consumption under aerobic conditions, however, provides a quantitative measure of

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degradation. The use of such respirometric data can allow the calculation of the degree and

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rate of biodegradation during aerobic composting process itself, which is very convenient.

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There have been several reports of the biodegradation of composites that contained

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bacterial cellulose7-9. A starch/BC composite had degradation measured by determining the

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weight loss after soil burial and it was found that composites achieved 69% degradation

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within 3 weeks of burial7. Bacterial cellulose contents and choice of culture medium were

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found to influence the degradation of poly(vinyl alcohol) (PVA)/BC composites when

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exposed to a single fungal strain in laboratory culture however in this study biodegradation

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was only studied qualitatively and no overall measurement of biodegradation was made9.

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The degradation of PHB has been reported in the literature, with highly variable results.

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For example, 0.3 – 0.4 mm films of PHB were found to degrade in compost in 30 days10,

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whereas injection-moulded dogbones were found to retain 96% of its initial weight after 151

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days in compost11. It has been found that the degradation rate of PHB composites is

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dependent of the type of material in the blends10.

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It must be noted that with most of these previous studies weight loss has been the primary

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method for measuring biodegradation, but as discussed previously this method can be

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problematic due to moisture absorption and difficultly recovering the material to determine

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the weight loss. One of the only papers we have located which uses respirometric methods to

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measure biodegradation of polyhydroxyalkanoates is that by Arcos-Hernandeza et al12

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There are a number of factors that can affect the rate of degradation. For example, nutrient

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supply, temperature, pH and moisture level can all change the rate of degradation, as can the

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microbial population and the surface area of the polymer13, which need to be kept consistent

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when comparing the degradation rate between samples. When a composite degrades, the rate



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of degradation will initially be influenced by the most biodegradable phase, which will come

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under microbial attack first. As a result, the composite will break down, providing more

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exposed surface area for the remaining biodegradable component of the composite to come

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under microbial attack6.

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Based on the potential for PHB and bacterial cellulose to be used in biodegradable

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composites, we produced composites of these two materials and measured the biodegradation

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using the precise method of evolution of carbon dioxide in conjunction with spectroscopic

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and diffraction methods to understand the changes in morphology occurring during the

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biodegradation process.

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EXPERIMENTAL

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Bacterial Strain

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A culture of bacterial cellulose-producing Gluconacetobacter xylinus ATCC 53524 was

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kindly provided by Mike Gidley, University of Queensland, Australia.

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Materials

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PHB (Tradename Enmat Y3000P, molecular weight 450,000 g/mol) was purchased from

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TianAn Biologic Materials Co. We produced bacterial cellulose in Yamanaka-mannitol

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media14 consisting of 5 wt% mannitol, 0.5 wt% yeast extract, 0.5 wt% (NH4)2SO4, 0.3 wt%

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KH2PO4 and 0.005 wt% MgSO4.7H2O, with the pH of media adjusted to 5.0 with HCl or

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NaOH and autoclaved at 121°C for 20 minutes. Cultures were grown in 600 mL media and

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were incubated for 7 days at 28° C under static conditions. Following incubation periods,

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pellicles were removed, rinsed to remove any residual media, and washed with 0.1 M NaOH

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at 80° C for 1 hour to remove bacterial cells. They were further repeatedly washed until a

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neutral pH was achieved, and the pellicles were dried at room temperature to constant weight.

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The pellicles were subsequently cryogenically ground to a fine powder (15±5µm) using a

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SPEX SamplePrep Freezer/mill 6870.


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Solution Blending and Casting

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Bacterial cellulose powder was sonicated in chloroform at room temperature for 60

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minutes. PHB was added to the cellulose/chloroform solution at 5 wt% and dissolved by

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mechanical stirring at 80° C for 3 hours. The blend was briefly sonicated, cast in glass petri

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dishes and stored at room temperature to allow the solvent to evaporate, producing films with

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thicknesses of approximately 20 µm.

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Biodegradation

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Films of neat PHB and PHB/BC with a bacterial cellulose content of 10 wt% were

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produced for biodegradation studies. Experiments were set up according to the Australian

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Standard AS 14855, using certified biodegradation facilities at CSIRO. The test specimens

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were cut into 2 × 2 cm2 pieces and were exposed to aerobic composting conditions with a

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continuous supply of airflow provided to each composting vessel; temperature of 58±2° C

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and moisture 50 – 55% using an in-house built respirometer unit. Each 3 L bioreactor vessel

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contained 300 g compost (sourced from Natural Recovery Systems, Bangholme Victoria) and

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50 g of the respective material, both on a dry weight equivalent. Microcrystalline cellulose

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powder (MCC) (20 microns, Sigma-Aldrich) was used as a positive reference during the

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biodegradation tests, and blank compost was kept in the same conditions to determine the

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amount of carbon dioxide given off by the compost alone. Neat bacterial cellulose powder

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was also examined in this way. The compost was examined in triplicate, with the films

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examined in duplicate and one vessel of neat bacterial cellulose examined, due to limited

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amounts of material.

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The theoretical amount of carbon dioxide (THCO2), in grams per bioreactor, was calculated by: ସସ

THCOଶ = M୘୓୘ × C୘୓୘ × ଵଶ

(1)



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where, MTOT is the total dry solids (in grams) in the test material at the start of the test;

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CTOT is the proportion of total organic carbon in the total dry solids of the test material (in

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grams per 100 grams); 44 and 12 are the molecular mass of carbon dioxide and the atomic

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mass of carbon, respectively.

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Percentage biodegradation (Dt ) was determined by:

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‫ܦ‬௧ =

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where, (CO2)T is the cumulative amount of carbon dioxide evolved in each bioreactor

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containing test material (in grams per bioreactor); (CO2)B is the mean cumulative amount of

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carbon dioxide evolved in the blank vessel (in grams per bioreactor).

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(େ୓మ)౐ ି(େ୓మ )ా ୘ୌେ୓మ

× 100 (2)

Following this, the cumulative amount of carbon dioxide evolved as a function of time was determined and graphed, with the test terminated after 60 days.

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Scanning Electron Microscopy

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Scanning electron microscopy (SEM) was performed under high vacuum using a field

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emission Philips XL30, with samples coated with iridium and viewed at an accelerating

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voltage of 5 kV.

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Fourier Transform – Infra Red

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Fourier transform – infra red (FTIR) spectroscopy was performed using a Perkin-Elmer

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Spectrum 100 Spectrometer with an Attenuated Total Reflectance (ATR) cell. Film samples

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were used for the FTIR measurements. Film samples were either the baseline PHB, PHB/BC

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or the fragments of the degraded samples removed from the compost (composted samples

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were washed in distilled water to remove compost materials and air-dried overnight prior

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scanning). Scans ranged between 4000 and 450 cm-1 wavenumbers (64 scans). Baselines

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were subtracted using the Spectrum software.

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X-ray Diffractometry


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A Bruker D8 Advance X-ray Diffractometer with CuKα radiation (40 kV, 40 mA)

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equipped with a LynxEye silicon strip detector was used for X-ray diffractometry (XRD).

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Each sample was scanned over the 2θ range 2° to 40° with a step size of 0.02 and a count

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time of 0.8 seconds per step.

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The samples were washed in distilled water to remove compost materials and air-dried overnight.

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Small Angle X-ray Scattering

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Radial symmetric transmission small angle X-ray scattering (SAXS) patterns were acquired

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on the two-dimensional detector of a Bruker Nanostar operating on a rotating anode CuKα

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alpha source for an acquisition time of 2 hours. Operational details of the instrument are

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found

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(http://www.ansto.gov.au/ResearchHub/Bragg/Facilities/Instruments/SAXS/BrukerSAXS/ind

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ex.htm). The patterns were converted to the 1-d intensity versus q, the scattering vector

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defined by:

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q=

at

4π

λ

( 2)

the

website:

sin θ

(3)

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where λ is the wavelength of the incident radiation (1.54 Å) and is the scattering angle

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using the program Fit2D 15and the scattering geometry. The scattering geometry is defined by

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a sample to detector distance of 72.35 cm and a detector consisting of 2048 × 2048 pixels 68

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µm2. Pixels around the beam stop and at the detector edges were masked out of the

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calculation to give an effective q-range of 0.01 to 0.45 Å-1.

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RESULTS AND DISCUSSION

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Observations of Films Degraded Over Time

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Neat PHB films and bacterial cellulose powder, along with PHB/BC blends, were

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incubated in the compost to determine their biodegradation time, and if the inclusion of the



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bacterial cellulose phase in the composite would increase or decrease degradation time.

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Biodegradation was determined by quantifying carbon dioxide produced during the

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experiment, compared to the total carbon content of materials and blank compost.

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After 1 week of composting, PHB and PHB/BC films were observed to still be intact,

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whereas the compost in the bioreactor containing the neat bacterial cellulose powder was

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covered with white mycelium indicating fungal growth (not shown).

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After 2 weeks of biodegradation, it was apparent (by visual observation) that the PHB/BC

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films were breaking down more rapidly than the neat PHB films (Figure 1). At 3 weeks,

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much of the neat PHB films remained as large pieces; however there were only a few of the

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large pieces of the PHB/BC blend visible, as most of them had disintegrated into extremely

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small fragments. This was also apparent at 4 weeks, when there was very little visible film of

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PHB/BC composite left, whereas relatively large PHB film pieces persisted, along with many

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smaller fragments. After 6 weeks, most of the PHB films had broken down, still leaving a

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few large, visible pieces (Figure 1).


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Figure 1. Degradation of PHB and PHB/BC films in compost over time with large pieces of

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film indicated by arrows

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Film samples were removed periodically from the compost for examination by SEM. The

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films degraded over time showing distinct holes and cracks in the films (Figure 2). The

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PHB/BC showed degradation at 1 week, with many small holes and cracks, whereas this was

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not seen in the PHB film until the second week of degradation.


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Figure 2. SEM of PHB and PHB/BC films during aerobic composting

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At 4 weeks, the PHB/BC films had degraded to an extent that a sample could not be

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collected for SEM, and the PHB film continued to degrade further (data not shown). This



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further demonstrates the increased rate of biodegradation in the films containing the bacterial

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cellulose phase.

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Aerobic Biodegradation

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The amount of CO2 produced from the bioreactors containing blank compost and compost

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with test samples was determined and values were expressed as means, with standard

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deviations, calculated from the triplicate compost and duplicate PHB and PHB/BC films

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tested (Figure 3). The cumulative CO2 produced by the blank samples was relatively low and

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increased gradually during the test. In comparison, compost with test samples produced

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higher cumulative CO2 levels during the initial 10 – 20 days (Figure 3). These results indicate

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that the test materials in the compost were actively metabolised by the microbes present,

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without a lag phase, producing high amounts of CO2 from the time that they were first

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exposed to the compost16. Moreover, neat bacterial cellulose produced lower cumulative CO2,

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when compared to the microcrystalline cellulose, however its rate of CO2 evolution was more

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rapid by comparison than MCC in the initial days of composting. The PHB/BC film produced

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similar amounts of CO2 as the neat microcrystalline cellulose during initial 10 days, thereafter

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the PHB/BC film produced significantly higher amounts of CO2 as compared to MCC. PHB

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films also produced higher cumulative CO2 overall as compared to MCC, however the values

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were relatively lower than PHB/BC (Figure 3).

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Figure 3. Cumulative levels of carbon dioxide evolved from bioreactors during composting

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The percentage biodegradation determined from the cumulative carbon dioxide is shown in

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Figure 4, with the gradient of each curve at a given time relating to the rate at which the

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samples degraded. The bacterial cellulose degraded at a very fast rate, indicating that its

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presence in biodegradable composites may encourage overall degradation to occur rapidly.

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This was confirmed with the PHB/BC films experiencing a faster degradation than PHB

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alone, as seen by an increase in the initial gradient, supporting the visual observations made

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of the films in the compost (Figure 1). In addition to their rapid biodegradation, the inclusion

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of bacterial cellulose fibres within a PHB matrix could also act as nucleation sites for

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biodegradation of the PHB itself, in effect opening up the structure and exposing more PHB

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surface to the biodegradation process.



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Figure 4. Calculated degree of biodegradation of test materials during aerobic composting

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Full degradation was difficult to reach in this experiment, as a certain amount of carbon

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will be converted into microbial biomass and other natural products, as well as into the

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compost itself6. However, it is apparent from the plateau in the production of CO2 (Figure 3)

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and the plateau in the degree of biodegradation shown here (Figure 4) that degradation, and

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subsequently CO2 production, was essentially complete.

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Due to the fast rate of degradation of neat bacterial cellulose and the potential for bacterial

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cellulose to act as an initiation site for biodegradation, the PHB/BC films degraded faster

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than the PHB films, due to the cellulose phase being the first point of microbial attack and

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creating greater surface area for attack on the PHB phase. PHB is stable in air, and is resistant

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to moisture, requiring exposure to specific environmental conditions and microbes for


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degradation17. Its capacity to degrade can be further enhanced if used in a blend with bacterial

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cellulose.

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Structural Examination of Degraded Films

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Samples were taken at weekly intervals and examined by FTIR. Analysis revealed the

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typical peaks of PHB, including peaks in the 1724 cm-1 region (C=O bonding) and peaks at

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1185 cm-1, ( C ̶ O ̶ C bonding) (Figure 5)18. Bacterial cellulose peaks could not be resolved

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due similarities in peak position with PHB.

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Figure 5. FTIR curves given by degrading PHB and PHB/BC films

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The 1185 cm-1 peak, representative of crystalline PHB, was found to increase as

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degradation occurred. Peaks present at 1185, 1228 and 1279 cm-1 are representative of

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crystallinity, with the 1185 cm-1 peak previously used to determine the crystallinity index of

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PHB18. The crystallinity index was therefore calculated as a result of the ratio of the peak at

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this position, and the peak at 1385 cm-1, which is known to be insensitive to the degree of

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crystallinity18. These crystallinity indexes are presented in Table 1. A crystallinity index of

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0.40 and 0.39 was determined for the PHB and PHB/BC, respectively, prior to degradation.

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Following degradation, the crystallinity index increased up to 0.58 and 0.60 at 2 weeks

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biodegradation for the neat material and the composite, respectively. Beyond 2 weeks the

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samples continued to degrade, the PHB showing a decrease in crystallinity and no data being

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available for the PHB/BC as it was degraded beyond the point of sampling at this time. Some

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variability in sampling is inevitable using this method due to variation in compost coverage

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across the sample. To minimise this effect, large sample collection and multiple sampling

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was used. The crystallinity indexes obtained here suggest that PHB and PHB in the PHB/BC

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blend become more crystalline as they degrade. This indicates that the amorphous region of

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the material is degraded first, only achieving a loss in crystallinity after significant

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degradation has taken place.

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Table 1. Crystallinity index values given from FTIR data for degraded films Film

Composting Time

Crystallin

Film

ity Index

Time

(weeks) PHB

0

Composting

Crystallin ity Index

(weeks) 0.40

PHB/

0

BC


0.39


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1

0.45

1

0.41

2

0.58

2

0.60

3

0.40

N/A

-

*two repeats of each FTIR scan were undertaken with standard deviation less than 0.05 for each sample.

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XRD patterns of semi-crystalline polymers are a linear sum of the contributions from

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amorphous and crystalline regions19. In the limit where instrumental broadening is small the

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peak widths are inversely proportional to crystallite dimensions in the direction normal to the

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crystallographic axis20. All XRD patterns obtained from the samples in this study consisted of

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relatively broad diffraction peaks superimposed on the broad halo of the amorphous PHB

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(Figure 6). The XRD of the degrading films revealed a clear increase in crystallinity with

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degradation of the PHB film, demonstrated by the relative increase in the crystalline (020)

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peak (2θ = 13.5°) (Figure 6). Increases in relative intensity of all major crystalline peaks were

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observed up to 2 weeks composting time. It also appears that some of the diffraction peaks,

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particularly the (020) which is clearly resolved, become sharper. Given that the peak width is

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inversely proportional to the width of the peak at half height20 this change in shape is

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indicative of some recrystallization process. As the time of composting increases, the

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contribution of the amorphous phase to the scattering pattern again increases.


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Figure 6. XRD peaks given by PHB and PHB/BC films with varying times and degrees of

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degradation



309 310

Major peaks for PHB ((110), (021), (121) and (200)) were observed and typically increased

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in relative intensity in the initial period of biodegradation. For the PHB/BC film, a similar

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highly resolved peak was observed, at 2θ = 13.5°, corresponding to the (020) peak of PHB. In

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the PHB/BC film an additional peak at 2θ = 23° was observed corresponding to the (200)

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peak of bacterial cellulose I14. This peak also increased in intensity throughout the

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biodegradation process. This possibly reflective of the relative resistance of the bacterial

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cellulose to degradation, although it must be noted that the interface between the bacterial

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cellulose and PHB can increase biodegradation as it acts as an initiation site for microbial

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attack. As amorphous PHB is degraded, bacterial cellulose becomes a higher fraction of the

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remaining material.

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The broad nature of the reflections affected the quantitative measurement of crystallinity

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from the XRD, due in part to the crystalline peak width variation during biodegradation, with

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increases of 0.13 and 0.08º 2θ in width of half maximum for the (002) peak being observed in

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PHB and PHB/BC respectively. Owen et al.21 have discussed peak broadening in

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crystallisation and melting behaviour studies of PHB where they observed increased

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crystalline peak resolution and peak broadening and suggested that this is due to a larger

326

crystallite size. We observe a similar effect in the early stages of biodegradation of PHB and

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PHB/BC. Despite the issues with XRD measurements, a qualitative increase in crystallinity

328

could be observed (see Figure 6). Thus both FTIR (quantitatively) and XRD (qualitatively)

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clearly show an increase in crystallinity; this is similar to increases in crystallinity previously

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observed during the biodegradation of poly(lactic acid) (PLA)-lignocellulose composite films

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22

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only be due to the preferential hydrolysis of amorphous regions, but also the ability of

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polymer chain alignment to occur from composting at high temperatures and loss in

. Way et al.22 proposed that this increase in the crystallinity in PLA composites may not


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molecular weight caused by degradation22. The increase in crystallinity from degradation is

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inconsistent, however, with findings in the poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

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copolymer, PHBV, which was found to have the same crystallinity before degradation, and

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after degrading in soil for 15 days, concluding that degradation occurred in both the

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crystalline and amorphous regions23. However, crystallinity was only determined at 0 and 15

339

days of degradation, so it is difficult to make a determination on which phase degraded first.

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It has previously been demonstrated that degradation can occur faster in soil than in

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compost16, so it may be that the PHBV samples degraded faster than the PHB degraded here

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in compost, as it was reported that the PHBV completely degraded after 30 days23. Here, the

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PHB/BC composites were found to be degraded to 80% at 30 days, whereas this level of

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degradation was not achieved in the PHB films until 48 days of composting.

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Based on the findings here, it is likely that degradation initially occurs in the amorphous

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region of the PHB. The increased rate of biodegradation in the PHB/BC composite suggests

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that it is the bacterial cellulose phase degrading first, due to its high rate of degradation, with

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the PHB (with an increased surface area) being quickly degraded after this time, having an

349

increased surface area due to the cellulose degradation.

350

For SAXS measurements made from different points within a film and different films there

351

was not a significant difference in the scattering curves apart from a constant scattering factor

352

which is consistent with samples of varying thickness but similar nano-structural structural

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attributes24. Figure 7 shows the typical SAXS curves for the PHB/BC and PHB samples. All

354

the curves consist of a decay with one or two small peaks superimposed. At the higher values

355

of q the scattered intensity gradually increases again as the features associated with wide

356

angle X-ray scattering (diffraction) are encountered. For more degraded samples, the

357

peaks/features become less well defined and may contain several very broad peaks. This

358

general form of a SAXS curve is typical of a semi-crystalline polymer such as PHB with the



Page 24 of 27

359

position of the peak being characteristic of a unit lamellar cell consisting of alternating

360

crystalline and amorphous polymer layers25, 26. A characteristic spacing, d, may be taken from

361

the position of the peak, qmax: ଶగ

362

݀=

363

using a simple Lorentz transformation of data, plotting q2.I versus q (inset Figure 7)27, to

364

௤೘ೌೣ

(4)

locate the position of the peak more precisely on the sloping baseline.

0.3

10000

10000

1000

0.1

100 0.1 q (Å-1)

10

1

0.2

intensity (a.u.)

1000

I.q2

intensity (a.u.)

0.2

100

10

Weeks 0 1 2

1

Weeks 0 1 2 3

0.1

365

0.1

q (Å-1)

q (Å-1)

366

Figure 7. SAXS curves as a function of composting time in weeks for PHB/BC film (left)

367

and PHB. The inset of the left hand side shows the Lorentz plot for determining d-spacing for

368

the time = 0 sample. The position of the peak is shown with an arrow.

369

In the case of the films at time 0, a single repeat spacing is observed for both samples. In

370

the case of the PHB/BC sample, the repeat distance is approximately 52 Å, and for pure PHB

371

it is at 49 Å. In the case of pure semi-crystalline polymers, the repeat spacing is function of

372

the processing used to form the film, and the molecular weight of the polymers used28. In this

373

case, where the method of forming the film is the same, the presence of the cellulose appears

374

to have a small effect on the nanostructure of the PHB. As the composting time increases, the

375

feature moves to lower values of q/larger length-scales (cf equation 4) and becomes less

376

pronounced. These peaks are not well defined and may, in some cases, contain more than one


Page 25 of 27


377

repeat spacing. This is not surprising in view of the heterogeneous nature of degradation in

378

compost.

379

Assuming that degradation does not occur within the crystalline phase since the crystalline

380

packing excludes enzymes responsible for degradation29 and a net increase in crystallinity of

381

PHB is initially observed, changes in the structure must arise in the amorphous region. It

382

would seem likely that changes in crystallinity and subsequent formation of lamellae occurs

383

as the result of degradation of bulk amorphous materials outside the original lamellae as the

384

backbone of the linear polymer is cleaved statistically along chain. This produces smaller

385

fragments, which are able to crystallise into new lamellae.

386

Overall, this study reports the comprehensive approach taken to measure and interpret the

387

biodegradation of PHB and PHB/BC films in compost. CO2 evolution was used as an

388

accurate in situ measure of biodegradation, in conjunction with other techniques of SEM,

389

FTIR, XRD and SAXS also employed to understand the morphological changes that occurred

390

during this process. Both PHB and bacterial cellulose were found to degrade rapidly, with

391

bacterial cellulose initially having the highest rate of degradation of the samples. The ability

392

of this material to quickly degrade caused a faster degradation rate in the PHB/BC composite

393

over PHB alone. In addition, the relative levels of crystallinity of the materials were found to

394

increase during degradation. These results demonstrate the ability for these natural materials

395

to be quickly degraded in a composting environment.

396

ACKNOWLEDGEMENTS

397

We wish to thank Mark Greaves of CSIRO for the SEM, and Liz Goodall and Aaron

398

Seeber for the XRD. This work was funded by a Julius Career Award from the CSIRO Office

399

of the Chief Executive.

400

REFERENCES



401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436

1. King, P. P., Biotechnology. An Industrial View. J Chem Technol Biot 1982, 32, (1), 2-8. 2. Holmes, P. A., Applications of Phb - a Microbially Produced Biodegradable Thermoplastic. Physics in Technology 1985, 16, (1), 32-36. 3. Owen, A.; Bergmann, A., On the fractal character of polymer spherulites: an ultrasmall-angle X-ray scattering study of poly[(R)-3-hydroxybutyrate]. Polym Int 2004, 53, (1), 12-14. 4. Xie, Y. P.; Noda, I.; Akpaiu, Y. A., Influence of cooling rate on the thermal behavior and solid-state morphologies of polyhydroxyalkanoates. J Appl Polym Sci 2008, 109, (4), 2259-2268. 5. Eichhorn, S. J.; Dufresne, A.; Aranguren, M.; Marcovich, N. E.; Capadona, J. R.; Rowan, S. J.; Weder, C.; Thielemans, W.; Roman, M.; Renneckar, S.; Gindl, W.; Veigel, S.; Keckes, J.; Yano, H.; Abe, K.; Nogi, M.; Nakagaito, A. N.; Mangalam, A.; Simonsen, J.; Benight, A. S.; Bismarck, A.; Berglund, L. A.; Peijs, T., Review: current international research into cellulose nanofibres and nanocomposites. Journal of Materials Science 2010, 45, (1), 1-33. 6. Shah, A. A.; Hasan, F.; Hameed, A.; Ahmed, S., Biological degradation of plastics: A comprehensive review. Biotechnol Adv 2008, 26, (3), 246-265. 7. Wan, Y. Z.; Luo, H. L.; He, F.; Liang, H.; Huang, Y.; Li, X. L., Mechanical, moisture absorption, and biodegradation behaviours of bacterial cellulose fibre-reinforced starch biocomposites. Composites Science and Technology 2009, 69, (7-8), 1212-1217. 8. Kibedi-Szabo, C. Z.; Stroescu, M.; Stoica-Guzun, A.; Jinga, S. I.; Szilveszter, S.; Jipa, I.; Dobre, T., Biodegradation Behavior of Composite Films with Poly (Vinyl Alcohol) Matrix. Journal of Polymers and the Environment 2012, 20, (2), 422-430. 9. Stoica-Guzun, A.; Jecu, L.; Gheorghe, A.; Raut, I.; Stroescu, M.; Ghiurea, M.; Danila, M.; Jipa, I.; Fruth, V., Biodegradation of Poly(vinyl alcohol) and Bacterial Cellulose Composites by Aspergillus niger. Journal of Polymers and the Environment 2011, 19, (1), 69-79. 10. Savenkova, L.; Gercberga, Z.; Nikolaeva, V.; Dzene, A.; Bibers, I.; Kalnin, M., Mechanical properties and biodegradation characteristics of PHB-based films. Process Biochem 2000, 35, (6), 573-579. 11. Mergaert, J.; Anderson, C.; Wouters, A.; Swings, J.; Kersters, K., Biodegradation of Polyhydroxyalkanoates. Fems Microbiol Lett 1992, 103, (2-4), 317-321. 12. Arcos-Hernandeza, M.; Laycock, B.; Pratta, S.; Donose, B.; Nikolić, M.; Luckmand, P.; Werkere, A.; Lanta, P., Biodegradation in a soil environment of activated sludge derived polyhydroxyalkanoate (PHBV)Polymer Degradation and Stability 2012, 97, (11), 2301-2312.

437 438 439 440 441 442 443 444 445 446 447 448 449

13. Byrom, D., The Synthesis and Biodegradation of Polyhydroxyalkanoates from Bacteria. Int Biodeter Biodegr 1993, 31, (3), 199-208. 14. Ruka, D. R.; Simon, G. P.; Dean, K. M., Altering the growth conditions of Gluconacetobacter xylinus to maximize the yield of bacterial cellulose. Carbohydrate Polymers 2012, 89, (2), 613-622. 15. Hammersley, A. FIT2D V9.129 Reference Manual V3.1; ESRF Internal Report: ESRF, 1998. 16. Dean, K.; Sangwan, P.; Way, C.; Zhang, X. Q.; Martino, V. P.; Xie, F. W.; Halley, P. J.; Pollet, E.; Avérous, L., Glycerol plasticised chitosan: A study of biodegradation via carbon dioxide evolution and nuclear magnetic resonance. Polymer Degradation and Stability 2013, 98, (6), 1236-1246. 17. Luzier, W. D., Materials Derived from Biomass Biodegradable Materials. P Natl Acad Sci-Biol 1992, 89, (3), 839-842.


Page 26 of 27

Page 27 of 27

450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478


18. Randriamahefa, S.; Renard, E.; Guérin, P.; Langlois, V., Fourier transform infrared spectroscopy for screening and quantifying production of PHAs by Pseudomonas grown on sodium octanoate. Biomacromolecules 2003, 4, (4), 1092-1097. 19. Riello, P.; Fagherazzi, G.; Canton, P.; Clemente, D.; Signoretto, M., Determining the Degree of Crystallinity in Semicrystalline Materials by Means of the Rietveld Analysis. J Appl Crystallogr 1995, 28, 121-126. 20. Garvey, C. J.; Parker, I. H.; Simon, G. P., On the interpretation of X-ray diffraction powder patterns in terms of the nanostructure of cellulose I fibres. Macromol Chem Physic 2005, 206, (15), 1568-1575. 21. Owen, A. J.; Heinzel, J.; Škrbić, Ž.; Divjaković, V., Crystallization and Melting Behavior of PHB and PHB/HV Copolymer. Polymer 1992, 33, (7), 1563-1567. 22. Way, C.; Dean, K.; Wu, D. Y.; Palombo, E., Biodegradation of sequentially surface treated lignocellulose reinforced polylactic acid composites: Carbon dioxide evolution and morphology. Polymer Degradation and Stability 2012, 97, (3), 430-438. 23. Gonçalves, S. P. C.; Martins-Franchetti, S. M.; Chinaglia, D. L., Biodegradation of the Films of PP, PHBV and Its Blend in Soil. J Polym Environ 2009, 17, (4), 280-285. 24. Glatter, O.; Kratky, O., Small angle X-ray scattering. Academic Press: 1982. 25. Ellar, D.; Lundgren, D. G.; Okamura, K.; Marchess.Rh, MORPHOLOGY OF POLYBETA-HYDROXYBUTYRATE GRANULES. Journal of Molecular Biology 1968, 35, (3), 489-&. 26. Strobl, G. R.; Schneider, M., Direct evaluation of the electron-density correlationfunction of partially crystalline polymers. Journal of Polymer Science: Polymer Physics Edition 1980, 18, (6), 1343-1359. 27. Cser, F., About the Lorentz correction used in the interpretation of small angle X-ray scattering data of semicrystalline polymers. J Appl Polym Sci 2001, 80, (12), 2300-2308. 28. Strobl, G. R., The Physics of Polymers: Concepts for Understanding Their Structures and Behavior. Springer: 2007. 29. Jendrossek, D.; Handrick, R., Microbial degradation of polyhydroxyalkanoates. Annu Rev Microbiol 2002, 56, 403-432.

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Bacterial Cellulose

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