5 RELATED STUDIES

ABOUT
BIOPLASTICS

Jomer Ruego
SSC 10-Newton
3 Foreign Studies
 Seaweeds can be a new source for bioplastics
Rajendran, N. , Sharanya Puppala, Sneha Raj M., Ruth Angeeleena B., and Rajam, C.

School of Bio Sciences and Technology, VIT University, Vellore, India

Received on:25-01-2012; Revised on: 24-02-2012; Accepted on:12-03-2012

ABSTRACT

The rapid growth of plastic production was a 20th century phenomenon on a historical scale. The low cost of plastics and its
versatility have paved a way for a wide range of applications. As the plastics are non-biodegradable and found to have toxic
effects on human, animals and environment, the bioplastics came into existence. Bioplastics are biodegradable and can be
derived from renewable biological sources. Bioplastics have same applications as plastics. Although there are different sources of
bioplastics like plants, animals and microbial sources, they have certain limitations such as non-availability of high biomass and
difficulties involved in cultivation. In such cases, seaweeds can serve as one of the alternatives for the production of bioplastics
because of its high biomass, its ability to grow in a wide range of environments and its cultivation in natural environment when
compared to other microbial sources which require a specific environment for their cultivation. In addition to the above benefits,
seaweeds are cost effective, minimize the impact on the food chain and are chemical-independent. Bioplastics from seaweeds are
reported to be more resistant to microwave radiation, less brittle and durable. The technology development for the seaweeds-
based bioplastics are still under the research phase and it is hoped that significant advancements would be made in the bioplastics
industries and can make seaweed bioplastics a reality in future. Fermentation and genetic engineering can take the lead in using
novel techniques to make bioplastics from seaweeds which would make them as a viable alternative. This review presents the
importance, advantages and applications of seaweeds as an alternative source for bioplastics.

Methodology
Bioplastic making from seaweeds

The component of seaweeds used in the making of bioplastics is polysaccha-

rides. Some of the polysaccharides of seaweeds are carrageenan, agar, floridean
starch and alginate. Quality control of polysaccharides extract begins at the
harvest. The seaweed is systematically gathered, quickly dried and then baled to
maintain its quality and freshness. At the manufacturing site, the dried seaweed is
mechanically ground and sieved to eliminate impurities such as sand and salt
which is followed by extensive washing to ensure additional quality. Seaweeds
undergo a hot extraction process to separate the polysac-charides which is a two-
step clarification process. First the dissolved polysac-charide mixture is
centrifuged to eliminate the dense cellulosic particles, filtered to remove the
smaller particles and then, the solution is concentrated

Fig. 4. Manufacturing process of bioplastic from seaweeds

(Source: http://www.fmcbiopolymer.com/Food/Ingredients/Carrageenan/
Manufacturing.aspx).

CONCLUSION

Currently application of bioplastics is in its infancy stage but holds signifi-cant promise in developing sustainable plastics for the
future. The price of fermentative PHA production per unit polymer is estimated to be $2/kg prices which are twice the price of
polyethylene. Even though bioplastics are expensive they are still considered as a viable option to improve environmen-tal
sustainability

Bioplastics from seaweed may also be expensive but they have gained ut-most importance in the recent times because of their
advantages over other biological sources which have already mentioned above. Seaweed based bioplastics play a vital role as an
environment friendly and biodegradable alternative compared to conventional plastics. Exploring the production of bioplastics
could play a major role in shaping the economics and viability of seaweed based products. The technology routes for the
production of sea-weed based bioplastics are still under research and the use of biotechnological and genetic engineering
techniques play a key role in conducting the feasibility and sustainability studies in seaweed based bioplastics. It is hoped that
significant advances made in the bioplastics industry in general will benefit seaweed based bioplastics industry as well and will
make seaweed based bioplastics a reality in the distant future.
 Microalgae as bioreactors for bioplastic production
Franziska Hempel1, Andrew S Bozarth2, Nicole Lindenkamp4, Andreas Klingl1, Stefan Zauner2, Uwe Linne3,
Alexander Steinbüchel4 and Uwe G Maier1,2*

Abstract

Background: Poly-3-hydroxybutyrate (PHB) is a polyester with thermoplastic properties that is naturally occurring and
produced by such bacteria as Ralstonia eutropha H16 and Bacillus megaterium. In contrast to currently utilized plastics and
most synthetic polymers, PHB is biodegradable, and its production is not dependent on fossil resources making this
bioplastic interesting for various industrial applications.

Methodology

Plasmid construction and P. tricornutum transfection

Plasmid pBHR68 [25] was used as template for amplifi-cation of phaA, phaB and phaC genes from R. eutropha H16. For in vivo
localization studies sequences were cloned upstream to the eGFP (enhanced green fluores-cent protein) sequence into the vector
pPha-NR, which is a derivative of pPhaT1 with endogenous nitrate reductase promoter/terminator flanking the multiple cloning
site [GenBank:JN180663]. The inducible nitrate reductase promoter system was established earlier in the diatom C. fusiformis by
Poulsen et al. 2005 [26]. Trans-fection proceeded as described previously [27] with the exception that cells were grown under
non-induced con-ditions with NH4+ as sole nitrogen source. For PHB synthesis in P. tricornutum, the sequence for phaC was
cloned into the vector pPha-NR (not containing eGFP), and sequences of phaA and phaB were inserted into the vector pPha-
DUAL[2xNR], which is a pPha-NR deriva-tive with two multiple cloning sites both under the con-trol of endogenous nitrate
reductase promoter [GenBank:JN180664]. Both plasmids were mixed and co-transfected under non-induced conditions.

Cell culture and induction of recombinant protein expression

Cells were grown in f/2 medium under standard condi-tions as described elsewhere (Apt et al. 1999) with either 0.9 mM NO3- or
1.5 mM NH4+ as the nitrogen source. For in vivo localization studies on GFP fusion proteins transfectants were grown in media
containing NO3- to induce recombinant protein expression. After 3 days, clones were analyzed by confocal laser scanning micro-
scopy. PhaA/phaB/phaC co-transfectants were first determined to have genomic integration by colony PCR for all three
sequences. Subsequently, positive colonies were grown in liquid culture containing NH4+ and allowed to reach exponential
phase, whereupon they were transferred to NO3- containing medium for varying time periods. For visualization of PHB granules,
cells were induced for 5 days and analyzed by electron and confocal microscopic analyses, respectively. For confocal
microscopy, cells were pre-incubated with the lipophilic dye Nile Red (0.5 μg/ml) for 24 h.

PHB analyses

For analyses on PHB production cultures were grown in NH4+ containing medium, washed in nitrogen-free medium and
transferred to NO3- containing medium for 7 days. Cells were harvested (1500 × g, 10 min), washed with phosphate buffered
saline (PBS) and lyophilized for 24 hours. The PHB contents of the cells were deter-mined upon methanolysis of 5 to 10 mg
lyophilized cells in presence of 2 ml methanol/sulfuric acid (85:15, v/v) and 2 ml chloroform. The resulting methyl esters of 3-
hydroxybutyrate were analysed by gas chromatography using an Agilent 6850 GC (Agilent Technologies, Wald-bronn,
Germany) as described previously [28,29].

Fluorescence and electron microscopy

In vivo localization of GFP fusion proteins was analysed with a confocal laser scanning microscope Leica TCS SP2 using a HCX
PLAPO 63x/1.32-0.6 oil Ph3 CS objective. GFP, chlorophyll and Nile Red were excited at 488 nm, and fluorescence was
detected at a bandwidth of 500-520 nm, 680-720 nm and 580-600 nm, respec-tively. For electron microscopic analyses, cells
were cen-trifuged at 2000 × g for 5 min followed by cryo-fixation and resin embedding. The samples were high-pressure frozen
in a Leica EM-PACT 2 and subsequently freeze substituted (Leica EM AFS 2; Leica, Vienna, Austria) with pure acetone
containing 2% (w/v) osmium tetrox-ide, 0.1% (w/v) uranyl acetate and 5% (v/v) H2O. Freeze substitution was carried out at -
90°C for 4 h, -60°C for 8 h, -30°C for 8 h and held at 0°C for 3 h with a heating time of 1 h in between each step. After washing
the samples with ice cold acetone for three times and infil-tration in Epon 812 (Ted Pella, Inc., USA) for 24 h, the resin was
polymerized at 60°C for 72 h. Ultrathin sec-tions were cut with a Leica Ultracut (Leica, Vienna, Austria), mounted on uncoated
400 mesh copper grids and post-stained with 2% (w/v) uranyl acetate for 20 min and 0.5% (w/v) lead citrate for 1 min.
Transmission electron microscopy was carried out on a JEOL 2100 TEM operated at 80 kV in combination with a fast-scan 2 k ×
2 k CCD camera F214 (TVIPS, Gauting, Germany).

Conclusions
Altogether, this study has demonstrated that microalgae like the diatom P. tricornutum have a great potential not only as
biosynthetic factory for recombinant pro-teins but also as photosynthetically fueled bioreactors for synthesizing
biotechnologically relevant polymers like PHB. Even though no enzyme engineering, no adap-tations to P. tricornutum specific
codon-usage, and no large-scale screening have been applied in these initial analyses, relatively high PHB levels of up to 10.6%
of algal dry weight have been obtained. Thus, in the future, there will be a focus on various targets for enhancing PHB
biosynthesis in P. tricornutum. Other subcellular compartments such as the plastids might yet be interest-ing sites for PHB
synthesis. Diatoms are naturally rich in lipids and silicate and already have applications in bio-technology [17]. Hence, inserting
and/or altering
 AGAR FROM MALAYSIAN RED SEAWEED AS
POTENTIAL MATERIAL FOR SYNTHESIS OF BIOPLASTIC FILM
SIEW-LING HII1, JIA-YEE LIM2, WAN-TECK ONG3, CHING-LEE WONG2,*

1Department of Food Technology, School of Engineering and Technology, University College of Technology Sarawak, 868
Persiaran Brooke, 96000 Sibu, Sarawak Malaysia 2School of Biosciences, Taylor’s University, Taylor’s University Lakeside
Campus, No. 1, Jalan Taylor’s, 47500 Subang Jaya, Selangor DE, Malaysia
3Department of Chemical Engineering, Faculty of Engineering and Science, Universiti Tunku Abdul Rahman, Jalan Genting
Kelang, 53300 Kuala Lumpur, Malaysia *Corresponding Author: chinglee.wong@taylors.edu.my

Abstract
The main aim of this study was to identify the potential use of agar extracted from red seaweed, Gracilaria salicornia, collected
from the coastal area of Malaysia as the raw material for synthesis of bioplastic film. Agar was extracted via two extraction
methods: (1) alkali extraction method and (2) photo bleaching extraction method. The yields of agar by both of the methods were
9 to 11 %. The alkali extracted agar (AEA) and photo bleached agar (PBA) were incorporated as the raw materials for the
formation of bioplastic films while sago starch and glycerol were added to increase workability. Physicochemical properties of
the two bioplastic films were characterised. FTIR analysis confirmed the presence of agar in both plastic films with the presence
of 3,6-anhydrogalactose residues and further indicated that the interactions of agar and sago starch were strong in both PBA and
AEA films. The results showed that tensile strength and percent elongation of PBA film (3.067 MPa, 3.270 %) was higher than
AEA film (2.431 MPa, 2.476 %). Thermogravimetric analysis (TGA; % residual weight) revealed that AEA film has higher
thermal stability (14.80 %) than PBA film (10.27 %) while rheological results proved that both films exhibited non-Newtonian
behaviors. The AEA film was completely decomposed after 30 days in the soil burial test. Results of current study show a wide
range of future possibilities and commercial applications of AEA and PBA bioplastic films.

Materials and Methods

Seaweed sampling and preliminary treatment

Specimens of red seaweed G. salicornia were collected at the beach of Port Dickson, Malaysia. The collected seaweed was
cleaned with tap water for several times and dried at 60 ºC in an oven. The dried seaweed specimens were subjected to size
reduction before stored in air-tight bags with silica gels.

Seaweed extraction method

Alkali treatment extraction

Alkali treatment extraction method was carried out according to the method of Chirapart et al. [17] with minor modifications.
Dried seaweeds (10 g) were treated with alkaline solution (500 mL of 5 %w/v NaOH) for 2 hours at 80 ºC. Following this, the
alkali-treated sample was rinsed properly and placed in deionized water (room temperature). The pH of sample was adjusted to a
range of 6.5 to 7.5. The sample was then heated at 120 ºC for 2 hours. The filtrate of sample was left to cool to room temperature
and frozen overnight to concentrate the agar gel. The frozen solidified agar was thawed and dried at 50 ºC for 24 hours.

Photo bleaching extraction

Photo bleaching extraction method was carried out according to the method of Li et al. [11] with minor modifications. The initial
extraction method is similar with alkali treatment extraction method with additional photo bleaching process. After pH of the
treated seaweed was adjusted to a range of 6.5 to 7.5, the seaweed samples were soaked in distilled water and left overnight under
fluorescent lamp prior to photo bleaching process for 8 hours. Following this, the seaweed were rinsed and concentrated
according to the process as described in the alkali treatment extraction method (Section 2.2.1).

Determination of agar yield

The yield of agar was determined based on the initial dry weight of the seaweed and the final dry weight of the agar extracted, as
shown in Equation 1.

dry weight of agar

Yield (% w/w) = initial dry weight of seaweed × 100 % (1)

Preparation of bioplastic film

Bioplastic films were prepared by film casting method. The alkali extracted agar (AEA) and photo bleached agar (PBA) were
used as raw material for the formation of bioplastic film. Sago starch and plasticizer (glycerol) were incorporated into film-
forming solution to increase workability. Sago starch (6.8 g) was first gelatinized and homogenized in 240 mL distilled water
using overhead stirrer in water bath (± 90 ºC). Agar powder was then added to the homogenized starch solution. Glycerol was
mixed to the film-forming solution and stirred for 5 minutes. The amount of dissolved components were added based on the
formulation from Wu et al. [18] to ensure the surface of bioplastic film produced is clear, smooth, flexible and without any phase
separation. Then, the film forming solution was casted on petri dish and dried at 50 ºC overnight.
Characterisation of bioplastic film
FTIR spectroscopy

The spectra of each casted film sample were recorded using FTIR spectrometer (Thermo Scientific Nicolet iS10). Each spectrum
was the average of 32 scans acquired at 2 cm-1 resolution. Transmittance mode was used to determine peak base lines and
heights. The peak heights were calculated and converted to absorbance.

Scanning electron microscopy (SEM)

Scanning electron microscope (Hitachi S3400N, Japan) with energy dispersive X-ray analysis (EDX) at 20 kV was used to obtain
scanning electron micrographs and elemental composition of the bioplastic film samples. The bioplastic film samples were
coated with Au/Pd up to 7 nm thick with high resolution sputtering instrument at a sputtering rate of 1.5 kV per minute. Then,
cross sectional image of the samples were observed using x1000 and x5000 magnification.

Mechanical properties

Tensile strength (TS, MPa) and elongation at break (%) of the bioplastic film samples were determined using tensile machine
(Tinius Olsen H10KS Universal, USA). The samples were cut into dumb-bell shape and fixed to the tensile machine at both ends
prior to the mechanical testing.

Thermogravimetric analysis (TGA)

TGA was carried out using a thermogravimetric analyser (Mettler Toledo TGA-SD815a, Switzerland). The bioplastic film
samples were cut into pieces (2.5 to 5 mg) and transferred to the sample holder. The testing temperature was from 30 to 800 ºC,
with a ramp temperature of 20 ºC and nitrogen gas flow rate at 30 mL/min. Curves of weight loss of bioplastic film samples
against temperature were plotted.

Rheological study

Constant shear rate test and dynamic oscillation test were conducted on AEA and PBA films using rheometer (Anton Paar
GmBH-MCR 301, Austria) equipped with a parallel plate with diameter of 0.5 mm and gap of 1 mm. Each sample of bioplastic
film was placed onto the bottom plate which equilibrated to 25 ºC and pressed by the top plate. Constant shear rate test was
carried out with shear rate at 0.01 s-1 for duration of 477 s. Dynamic oscillation test was conducted in strain sweeping mode
where strain amplitude was changed from 0.01 % to 100 % with the shear frequency of 1 Hz. G’ (measure of elastic response)
and G” (measure of viscous response) were monitored along the sweep amplitude test.

Soil burial test

Biodegradability of the bioplastic films were examined with soil burial test. The samples were buried in locations with different
type of soils and left for 30 days. The initial weight (Mo) and the final weight (M1) were recorded. The percentage of weight loss
after 30 days is calculated by using Equation 3.

Mo – M 1
Weight loss (%) = × 100 % (2)
Mo

Conclusions

In the present study, agar extracted from different extraction technique were rigid, brittle with uneven surface and have an
average yield of 9 to 11 %. FTIR spectra of AEA and PBA bioplastic films confirmed that there are chemical interactions
between agar, sago starch and glycerol with PBA having the stronger interaction effects. The SEM images and mechanical tests
further revealed the PBA bioplastic film is with better mechanical properties (higher tensile strength and percent elongation)
which have denser and packed structure. The AEA bioplastic film exhibited excellent biodegrability with the highest weight loss
of 99.29 % in comparison to PBA bioplastic films with 43.27 % weight loss within 30 days of soil burial test. TGA results
indicated that thermal stability of AEA bioplastic film is better than PBA bioplastic film. Rheological analysis of the biopolymer
blends showed that all the bioplastic blends produced are non-Newtonian fluid and thus performed like a weak gel system.

As conclusion, there are a lot of explorations and optimizations to be made to both of the bioplastic films but there is no doubt
that agar extracted from G. salicornia are able to bring future potential to a wide range of bioplastic applications in the industry.
2 Local Studies
 Gabi Starch as Plastic
Jimreen BayAn Coligman

Background Of The Study

Gabi, or taro, is prized chiefly on account of its large corms, or underground stems, which may be a staple food in some areas
but in our own locality it is usually preferred as “Food for the pigs” due to a property which is known to be harmful for human
consumption when not properly cooked. It has high starch content, is very nutritious and has many medicinal and non-medicinal
uses since the beginning of time. Pure Starch is a white, tasteless and odorless powder that is insoluble in cold water or alcohol. It
consists of two types of molecules; the linear and helical amylase and the branched amyl pectin. Thus, it is the most suitable
biodegradable agent.
Plastic is all around us. It is useful, lightweight, durable, strong and relatively inexpensive. In recent years there have been
plastic issues causing downfalls in many urban countries. Worldwide we produce about 100 million tons of petroleum plastic per
year. Most of this ends up in landfills, rivers, oceans, and lakes, where it pollutes the ecosystems for hundreds of years. Over 540
billion pounds of oil-based plastic are produced every year and to make these plastics we use about 7 million barrels of oil per
day. Plastics are not biodegradable because the polymer chains are too tight and large to be broken down. Therefore it is
significant to promote biodegradable plastics especially in our time.
Even if the government or those so called experts say that we should eliminate the use of plastics, the fact is we really cannot
because of its versatility and let`s face it, it has become a necessity, an accessory and even a form of advertisement for some
businesses which helps the economy to grow.
METHODOLOGY
Materials
The materials used in the study were categorized according to their usage. The Independent variables were the glycerol and
vinegar while the dependent variables were the starch and water.
Devices manipulated were graduated cylinder, beakers, ruler, and measuring spoons. Laboratory apparatuses utilized were
stirring rod, mortar and pestle, funnel were borrowed from the BSU-SLS Chemistry Laboratory. Other implements such as plates,
glasses and strainer were borrowed from the BSU-SLS Food department. And other materials such as grater, knife, and gloves
were supplied by the researchers.

Table 1. Formulas
Formula 1Formula 2Formula 3
Starch 1 tbsp. 1.5 tbsp. 2tbsp.
Water 60 ml 60 ml 60 ml
Lemon 2 tsp. 2 tsp. 2 tsp.
Glycerol 1 tsp. 1 tsp. 1 tsp.
Polyvinyl Acetate Resin Glue 15 ml 20 ml 25 ml

Procedures

Preparation of Materials
The glycerol was obtained from the Department of Chemistry of Benguet State University while the hydrochloric acid was
acquired in the BSU-SLS Chemistry Laboratory. The lemon and Galiang were bought from the La Trinidad Public Market. Other
materials such as measuring devices and apparatuses were borrowed from the BSU-SLS Chemistry Laboratory and BSU-SLS
Food Department.

Extracting the Starch

The Galiang was peeled using a paring knife and was washed through a running water. Then the peeled Galiang was grated using
a grater and was placed into the mortar. About 100 ml of water was added to the mortar, and the Galiang was grinded carefully.
The Galiang-water mixture was pour through the strainer into the funnel, to avoid spillage, then finally into the beaker, leaving
the Galiang behind the strainer. Then the grinding and pouring of the mixture was repeated twice more. Then the mixture was left
overnight to let the starch settle in the beaker.
The water was decanted from the beaker, leaving behind the white starch that has settled in the bottom. About 50 ml of water was
put in with the starch and was stirred using a stirring rod. The mixture was left for 2 hours to settle and then the water was
decanted, leaving the starch behind. The slurry starch was sun dried.

Making the Plastic sheet

In order to determine the right combination, three batches with different proportions were prepared.
T1 =1 tbsp. of starch, 60 ml of water, 2 tsp. of lemon, 1 tsp. of glycerol, 15 ml resin glue
T2 =1.5 tbsp. of starch, 60 ml of water, 2 tsp. of lemon, 1 tsp. of glycerol, 20 ml resin glue
T3 =2 tbsp. of starch, 60 ml of water, 2 tsp. of lemon, 1 tsp. of glycerol, 25 ml resin glue
Measured amounts of the ingredients were added everything to the pot. The hot plate was turn on to medium and the mixture was
stirred until it turns from cloudy white to clear, until a sticky paste was formed. Then the heat was turned up a little and was
stirred rapidly until it was completely clear. Then the mixture was quickly poured onto the cooling sheet, and was spread to let it
dry.

Preparation for the tests

The plastic sheets formed were rolled into the laminating machine to create an even thinness for testing. Then the samples
produced were cut into strips with the dimension of 2cm and 4cm.
Testing the Plastic Sheets

Several tests were conducted to determine the mechanical properties of the samples.
1. Effects of strong acids
The plastic strips were immersed in concentrated hydrochloric acid for 30 minutes. Changes in length, width and appearance
were noted. The purpose of this is to determine if the treatments can be dissolve in strong acids like Hydrochloric acid.
2. Tensile test
The plastic strips were hooked to a spring balance and were pulled until they tore apart. The readings on the spring balance when
the strips broke were recorded. The purpose of this is to determine the treatment’s durability or resilience.
3 Organic solvent tests
The plastic strips were immersed in ethanol for 48 hours. Changes in appearance were noted. The purpose of this is to determine
if the treatments can be dissolved in organic solvent like ethanol.
4. Biodegradable test
The plastic strips were stapled to a piece of cardboard and was buried in a can of soil. The strips were unearthed after a week and
their appearance were recorded. The purpose of this is to determine if the treatments can degrade in the soil.
5. Water Resistance
The plastic strips were immersed in water for 5 days. Changes in appearance was observed and noted. The purpose of this is to
determine if the treatments can resist water.

SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS

Summary And Conclusions

The Galiang plant has the potential to serve as an alternative in making plastics but not as effective as the control in
some cases.

1. In the Tensile Stress Test the top 3 were Treatment 0.2: Plastic cover is the best treatment, with the measurement of
9.25 cm, followed by treatment 0.1: Plastic bag with 7.42 cm then lastly treatment 1 with 4.17 cm
2. In the Strong Acid Test, Hydrochloric Acid. Treatment 1 is the best treatment, which was dissolved completely,
followed by treatment 2 with the area of 0.44 cm2, then lastly treatment 3 with 1cm2 areas.
3. In the Organic Solvent Test, Ethyl Alcohol. Treatments 1, 2 and three became brittle, making it easy to be torn while
treatments 0.1 and 0.2 were unchanged.
4. In the Water Resistance Test. Treatment 0.1: Plastic Bag and Treatment 0.2: Plastic Cover were the best treatments
making them water-resistant, while treatments 1, 2 and 3 are water-soluble.
5. In the Biodegradable Test. Treatments 1, 2 and three have positive results making them biodegradable, treatment 1 had
the most successful results followed by treatment 2 then treatment 3 while control 0.1 and 0.2 remain unchanged.
Favorable results were obtained in the Strong Acid Test. The samples were found to be water-soluble. However, the
samples would not dissolve in ethanol, an organic solvent as for the tensile stress the treatments. All the experimental treatments
were biodegradable.

It is therefore concluded that the Galiang plant may be used as an additive in making biodegradable plastic.
 Bioplastic based on starch and cellulose nanocrystals from rice straw
Melissa B Agustin1 , Bashir Ahmmad2 , Shanna Marie M Alonzo1 and Famille M Patriana
1 Department of Chemistry, College of Arts and Sciences, Central Luzon State University, Science City of Mun˜oz, Nueva Ecija,
Philippines 2 Graduate School of Science and Engineering, Yamagata University, Yonezawa, Japan

Introduction

The demand for the production of environmentfriendly material is increasing. The rising concern towards environmental
problems brought by petroleum-based products inspired the development of the so called ‘‘green’’ materials. The guiding
principles for the production of these materials are sustainability, industrial ecology, eco-efficiency, and green chemistry.1,2 One
of the ‘‘green’’ materials emerging in the market nowadays is biodegradable plastics or bioplastics. Bioplastics are derived from
agricultural resources and biomass feedstock that are renewable and therefore comply with materials that are eco-efficient and
sustainable. It has been reported that the worldwide production capacity of bioplastics will increase from 0.36 Mt in 2007 to 3.45
Mt in 2020.3 Among the biopolymer matrices being utilized for the production of bioplastics, starch is considered the most
widely used material. Starch-based plastics have been projected to comprise the largest production capacity amounting to 1.3 Mt
in 2020 while the remaining production is based on polylactic acid (PLA), polyhydroxyalkanoates (PHA), bio-based
polyethylene, and others.3 The large contribution of starch-based plastics in the market can be accounted for its several cited
advantages such as high abundance, low cost, and renewability. However, starch alone is not a true thermoplastic. It has to be
processed in the presence of heat and mechanical treatment together with a plasticizer, most often glycerol. This process produces
thermoplastic starch (TPS). Pure TPS cannot meet the different properties needed for a particular application. It has to be
combined with other materials often a filler to modify its properties. Generally, reinforcement with filler enhances the mechanical
properties of starch and reduces the hydrophilic character.4 In recent years, the use of bio-based and nano-sized fillers has
attracted substantial research interest. Several advantages of using nano-sized fillers from natural resources have already been
cited in different works and these include low density, renewable character, great abundance, high specific resistance, excellent
mechanical properties, biodegradability, low-cost, low abrasive nature, and reactive surface for easy modification.5–8 Moreover,
it was also noted that the nanoscale dimensions of the filler keeps the transparency of the material if inherent for the neat matrix.9
One of the most investigated bio-based fillers is cellulose nanocrystals (CNCs); cellulose crystallites with at least one dimension
equal or less than 100 nm. CNCs can be isolated from the native cellulose by acid hydrolysis. The amorphous portion of the
cellulose can be easily hydrolyzed in strong acidic condition leaving the individual crystallites. Sulfuric acid is commonly used
because the introduction of sulfate esters on the surface of the individual crystallites stabilizes the resulting suspension of CNCs
by electrostatic repulsion.10 Depending on the source and reaction condition during hydrolysis, the size and properties of the
isolated CNCs also vary.11 In the present paper, our group reports the isolation and characterization of CNCs from rice straw and
its application as reinforcing filler in starch-based bioplastic. Rice straw is one of the most abundant agricultural residues in the
Philippines. The Philippine Rice Research Institute cited that 11.3 million tons of rice straw every year is being left on the field
during rice production.12 This study therefore seeks to find a novel application for rice straw. Although isolation of CNC from
rice straw was reported by Lu et al.13 using toluene solution and high concentration of sulfuric acid at high temperature, this
work employed relatively milder condition, i.e. lower acid concentration and temperature.

Materials and methods

Materials
Rice straws (R216 variety) were obtained from a farm in Science City of Mun˜oz, Nueva Ecija, Philippines. Technical grade
sodium hydroxide and sodium hypochlorite for the delignification and bleaching process were purchased from Alyson’s
Chemical Enterprise. Analytical grade glycerol, sulfuric acid and acetic acid were obtained from Ajax Chemicals. Food-grade
cornstarch with an amylose content of 31% supplied by Food Industries Inc. was used for the bioplastic preparation.
Preparation of cellulose fibers by delignification and bleaching
The method described by Elanthikkal et al.14 with slight modification was employed in the preparation of cellulose fibers. Fifty
grams of the cut rice straws was transferred into a 2-L beaker and treated with 700 mL of 10% w/v NaOH solution. The mixture
was heated with occasional stirring for 2 h at a temperature range of 60–65C removing the lignin, hemicellulose and other pectic
substances. After heating, the mixture was filtered and washed several times to separate the insoluble pulp and remove the excess
NaOH. The insoluble pulps which constitute the cellulose fibers were bleached with 500 mL of 1% v/v sodium hypochlorite
solution buffered to a pH of 5 by an acetate buffer. The bleached fibers were washed at least thrice with distilled water or until
the pH of the washing becomes neutral. The cellulose fibers were air dried and weighed.
Isolation of cellulose nanocrystals by acid hydrolysis
Ten grams of the bleached fibers were hydrolyzed in 100 mL 50% v/v H2SO4 at 30C for 3 h with constant stirring. Hydrolysis
was stopped by the addition of 100 mL cold 20% NaOH solution. The suspension was then centrifugated at 8500 r/min for 10
min obtaining the coagulated CNC. The coagulated CNC was suspended again in cold distilled water, and the centrifugation
process was repeated until the pH of supernatant reaches around 2–3. The resulting suspension was dialyzed against distilled
water to remove the excess sulfuric acid by using SpectraPor 4 dialysis membranes (12–14 kDa MWCO). When the pH of the
water outside the membrane reaches around 6–7, the suspension was collected. Every 200 mL of the CNC suspension underwent
sonication using Cole Palmer 4710 series ultrasonic homogenizer for 10 min in an ice bath at atmospheric condition. The
collected suspension was stored in a refrigerator and a portion of it was freeze dried for analysis that required dry samples. The
percent CNC in the suspension was determined using gravimetric method.
Characterization of the isolated CNCs
The FTIR spectrum of the isolated CNC was recorded from 4000 to 400 cm1 on a Perkin-Elmer Spectrum One spectrometer
equipped with attenuated total reflectance (ATR) accessory. The spectrum was collected from a total of 16 scans at a resolution
of 4 cm1 . A JEOL JEM 1010 transmission electron microscope (TEM) was used to determine the shape and size of the CNCs.
The CNC suspension was prepared in methanol at 100 mg/mL concentration and was diluted 5-fold. A drop of suspension was
placed onto carbon-coated grid at room temperature and then examined using TEM operating at 100 kV. The diameter of the
isolated CNCs was determined by using the software, SemAfore. For comparison, scanning electron micrographs of raw and
delignified rice straw were also taken. The x-ray diffraction patterns of the untreated and delignified rice straw, and the isolated
CNC were measured using the Rigaku (ultimate VII) X-ray diffraction instrument operated at a target voltage of 40 kV and
current of 40 mA. All samples were scanned at 2y ranging from 4 to 80 at a rate of 10/min. The crystallinity index was obtained
using the following equation15 Crystallinity index ¼ Imax Iam Imax x100% where Imax is the maximum intensity, Iam is the
minimum intensity that lies between the two most welldefined peaks.
Preparation of starch-CNC bioplastic film by solution casting
Ten grams corn starch was dissolved in 200 mL distilled water and heated at 60C for 15 min with constant stirring. CNC was
then added in the form of a suspension to avoid agglomeration. The amount of CNC per 100 g (dry weight) starch was varied to
identify the optimum starch to CNC ratio. Glycerol (3 g) was then added and the mixture was stirred for two h at a constant
temperature of 70C. The mixture was casted and cooled on glass plates and then dried at room temperature. Overall, bioplastic
films with starch to CNC ratios of 100:0, 100:2.5, 100:5, 100:10, and 100:15 were prepared.
Characterization of starch–CNC bioplastic film
The morphology of the fracture surface (cross-sectional surface) of the starch-CNC films were visualized using a scanning
electron microscope (SEM, JSM 5800). The films were frozen in liquid nitrogen and cut into small portions which were then
mounted vertically using a small metal support on the sample holder. The samples were sputter coated with gold using a sputter-
coater and viewed under the scanning electron microscope. Tensile strength and modulus were determined using the ASTM
D882-02 method. Samples with dimensions of 10 cm in length, 1.5 cm in width, and 0.20

0.04 mm in thickness were cut manually and conditioned at a relative humidity of 50

5% and temperature of 23

2C for 24 h. Films were then stretched by Instron 5585H tensometer using 50 N load cell at a crosshead speed of 12.5 mm/min.
The average values from five measurements were obtained and statistical significance of differences in means was evaluated
using the least significance difference analysis at 95% confidence level. Moisture uptake test was determined at conditions with
two different relative humidity values: 40% and 70%. Films were cut into 2 2 cm2 sheets, weighed, and oven dried at 50C for 24
h. Then, these were placed inside a desiccator with silica gel. The humidity was kept constant at 40% and monitored using a
hygrometer. The films were re-weighed and placed back in the desiccator and this process was repeated until a constant weight
that agrees within

0.0005 g is achieved (Wo). The silica gel was replaced by a saturated solution of sodium chloride to increase the humidity to
70%. The films were weighed again and placed back in the desiccator and this was repeated until a constant weight that agrees
within

0.0005 g was reached (Wh). The moisture uptake was reported as percentage and was calculated using the equation % Moisture
uptake ¼ Wh Wo Wo x 100: Thermogravimetric analysis (TGA) was carried out using TA Instrument Q50. The weight loss of
the bioplastic films due to thermal degradation was monitored as a function of temperature. Five milligrams of the film placed in
a platinum pan was heated initially to 110C for 10 min to remove bound moisture. The temperature was then ramped to 600C at a
heating rate of 20C/min under the flow of nitrogen gas.

Conclusions

Short, rod-like cellulose nanocrystals with a narrow width distribution of 10–12 nm and crystallinity of 76.1% can be isolated
from rice straw at a relatively milder condition (30C and 50% H2SO4) but longer hydrolysis time (3 h). The isolated CNCs can
be used as reinforcing filler for starch-based bioplastics. Tensile strength and modulus significantly increased with increasing
CNC load. Elongation at break, however, decreased. The moisture resistance of the starch-based bioplastics can also be enhanced
by CNC addition, which can be related to the rigid network of crystalline cellulose that hinders the absorption of water. However,
the inherent low thermal stability of sulfuric acid-hydrolyzed CNC decreased the thermal stability of the bioplastic. If aiming for
thermally stable CNCreinforced bioplastic, thermal stabilization of sulfuricacid hydrolyzed CNC should be considered for future
work.