Bioresource Technology Reports 6 (2019) 81–84

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Bioresource Technology Reports

journal homepage: www.journals.elsevier.com/bioresource-technology-reports

Production of crude bioplastic-beads with microalgae: Proof-of-concept T

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Naohiro Kato
Department of Biological Sciences, Louisiana State University, USA

A R T I C LE I N FO A B S T R A C T

Keywords: The demand for plastics that are produced from biological resources, easily recyclable and biodegradable, has
Chlamydomonas increased in recent years. However, their application in plastic-bead industries, such as recreational shooting
Triacylglycerol sports, fishing, and disposable ornaments, has been limited due to high cost of the production. One of reasons for
Crude bioplastic the high cost is involvement of extraction and purification processes of biopolymers, the raw materials produced
Mardi Gras beads
in organisms for bioplastic production. It was previously found that Chlamydomonas reinhardtii produces a large
amount of triacylglycerol, the ingredient of biopolymers, following centrifugation (Kato et al., Biteb, doi:https://
doi.org/10.1016/j.biteb.2018.10.003). This article reports that the triacylglycerol produced in Chlamydomonas
reinhardtii can be directly molded, without extraction and purification, into a 7 mm bead that withstands
compressive stress to 1.7 megapascals. With further research and development, the method developed in this
study may help to produce disposable bioplastic-beads economically in industry.

1. Introduction
Disposable plastic beads are widely used in a number of applica-
tions. Examples of markets for plastic beads include pellets for recrea-
tional shooting sports, beads for fishing rulers, and beads for disposable
ornaments such as bead necklaces. The market for plastic beads in the
U.S. was estimated to be about $80 million in 2015. In recent years, the
demand for biologically produced and degradable plastic beads (bio-
plastic beads) has increased due to increasing public concern about the
environment, climate change, and limited fossil fuel resources (Endres,
2017). Although the raw materials for bioplastics, biopolymers, are
commercially available, these biopolymers are not economically com-
petitive with petroleum-based polymers due to their high cost of ex-
traction and purification (Endres, 2017; Rujnić-Sokele and Pilipović,
2017). Unlike other bioplastic markets that may reach a value of $324
billion annually in just over a decade, and already filled with major
chemical companies, the use of bioplastics in the bead industries is
limited. An example of the limitation is Mardi Gras beads, plastic
necklaces thrown during the annual Carnival celebration in Louisiana
and elsewhere in the U.S. The market for Mardi Gras beads is estimated
about $50 million annually. It is a tradition in Louisiana that riders of
Mardi Gras parades (krewes) throw the beads to crowds of onlookers.
Although Mardi Gars beads are historically made of glass, recent Mardi
Gras beads are made of petroleum-based polyethylene or polystyrene,
known as mold-on-thread (MOT) beads in the industry. An analysis
estimates that over 100,000 metric tons of plastic ornaments, including
MOT, are imported to New Orleans, the largest city in Louisiana, each
year for the celebration, but many of them end up in landfills. In 2018,
more than 46 tons of the plastic ornaments clogged a five block stretch
of the parade route, causing the New Orleans City Council to spend $22
million to clean and repair thousands of catch basins. Local organiza-
tions encourage recycling the beads and throwing eco-friendly beads
(i.e., paper beads) to conserve resources while preserving the tradition.
However, the impact of the efforts has been limited (Galofaro, 2012).
Because people on the parade route seek krewes to throw many Mardi
Gras beads, inexpensive Mardi Gras beads are widely used (Remon,
2014). Although some entities have tried to make a profitable business
in biodegradable Mardi Gras beads, success has been limited. For ex-
ample, a private company in New Orleans, which invented and mar-
keted biodegradable beads with recycled paper in 2012, is not viable as
of 2018 due to lower product sales than anticipated, attributed to low-
cost competition from imports. One of the solutions to reduce the
production cost of the bioplastic beads is to mold crude bioplastics, a
whole biomass accumulating the biopolymers. A study showed that a
biomass of bacteria accumulating PHA (polyhydroxyalkanoate), the
biopolymer, can be molded without the extraction and purification of
PHA (Ivanov et al., 2015). It would reduce the cost of bioplastic pro-
duction. However, the cost to cultivate the bacteria cannot compete
with the price of petroleum-based polymers.
Triacylglycerol (TAG) is an esterified molecule derived from a gly-
cerol and three fatty acids and can be a raw material for bioplastics
(Galià et al., 2010; Seniha Güner et al., 2006). Studies have shown that

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226 Life Sciences Building, Louisiana State University, Baton Rouge, LA 70803, USA.
E-mail address: Kato@LSU.edu.

https://doi.org/10.1016/j.biteb.2019.01.022
Received 14 October 2018; Received in revised form 31 January 2019; Accepted 31 January 2019
Available online 07 February 2019
2589-014X/ © 2019 Elsevier Ltd. All rights reserved.
N. Kato
TAG can be crosslinked through the coupling of free radicals that are
formed from the decomposition of hydroperoxide, typically when TAG
include unsaturated fatty acids (Meier, 2007). TAG production in mi-
croalgae has been widely studied for industrial use because microalgae
are capable of producing TAGs efficiently (Ghasemi et al., 2012).
Among the microalgae, Chlamydomonas reinhardtii (taxonomically
classified in the Chlorophyta division) has been studied as a model
organism (Harris, 2001). The studies on Chlamydomonas found that it
produces TAG when it is grown in stressful environments, such as ni-
trogen (N) depletion (Siaut et al., 2011), iron deficiency (Urzica et al.,
2013), high salinity (Siaut et al., 2011), or high temperature (Legeret
et al., 2016). Most recently, Chlamydomonas was found to accumulate
TAG following centrifugation (Kato et al., 2018). Similarly, starch, a
polymeric carbohydrate, can be a raw material for bioplastics (van
Soest et al., 1996). Starch can be cross-linked by reinforcing the hy-
drogen bonds (Chen et al., 2015). It is found that Chlorella vulgaris, also
classified in Chlorophyta division, produces starch very efficiently
under N depletion (Yao et al., 2012).
In a previous study (Kato et al., 2018), it was shown that Chlamy-
domonas reinhardtii can simultaneously produce both TAG and lutein,
the carotenoid used in pharmaceutical, dietary supplement, and food
industries, (Abdel-Aal et al., 2013). Here, use of algal biomass to pro-
duce crude bioplastic-beads is considered. It is rationalized that TAG
and/or starch accumulated microalgae can be used as crude bioplastics.
A concept is to produce crude bioplastics economically. By ex-
tracting high value compounds prior to molding the crude bioplastic
materials, the producer would be able to have two independent profits.
A profit generated by the high-value compounds could further com-
pensate the cost of the crude plastic production to compete with the
price of petroleum-based polymers.
2. Materials and methods
2.1. Microalgae strains and culture conditions
Chlamydomonas reinhardtii strain D66 was obtained from Dr. James
Moroney (Louisiana State University, Department of Biological
Sciences). Chlorella vulgaris was obtained from Culture Collection of
Autotrophic Organisms at the Institute of Botany of the Czech Academy
of Sciences (Stock#: CCALA 924). The Chlamydomonas and Chlorella
strains were cultured in 250 mL flasks containing 100 mL TAP (Tris-
acetate-phosphate) medium (Gorman and Levine, 1965) and BBM
(Bold's Basal Medium) (Bold, 1949), respectively. The cultures were
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Bioresource Technology Reports 6 (2019) 81–84
Fig. 1. Algal beads maintain strength.
(A) Appearance comparison of beads. Plastic bead: a
bead in Mardi Gras beads (mold-on-thread beads)
thrown during a Mardi Gras parade was detached.
Plastic bead paint removed: Paint coated on the
plastic bead was removed. Algal bead: algal biomass
subjected to centrifugation-induced triacylglycerol
production was molded manually. Algal bead coated
with acrylic paint: the molded bead was painted with
paint blush manually.
(B) Compressive strength of the algal beads cross-
linked and molded with 10% (w/d.w. biomass) am-
monium persulfate and 10% (w/d.w. biomass) gly-
cerol, respectively. Dashed red line: The result of an
algal bead (5.5 mm in diameter, no paint) molded
with 10% (w/d.w. biomass) ammonium persulfate,
the radical producer. Blue solid line: The result of an
algaplastic bead (6.95 mm in diameter, no paint)
molded with 10% (w/d.w. biomass) glycerol, the
plasticizer. (For interpretation of the references to
colour in this figure legend, the reader is referred to
the web version of this article.)
kept at 23 ± 2 °C under fluorescence light (60 μmol per m2 s) and
constantly shaken on an orbital shaker at 180 rpm. Every 7 days, 100 μL
of the culture solution was transferred to a freshly prepared 100 mL
medium.
2.2. Bead molding
Chlamydomonas and Chlorella were cultured in 2 L flasks containing
1 L culture medium, TAP and BBM, respectively. The cultures were kept
at 23 ± 2 °C under fluorescence light (60 μmol per m2 s) and constantly
shaken on an orbital shaker at 180 rpm. The culture was centrifuged in
4 × 250 mL centrifuge bottles (Nalgen) at 4000g for 5 min. The bottles
with the cap were left on a laboratory bench at room temperature after
the supernatant was decanted. Chlamydomonas strain subjected to the
centrifugation-induced TAG production accumulates 55 ± 17 μg per
106 cells (equivalent to about 30% dried weight) (Kato et al., 2018). On
the other hand, Chlorella vulgaris does not accumulate TAG through the
process (Kato et al., 2018). It was, however, rationalized that the
compounds in the Chlorella biomass such as starch can contribute to the
crosslinking of the molecules within the mixed biomass. The biomass
(0.1 g each) of Chlamydomonas and Chlorella were mixed on a micro-
scope slide (Corning 294875X25) and 10% (weight per dry weight
biomass) of ammonium persulfate or glycerol was mixed. To enhance
the radical production and crosslinking among the molecules in the
biomass, but not to release starch and TAG, the biomass containing the
additive was autoclaved at 121 °C for 20 min. The biomass was then
molded into beads manually. The beads were left at room temperature
overnight.
2.3. Compressive strength test on beads
Compressive strength test was conducted with Material Testing
System Model 810 (MTS Systems Corporation). The vertical compres-
sion load was applied to each bead with a compression rate at
0.02 mm s−1. The applied force in megapascal (MPa) was continually
monitored with the TestWare-SX software.
2.4. Carotenoids extraction by ethanol and the analysis
Centrifuged Chlamydomonas from which supernatant was decanted
(0.1 g dry weight biomass) was mixed with 10 mL of 95% ethanol in a
15 mL tube. The tube was then continuously rotated for 24 h at room
temperature. The Chlamydomonas biomass was collected by centrifuge
N. Kato
Fig. 2. Production of a crude bioplastic with Chlamydomonas biomass after
extracting carotenoids. TLC (thin layer chromatography) analysis of the ex-
tracted carotenoids. Commercially valuable carotenoids are indicated with an
arrow. A crude bioplastic produced from the carotenoids-extracted biomass is
shown in a window. Bar = 5 mm.
and used for molding. The supernatant was evaporated and re-
suspended in 20 μL of ethanol for the analysis. The contents of car-
otenoids in the solutions were analyzed by thin layer chromatography
as described in Kato et al. (2018).
3. Results and discussion
To examine the appearance and physical strength of the molded
beads, they were compared to petroleum-based Mardi Gras beads
thrown by a krewe during a Mardi Gras parade in Louisiana (Fig. 1A).
Individual beads were detached from the threaded Mardi Gras
beads. The beads were then compared to the algal beads that were
molded manually. The appearance between the beads was almost in-
distinguishable although the shape of the algaplastic beads was not
perfectly spherical due to manual molding. I also found that the algal
beads can be painted manually. Compressive strength test revealed the
point (1.70 MPa, 0.38 mm extension) where the algal bead cracks
(Fig. 1B). The same test with the petroleum-based Mardi Gras bead
revealed the cracking point is 12.08 MPa at 1.56 mm extension (Data
not shown). These suggest that the strength of the algal bead is about
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Bioresource Technology Reports 6 (2019) 81–84
14% of that made from the petroleum and similar to mud bricks that are
used for non-industrial constructions (Venkatarama Reddy and Gupta,
2006). This indicates that the beads made with the algal biomass
maintain certain physical strength when molded with ammonium per-
sulfate. When the bead is molded with 10% (w/d.w. biomass) of gly-
cerol (Fig. 1B) the stress and strain are linearly correlated, but the slope
is much lower than that with ammonium persulfate. This indicates that
the bead is elastic and suggests that adding a different additive in the
biomass could change the physical characteristics of the final products.
To examine whether Chlamydomonas biomass can be molded after
carotenoids are extracted, carotenoids were removed from the biomass
by an ethanol extraction prior to the bead molding. The bead was
successfully produced from the carotenoids-extracted biomass (Fig. 2).
However, the strength of the bead is not conclusive, largely varying
in beads. This is most likely due to co-extraction of TAGs with car-
otenoids by the ethanol extraction (dos Santos et al., 2015). Without
main ingredients that allows crosslinking within the biomass, it may be
difficult to sustain the strength. Extraction of the carotenoids prior to
molding crude bioplastics remains as the future scope.
The beads generated with crude bioplastics in this study may not be
strong enough for other industrial use. However, they may be sufficient
for use in applications such as MOT beads with further research de-
velopment. With respect to degradability of the bead, it was found that
the algal bead coated with acrylic paint (Fig. 1A) is not decomposed in
water for at least 7 days but would undoubtedly be shorter than the
time required for breakdown of plastic beads. It would be a future scope
of the bead production toward industrial uses.
Although a cost of the microalga-biomass production highly de-
pends on the scale and alga strain, National Renewable Energy
Laboratory reports that minimum biomass selling price to maintain the
facility is $0.5−1 kg (Davis et al., 2016). In this study, ammonium
persulfate was used as crosslinking reagent, which would cost
$4.0−1 kg for the production. In addition, operation cost of autoclave
and molding machines would cost $1.0−1 kg. This estimated produc-
tion cost is not competitive to plastic beads currently available in the
market. On the other hand, Extraction and purification cost of car-
otenoids from the biomass would cost $10–20 per kg while the purified
carotenoids would be sold $100 or more per kg. The profit from the
carotenoid production would compensate the high cost of crude bio-
plastic production. Further research and development are required to
reduce the cost of the production while increase the quality of the algae
based crude bioplastics.
4. Conclusion
This study shows crude bioplastic-beads can be produced from
Chlamydomonas. This technology may be applicable to other types of
microalgae that also produce commercially valuable compounds and
cross-linkable compounds, simultaneously.
Acknowledgements
This project was supported, in a part, by Board of Supervisors of LSU
LIFT2 program, and LSU Economic Development Assistantship. I thank
Dr. Guoqiang Li and his laboratory members at LSU for helping me to
test compressive strength of the beads. I also thank Dr. William Doerrler
at LSU for critical reading of the manuscript.
Conflict of interest
None.
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