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Journal of Polymers and theEnvironmentformerly: `Journal of EnvironmentalPolymer Degradation' ISSN 1566-2543 J Polym EnvironDOI 10.1007/s10924-013-0609-8

Characterization and Application inBiocomposites of Residual MicroalgalBiomass Generated in Third GenerationBiodiesel

Claudio Toro, Murali M. Reddy, RodrigoNavia, Mariella Rivas, Manjusri Misra &Amar K. Mohanty

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ORIGINAL PAPER

Characterization and Application in Biocomposites of ResidualMicroalgal Biomass Generated in Third Generation Biodiesel

Claudio Toro • Murali M. Reddy • Rodrigo Navia •

Mariella Rivas • Manjusri Misra • Amar K. Mohanty

� Springer Science+Business Media New York 2013

Abstract This research paper provides a brief discussion

about the relevance of third generation biodiesel co-prod-

ucts diversification. This diversification can be performed

through the utilization of residual microalgal biomass

(RMB) after oil extraction process. The present work

analyses the use of RMB as potential filler for biocom-

posite production by means of understanding the chemical

composition, the thermal stability as well as the protein

content of RMB. Thermogravimetric analysis revealed the

processing window of the RMB for biocomposite produc-

tion and its dependence on its purity, especially on residual

fat content. Biocomposites of RMB and poly(butylene

succinate) (PBS) were prepared by melting processing

technique using extrusion followed by injection-molding.

Tensile, flexural and impact properties of the processed

samples were evaluated. Scanning electron microscopy of

fractured sections of the biocomposites was also used to

examine the dispersion of RMB in PBS matrix. Finally,

this study shows a competitive alternative to produce PBS-

RMB biocomposites by replacing PBS by RMB in the

range between 20 and 30 %. However, further studies are

necessary to improve the compatibility of RMB with PBS

to obtain competitive mechanical properties, compared to

neat materials through, for instance, block co-polymers.

Keywords Microalgae � Residual microalgal biomass �Biomposites � Biofuel production � Poly(butylene

succinate)

Introduction

Among third generation feedstocks for the development of

bioproducts and biofuels, microalgae are one of the best

alternatives available. Microalgae can be grown in both

treated wastewater and saltwater and have significant

potential to produce bioproducts and lipid compounds

[1–4]. Therefore during the last few years, the microalgae

have been studied and used for biofuels and bioproducts

production. However, the big challenge is still making the

fuel production process from microalgae more efficient and

sustainable. Several authors suggest that the feasibility of

producing biofuels from microalgal biomass is directly

related to the ability to produce large-scale biomass [1, 5, 6].

According to Chisty (2007) if the biomass production

capacity is 10,000 ton year-1 the production cost per

C. Toro

Desert Bioenergy S.A., Piso 10, 2329 Vitacura, Santiago, Chile

Present Address:

C. Toro (&)

Centro de Investigacion de Polımeros Avanzados (CIPA),

Concepcion, Chile

e-mail: c.toro@cipachile.cl

M. M. Reddy � M. Misra � A. K. Mohanty

Department of Plant Agriculture, Bioproducts Discovery and

Development Centre, University of Guelph, Crop Science

Building, Guelph, ON N1G 2W1, Canada

R. Navia

Departamento de Ingenierıa Quımica y Nucleo Cientıfico

Tecnologico en Biorrecursos, Universidad de La Frontera,

Casilla 54-D, Temuco, Chile

M. Rivas

Centro de Investigacion Cientıfica y Tecnologica para la Minerıa

CICITEM, Universidad de Antofagasta, Antofagasta, Chile

M. Misra � A. K. Mohanty

School of Engineering, University of Guelph, Thornbrough

Building, Guelph, ON N1G 2W1, Canada

123

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DOI 10.1007/s10924-013-0609-8

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kilogram biomass ranges between US$0.47 and US$0.60.

Likewise Moazami et al. (2012) estimated the possibility of

producing 130 ton h-1 year-1 of microalgal biomass from

the marine strain of Nannochloropsis sp. (PTCC 6016)

which has the capability to produce 60,000 L of biodiesel

per year. With a large-scale microalgal biomass production,

a high residual microalgal biomass (RMB) generation is

expected. RMB may range between 30 and 60 % of the dry

processed biomass since lipids percentage in microalgae can

vary between 25 and 75 % depending on the strain and

growing conditions [1]. This biomass can contain small

remaining quantities of oils, and high quantities of proteins,

carbohydrates and ashes. Consequently, it has huge potential

as animal feed, anaerobic digestion for biogas production

and power generation as well as for composite materials,

among others. In spite of all these characteristics, the animal

feed industry is not prepared to use such huge quantities of

residual biomass. Furthermore, some of the co-products

generated in the biofuels industry are currently projected as

a source of animal feed and of course a very strong com-

petition for RMB. Moreover, the biogas production has seen

some interesting advances [7, 8] but it has some intrinsic

problems because of the large hydraulic retention times.

Besides, the total conversion of biomass are yet under

constant development [9]. Electrical-power generation by

direct combustion of biomass is a conservative alternative

for industrial residual heat recovery. However, this use

generates high emission levels which make this option

something unfeasible. The manufacture of composite

materials sounds as a hopeful alternative for the use of RMB

as several plant fibers and wood based fillers have been

already used in polymer matrices with good results [10–13].

In this work, samples of RMB of Botryococcus Braunii

(an interesting microalga for biofuel production due to its

high growth rate and high oils and chemicals content

[14–17]) have been used as filler for poly(butylene succi-

nate) (PBS), a biodegradable plastic, in biocomposites

fabrication. Nowadays biodegradable plastics are auspi-

cious materials as they can cover a wide range of appli-

cations due to their attractive properties [18–20]. They are

specially appreciated in agricultural applications where the

use of non-biodegradable plastics involves removing large

amounts of material after harvesting periods. Examples of

these products are mulch films and compostable bag

applications [21, 22]. However, these kinds of polymers are

available at higher cost than petroleum derived polymers.

For instance, the cost per kilogram of PBS is approximately

US$4, and therefore, even if RMB cost would be calculated

as virgin biomass [US$0.47 according to the estimated cost

by Chisty (2007)], 20 % replacement of RMB would mean

about 17 % savings.

Experimental Section

Microalgae Sample Supply

The microalgae used in this experiment, B. Braunii, were

cultivated in the Microbial Ecology Laboratory of the

University of Antofagasta, Chile. The microalgal suspen-

sion was harvested by continuous-flow centrifugation. The

dark-green microalga slurry was dried at 45 �C for 24 h.

Later, dry microalga was ground to obtain a fraction of fine

powder. This microalga was defatted by Soxhlet extraction

with petroleum ether, and the powder thus obtained

(moisture content 7 %) was preserved in plastic boxes for

further use.

Oil Extraction from Microalgae

Oil extraction of microalgal biomass was carried out using

petroleum ether (40–60 �C boiling point, supplied by

Merck) in a Soxhlet extractor. Thus 300 mL petroleum

ether was poured into a round bottom flask. Then 5 g of the

sample was placed in a thimble and was inserted in the

center of the Soxhlet extractor. The temperature was set at

60 �C. The extraction time was 6 h at a drip rate of

180 drops min-1. When the extraction was finished, the

solvent was evaporated by Rotary evaporator (Stuart

Rotary evaporator RE300) and the extracted lipids were

left in a desiccator until they reached constant weight. Oil

content was determined on a gravimetric basis and

expressed as percent of weight. Subsequently, defatted

microalgae biomass was dried at 80 �C under vacuum

overnight.

Protein Extraction

Protein extraction from defatted microalgal biomass was

carried out by alkaline extraction using water as solvent

medium and NaOH 4 M to adjust pH. Thus, the solvent/

cake ratio used in the extraction experiments was 90 and,

adding small aliquots of a 4 M NaOH solution, the pH was

adjusted to 12. The extraction was left for 1 h at 40 �C.

After that, the soluble protein fraction was separated from

the residual microalgal biomass by centrifugation. Subse-

quently, protein extraction yield was determined in the

soluble fraction as yp = (CpV/wd) 100; where Cp is the

protein concentration determined by Lowry method [23],

V is the solvent volume and wd is the dried defatted mic-

roalgal biomass weight. After protein extraction, residual

microalgal biomass (RMB) was obtained. The flour was

packed in polyethylene bags and stored at room tempera-

ture until use.

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Chemical Composition Analysis

The chemical characterization of microalgal and defatted

microalgal biomass was determined through a proximate

analysis which considers crude fiber content, protein con-

tent, oils content and nitrogen free extract content (calcu-

lated as the difference between 100 % and the sum of oils,

protein, crude fiber and ash). Oils, protein, crude fiber, and

ash content of the samples were analyzed according to the

Association of Analytical Communities (AOAC, Official

Methods) [24]. The chemical characterization of RMB was

determined after protein extraction, recalculating the

remaining quantity of components from the defatted mic-

roalgal biomass composition after extracting 49 % of

protein, based on total protein content.

FT-IR Spectroscopy

The identification of functional groups of starting materials

and injection moulded biocomposites was measured in a

Thermo Scientific NicoletTM 6700 FT-IR spectrometer,

USA. The spectrum was recorded at room temperature with

a resolution of 4 cm-1 and 32 scans per sample. Spectral

outputs were recorded in transmittance mode as function of

wave number.

Injection Moulded Biocomposites from RMB and PBS

Residual microalgal biomass (RMB) and PBS 1020 (sup-

plied for Showa High Polymers, Japan) were dried in

vacuum oven overnight at 80 �C and stored in Zip-loc bags

prior to processing. Processing experiments were carried

out in a micro twin-screw extruder with injection molding

system (TS/I-2, DSM, Netherlands). The composition of

the blends were fixed at 70/30 and 80/20 wt% PBS/RMB.

The micro twin-screw extruder is equipped with co-rotat-

ing screws which have a length of 150 mm, with L/D of 18

and net capacity of 15 cm3 and an attached injection

molding unit capable of 120–140 psi injection force. The

materials were melted at 130 �C and at 150 rpm screw

speed for 2 min (residence time) and after extrusion the

melted materials were transferred through a preheated

cylinder to the mini injection molding to obtain the desired

specimen samples for tensile testing, flexural testing and

impact testing analysis.

Tensile and Flexural Testing

Tensile and flexural testing was done on a Universal Test

System (UTS) load frame Instron according to ASTM

D638 and ASTM D790, respectively. Testing was done

with a cross-head speed of 5 mm min-1 for tensile tests.

Flexural test was stopped when the sample reached the 5 %

deflection or the sample was broken before 5 %. Four

samples were tested for each category and the average

values are reported.

Thermogravimetric Analysis (TGA)

The thermal stability of RMB, PBS 1020 and injection

moulded biocomposites (RMB ? PBS 1020) was mea-

sured by using a TGA Q500 (TA Instruments, USA)

thermo-gravimetric analyzer. Each sample was run from

room temperature (*20 �C) to 800 �C at a rate of 20 �C

min-1 in a nitrogen environment. The comparison of

thermal stability of starting materials and final biocom-

posites was performed to determine the effect of the

extrusion and injection process on the final composite

material.

Scanning Electron Microscopy (SEM)

Fractured samples from impact test were sputter coated

with gold/palladium (EmitechK550) under an argon flow.

Scanning electron micrographs were obtained using a

Hitachi S-570 scanning electron microscope at an accel-

eration voltage of 15 kV.

Dynamic Mechanical Analysis (DMA)

DMA of the PBS and the PBS/RMB blends were per-

formed using a DMA Q-800 (TA Instruments, USA) on

tension film clamp. Samples were analyzed from -60 �C

to 100 �C at a heating rate of 3 �C min-1, 1 Hz frequency

strain mode and 15 lm of amplitude.

Heat Deflection Temperature (HDT)

A dynamic mechanical analyzer (TA Instruments DMA

Q800), operated in the DMA controlled force mode with

three point bending clamps, was used to determine the heat

deflection temperature according to ASTM D648, under a

load of 0.455 MPa. The heating rate was 2 �C min-1 and

data was collected from 15 to 100 �C.

Results and Discussion

Chemical Composition Analysis

Chemical composition of microalgal biomass, defatted

microalgal biomass and RMB is shown in Table 1.

As shown in Table 1, microalgal biomass after oil and

protein extraction, i.e. RMB, is mainly composed by pro-

teins, nitrogen free extract and ashes. The remaining frac-

tions are a little percentage of fat and fibers. The presence

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of high protein content can be explained due to the rela-

tively low protein extraction yield (49 % based on total

protein content). The percentage of nitrogen free extract is

mainly due to a high quantity of polysaccharides normally

present in B. braunii strains [14, 16]. Regarding ash

content, it is commonly present in microalgal biomass

because of the high quantity of nutrients present in culture

media. These nutrients, basically including NPK sources,

remain in the microalgal sludge after the harvesting

process.

Thermal and FTIR Analysis of PBS and RMB Based

Biocomposites

The weight loss curves with the increase in temperature of

RMB, PBS and PBS-RMB biocomposites are presented in

Fig. 1a. It can be seen that RMB displayed a lower thermal

stability compared to PBS and also a multi-step degrada-

tion profile, indicating a multi-component system com-

posed of protein, carbohydrates, cellulose and residual fat.

This fact agrees with the chemical analysis were these

compounds were identified. On the opposite, PBS dis-

played single step degradation at 320 �C, while the deg-

radation temperatures of the composites have shifted

towards RMB degradation temperatures. Since dried sam-

ples were used for the experiments, no significant weight

loss was observed up to 100 �C in all the materials

analyzed.

Figure 1b shows DTGA curves obtained for different

composites (0, 20 and 30 % RMB). The temperature at the

maximum degradation rate was shifted slowly to lower

values as the filler content increased because of the

decrease in PBS, which is much more thermally stable than

RMB. In addition, the presence of RMB filler decreased the

thermal stability compared with that of the raw matrix,

Table 1 Chemical composition of the microalgae biomass, defatted

and residual (after extraction of oils and proteins)

Constituent (%)a Microalgae

biomass

Defatted

microalgae

biomassb

Residual

microalgae

biomass (RMB)c

Protein 50.9 48.0 31.9

Fat 17.4 4.0 5.2

Nitrogen free extract 14.3 22.6 29.5

Fiber 6.8 4.4 5.8

Ash 10.6 21.1 27.6

a The constituents are expressed as g 9 100 g21 of dry matterb Percentage of constituents after oil extractionc Percentage of constituents after protein extraction

Fig. 1 a TGA and b DTGA curves of RMB, PBS, PBS/RMB 80/20

and PBS/RMB 70/30 Composites

Fig. 2 FTIR spectrum of RMB, PBS, PBS/RMB 80/20 and PBS/RMB

70/30 Composites

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Fig. 3 SEM Micrographs of: (i) neat PBS a 92,500 and b 95,000, (ii) PBS ? RMB (80/20) c 92,500 and d 95,000, and (iii) PBS ? RMB (70/30)

e 92,500 and f 95,000

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because of the lower degradation temperature of the filler.

The same behavior was observed with the work of [25] and

[26] for composite materials. The thermal stability of the

developed composites follows the sequence: PBS [ PBS/

RMB 80/20 [ PBS/RMB 70/30 [ RMB.

Figure 2 shows the FTIR spectra of microalgal biomass

and its PBS composites. Residual algal biomass (RMB) is

obtained after oil and protein extraction and its chemical

composition mainly consists of proteins and carbohydrates,

moisture and little traces of cellulose. The FTIR spectrum

of RMB in Fig. 2, showed the presence of N–H and C–N

bonds at 1,620 and 1,050 cm-1 respectively, which cor-

respond to protein groups. RMB also shows the presence of

polysaccharides which have a characteristic absorption at

812 cm-1, corresponding to a C–H stretching peak of

mannose. In addition a peak at 1,110 cm-1, could be

typical for D-glucose in pyranose form, fused with a broad

peak assigned to C–N bond [27]. There is also a weak

narrow peak at 2,800 and 3,000 cm-1 which could corre-

spond to aliphatic fats and a weak broad peak around

3,100–3,700 cm-1 which could confirm a little moisture

content.

Regarding to RMB-PBS composites and pure PBS, as

can be seen in Fig. 2, they have very similar spectra

demonstrating that there is no important chemical inter-

action between RMB and PBS during the biocomposites

production process.

Material Properties of PBS and RMB Based

Biocomposites

Scanning Electron Microscopy (SEM) micrographs of PBS

and PBS-RMB biocomposites are given Fig. 3. The frac-

ture surfaces of the composites after tensile tests were used

for morphological analysis. It can be seen that for PBS

without any filler, the matrix is oriented in the direction of

the flow. This morphology changed with the addition of

RMB into the system. In fact, at a 20 % load, we can see

that the filler is dispersed within the matrix and no voids or

pullouts are observed. One reason for the observed phe-

nomenon is that the biomass filler was not used in fibrillar

shape. However, when increasing RMB content to 30 %, a

clear cellular morphology was observed along with the

uniform dispersion of the filler. Also, the morphology

shows that there is a poor interfacial adhesion between the

matrix and RMB. This situation agrees with the FTIR

analysis, indicating the need to use a compatibilizer for the

filler.

Understanding the mechanical properties of the bio-

composites is very important as it reveals the information

about the polymer-filler interaction and also helps knowing

the stress transformation phenomena in the system. In this

work, we have used residual algal biomass (RMB) as filler

for polybutylene succinate (PBS) matrix. The stress–strain

behavior of the PBS and PBS-RMB biocomposites are

shown in Fig. 4a indicating that the break behavior is

almost the same in both neat PBS and PBS-RMB com-

posites. In general, the stress–strain behavior of these

biocomposites is nonlinear, mainly because of the polymer

matrix deformation. As the filler loading increased, the

tensile stress and strain of all biocomposites dramatically

decreased when compared with those of neat PBS. This is

0

5

10

15

20

25

30

35

40

0 0.05 0.1 0.15 0.2

Ten

sile

Str

ess

(MP

a)

Tensile Strain (%)

PBSPBS/RMB (80/20)PBS/RMB (70/30)

0

200

400

600

800

1000

0

10

20

30

40

PBS PBS/RMB (80/20)

PBS/RMB (70/30)

Fle

x M

od

ulu

s (M

Pa)

Fle

x S

tren

gth

(M

Pa)

Flex Strength (MPa)

Flex Modulus (MPa)

(a)

(b)

Fig. 4 a Tensile (Stress–strain curves) and b flexural properties of

neat PBS, PBS/RMB (80/20) and PBS/RMB (70/30) Composites

Table 2 Tensile and impact properties of PBS and its algal residual biomass composite

Material Tensile modulus (MPa) Tensile strength (MPa) Percent elongation Impact strength (J/M)

PBS 583 ± 5 33.3 ± 0.4 14.2 ± 2.14 29.30 ± 2.26

PBS ? RMB (80/20) 689 ± 14 21.6 ± 0.7 8.6 ± 0.80 28.68 ± 2.67

PBS ? RMB (70/30) 780 ± 16 18.7 ± 1.0 7.6 ± 0.58 31.61 ± 1.72

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attributed to poor compatibility between the polar hydro-

philic microalgae powder and the nonpolar hydrophobic

PBS matrix. The content of microalgal powder in the

biocomposites reduced their ductility and increased their

brittleness under tensile deformation.

The tensile and impact properties of the PBS-RMB

biocomposites are presented in Table 2. It can be seen that

the addition of RMB has helped improving the tensile

modulus, while strength and percent elongation decreased

by increasing the filler content. However, Izod impact

strength did not decrease with RMB loading. Improvement

in the impact strength can be explained by the change in

morphology as observed in SEM micrographs where a

cellular type structure was observed with an increase in the

filler loading. It is well known that the cellular morphology

helps improving the shock absorption phenomena during

impact strength testing and thereby increasing the impact

strength values.

In general, stress is assumed to be transferred from the

matrix to the fiber by a shear transfer mechanism. If the

fiber length is too short, the matrix cannot grip it to take the

strain. Then, they are pulled out, instead of being broken

under tension. Achieving high mechanical strength of

composites requires a strong fiber–matrix-interface, which

enables stress transfer from the matrix to the fiber. Fur-

thermore, a uniform distribution of the fiber within the

polymeric matrix is necessary to obtain the full mechanical

advantages of them. Additionally, all of the developed

composites have displayed a decreased percent elongation.

The prepared composites had a poor interfacial adhesion

between the filler and the matrix and thereby displaying the

reduced tensile properties. However, this may be improved

with a compatibilizer or surface modification of the fillers.

In the current work, we have sought the understanding of

RMB as filler for polymeric systems hence further com-

patibility or surface modification is beyond the scope of

this study.

Flexural data (Fig. 4b) showed contradicting results for

all PBS-RMB biocomposites compared to their tensile

properties. Flexural strength maintained relatively constant

while flexural modulus increased with the increase in filler

content. This phenomenon was also observed in biopol-

yester-proteineous meal based biocomposites [28]. This

could be attributed to multiple cracking observed and also

due to the increased brittleness in composites [29].

The storage modulus of the PBS-RMB biocomposites as

a function of the temperature is shown in Fig. 5a. It can be

seen that the storage modulus of all the biocomposites

decreased by increasing the temperature. This behavior

may be due to the softening effect of the PBS matrix at

high temperatures due to polymer chain mobility [30].

However, storage modulus values of PBS-RMB biocom-

posites are higher than that of the PBS matrix itself. This

could be explained by the fact that RMB particles are

0

0.02

0.04

0.06

0.08

0.1

0.12

-80 -30 20 70 120

Tan

Del

ta

PBS PBS-RMB7030 PBS-RMB8020

(a)

(b)

Temperature (°C)

Fig. 5 Dynamic mechanical analysis of neat PBS, PBS ? RMB (80/

20) and PBS ? RMB (70/30), a Storage modulus and b Tan delta

0

20

40

60

80

100

PBS PBS/RMB (80/20) PBS/RMB (70/30)

Hea

t D

efle

ctio

n T

emp

erat

ure

(°C

)

Fig. 6 Heat deflection temperature of neat PBS, PBS ? RMB (80/

20) and PBS ? RMB (70/30)

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stiffer than PBS. The increased storage modulus with

increasing RMB loading in biocomposites could be

explained by physicochemical interactions, intramolecular

bonds, and a crystalline structure of the composite, which

improves the reinforcement provoked by RMB particles

that allowed stress transfer from the matrix to the filler

[31].

Figure 5b shows that the height of the tan d peak

decreased with the presence of RMB filler at 30 %. This

effect could be explained by the fact that there is no

restriction to the chain motion in PBS matrix, but the

addition of RMB filler hindered the chain mobility which

produces a sharpness and height of the tan d peak reduc-

tion, proving the presence of a rigid filler in the biocom-

posite system.

Heat Deflection Temperature (HDT)

The heat deflection temperature, HDT, defined as the

temperature at which a material deflects 0.25 mm under a

load of 0.455 or 1.82 MPa, is an important benchmark for

composites application. It tells us about the upper limit of

the temperature for the material to be used. Figure 6 shows

the HDT of PBS and its RMB composites obtained as

described in ‘‘Dynamic Mechanical Analysis’’. It can be

seen that the RMB addition to the matrix hasn’t alerted the

HDT of the matrix. And the reason for this behavior could

be the fact that RMB is mainly a proteineous material and

does not offer resistance like cellulosic fibers or mineral

fibers. Also, this suggests that the nature of the filler was

not compatible with the polymeric matrix.

Conclusions

Chemical composition analysis showed that protein is the

major fraction in RMB which presented a very narrow

processing window for biocomposites fabrication, only

with polymers with low melting point. PBS/RMB based

biocomposites displayed the same impact strengths as PBS

due to morphology change. SEM indicated that RMB

yields a cellular morphology type with PBS which helps

observing the energy from impact testing. DMA revealed

that rigidity improved with RMB as a result of polymer

chain mobility restriction. Finally, HDT didn’t show any

improvement compared to PBS. Compatibility of the bio-

composites might lead to improvement in the material

properties.

The present study has shown that the residual microalgal

biomass is potential filler for biocomposites fabrication; it

is also inexpensive, capable of replacing between 20 to

30 % of the polymer matrix, reducing production costs and

increasing the competitiveness of bioplastics.

Acknowledgments This work was supported by Desert Bioenergy

S.A. and the Chilean projects No 78110106 and VI2010-061 funded

by CONICYT-CHILE. Project DI10-7001, Universidad de La Fron-

tera. Ontario Ministry of Agriculture, Food and Rural Affairs

(OMAFRA)-2009 New Directions & Alternative Renewable Fuels

‘Plus’ Research Program-SR9223 and Hannam Soybean Utilization

Fund (HSUF)-2008.

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