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Pretreatmentofmicroalgalbiomassforenhancedrecovery/extractionofreducingsugarsandproteins

ARTICLEinBIOPROCESSANDBIOSYSTEMSENGINEERING·OCTOBER2015

ImpactFactor:2·DOI:10.1007/s00449-015-1493-5

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MarwaEldalatony

HanyangUniversity

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AkhilNKabra

HanyangUniversity

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Jae-HoonHwang

ArizonaStateUniversity

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Byong-HunJeon

HanyangUniversity

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ORIGINAL PAPER

Pretreatment of microalgal biomass for enhancedrecovery/extraction of reducing sugars and proteins

Marwa M. Eldalatony1 • Akhil N. Kabra3 • Jae-Hoon Hwang2 • Sanjay P. Govindwar3 •

Ki-Hyun Kim4• Hoo Kim1

• Byong-Hun Jeon1

Received: 3 August 2015 /Accepted: 19 October 2015

� Springer-Verlag Berlin Heidelberg 2015

Abstract Microalgae species including Chlamydomonas

mexicana, Micractinium reisseri, Scenedesmus obliquus

and Tribonema aequale were cultivated in batch cultures,

and their biochemical composition was determined.

C. mexicana showed the highest carbohydrate content of

52.6 % and was selected for further study. Sonication

pretreatment under optimum conditions (at 40 kHz,

2.2 Kw, 50 �C for 15 min) released 74 ± 2.7 mg g-1 of

total reducing sugars (TRS) of dry cell weight, while the

combined sonication and enzymatic hydrolysis treatment

enhanced the TRS yield by fourfold (280.5 ± 4.9 mg g-1).

The optimal ratio of enzyme [E]:substrate [S] for maxi-

mum TRS yield was [1]:[5] at 50 �C and pH 5. Combined

sonication and hydrolysis treatment released 7.3 %

(27.1 ± 0.9 mg g-1) soluble protein of dry cell weight,

and further fermentation of the dissolved carbohydrate

fraction enhanced the soluble protein content up to 56 %

(228.4 mg g-1) of total protein content. Scanning and

transmission electron microscopic analyses indicated that

microalgae cells were significantly disrupted by the com-

bined sonication and enzyme hydrolysis treatment. This

study indicates that pretreatment and subsequent fermen-

tation of the microalgal biomass enhance the recovery of

carbohydrates and proteins which can be used as feedstocks

for generation of biofuels.

Keywords Sonication � Enzymatic hydrolysis �Fermentation � Biofuel � Chlamydomonas mexicana � Totalreducing sugars

Introduction

Bioethanol, a promising biofuel that can substitute gaso-

line in combustion engines [1], has been produced at

commercial scale via fermentation of different carbohy-

drate-rich feedstocks such as sugarcane and corn (first

generation) and waste lignocellulose biomass (second

generation) [2, 3]. However, research on the microalgal

cellulosic based material (third generation) has gained

much attention as they have lacked lignin and can accu-

mulate large quantities of carbohydrates [4]. The main

challenge in bioethanol production from microalgal bio-

mass is to efficiently release fermentable sugars from

microalgal cells [5, 6].

The conversion of native cellulose from the microalgae

biomass to fermentable sugar is extremely slow, since the

cellulosic material is well protected by the cell matrix [7].

Pretreatment of the biomass enhances the rate of hydrolysis

to fermentable sugar as it increases the surface area,

enhances the sugar solubility, improves the substrate

digestibility and weakens the cell wall for enzymes to be

accessible [8, 9]. Several methods for algal cell disruption

including alkaline or acid reagents, bead beating, micro-

wave (220–1100 W/60 min), osmotic shock (with NaCl),

high-pressure homogenization and autoclaving (at 121 �C)

& Byong-Hun Jeon

bhjeon@hanyang.ac.kr

1 Department of Natural Resources and Environmental

Engineering, Hanyang University, Seoul 133-791,

South Korea

2 Swette Center for Environmental Biotechnology,

The Biodesign Institute at Arizona State University,

P.O. Box 875701, Tempe, AZ 85287-5701, USA

3 Department of Biochemistry, Shivaji University, Vidyanagar,

Kolhapur, Maharashtra 416004, India

4 Department of Civil and Environmental Engineering,

Hanyang University, Seoul 133-791, South Korea

123

Bioprocess Biosyst Eng

DOI 10.1007/s00449-015-1493-5

have been evaluated [10–13], but such methods are eco-

nomically infeasible as they require high temperatures and

addition of beads or chemicals [14]. Alternatively, ultra-

sonication technique has been considered as an emerging

technology with the potential to reduce reaction time and

chemical loading, and the capacity to modify the surface

structure of biomass with beneficial effects on the sac-

charification process [15]. Sonication has been extensively

used for cell lysis and homogenization, and has been

considered as a cost-effective pretreatment for disrupting

the rigid cell envelopes of microalgae [16, 17], but it has a

low efficiency to accumulate carbohydrates residues for

bioethanol production compared to enzymatic hydrolysis

[18]. Enzymatic hydrolysis has been reported as an effi-

cient method for the hydrolysis of microalgal cell wall [19,

20] and has been considered advantageous over acid

hydrolysis because of its higher selectivity and production

of low toxic hydrolysates compared to acid hydrolysis [21].

Fu et al. [22] reported a higher conversion yield (80 %) of

polysaccharides to fermentable sugars using enzymatic

hydrolysis [23, 24]. Carbohydrases have been used for

extraction of plant proteins at neutral and slightly basic pH

levels [25]. Microalgal soluble proteins after carbohydrate

fermentation process can be used for higher alcohol pro-

duction, which make the overall process cost effective with

increased utilization of the microalgal biomass towards

bioenergy [26].

The main objective of this work was to maximize the

release of reducing sugars and protein. A combined soni-

cation and enzymatic hydrolysis treatment was conducted.

Sonication with respect to temperature and time, and

enzymatic hydrolysis for [E]:[S] ratio, temperature and pH

were optimized. Dissolved protein concentration was

monitored during the sonication and enzymatic hydrolysis

treatments. Fermentation process was also evaluated for the

release of soluble proteins. Scanning electron microscope

(SEM) and transmission electron microscope (TEM)

analysis were used to observe the cell integrity after the

pretreatments.

Materials and methods

Cultivation of different microalgae strains

Chlamydomonas mexicana (YSL008), Micractinium reis-

seri (YSL004), Scenedesmus obliquus (YSL014) and Tri-

bonema aequale (YSR021) were isolated from an effluent

of a municipal wastewater treatment plant (Wonju, Water

Supply and Drainage Center, South Korea) and were sub-

mitted to the GenBank under accession numbers of

FR751193, FR751188, FR751171 and FR751201, respec-

tively. The molecular identification of the strains was

performed following the earlier protocol [27]. Microalgae

were cultivated in 5 L Erlenmeyer flasks with 4 L working

volume of Bold’s Basal Medium [28]. Cultures were kept

under white fluorescent light illumination at 60 lmol

photon m-2 s-1 and 26 ± 2 �C for 20 days. Cultures were

mixed using a magnetic stir plate and sparged with sterile

air (0.2 lm filters) at a flow rate of 0.6 L min-1.

Biochemical characterization of microalgae strains

The carbohydrate concentration was determined using

phenol–sulfuric acid method [29]. 10 mg was reacted with

5 mL of sulfuric acid and 1 mL of phenol (5 %) in a water

bath. The mixtures were incubated for 5 min at 90 �C, thenthe absorbance measurements (490 nm) were compared to

a standard curve based on glucose. The concentration of

individual sugars was determined using an ICS-5000 bio-

liquid chromatography (Dionex, USA) with CarboPac PA1

column [30]. Lowry method was used to determine the

protein content in the microalgae strains using bovine

serum albumin (BSA) as a standard protein substrate [31].

Amino acids were analyzed after acid hydrolysis of the

samples with 0.02 N HCl at 110 �C for 22 h. Wako (019-

08393) amino acids mixture standard solution, type H was

used as a standard solution. Ammonia content was also

presented as it comes from the degradation of some amino

acids (e.g., glutamine, asparagine) during acid hydrolysis

[32].

Optimization of sonication pretreatment

The microalgal biomass (C. mexicana) was harvested at the

end of the exponential phase using a centrifuge (Hanil

science industrial, 3000 rpm for 10 min), and was soni-

cated for 15, 30, 45 and 60 min at 30, 40 and 50 �C in a

sonicator (Branson 8510-DTH sonicator, Danbury, Con-

necticut, USA) at a constant frequency of 40 kHz with

maximum power output of 2.2 Kw [16]. At selected time

intervals, a 1.5 mL sample was withdrawn, filtered (syringe

filter, PES Membrane, 0.22 lm, 25 mm) and used for TRS

and protein analysis using microplate reader (Infinite M200

PRO Microplate Reader, NanoQuant from Tecan).

Optimization of enzymatic hydrolysis pretreatment

Cellulase from Trichoderma reesei ATCC 26921 was

purchased from Sigma-Aldrich, as a lyophilized powder

with concentration C1 Unit per mg of solid, and was used

for hydrolyzing C. mexicana biomass. One unit of cellulase

liberates 1.0 lmol of glucose from cellulose (substrate) in

1 h at pH 5.0 and temperature 37 �C [24]. The enzyme

cellulase was dissolved in sterile de-ionized (DI) water in

the presence of 0.15 % polyhexamethylene biguanide

Bioprocess Biosyst Eng

123

(PHMB) at 5 mg mL-1 concentration before the experi-

ments. PHMB was used as a disinfectant and antiseptic

agent. Hydrolysis of sonicated microalgae biomass was

performed in a 50 mM Sodium Acetate Buffer solution.

The sonicated biomass was incubated with the enzyme

solution at different [E]:[S] ratios (1:25, 1:10 and 1:5), pH

(3–6) and temperatures (20–60 �C). Each hydrolysis pro-

cess was conducted at a working volume of 50 mL in a

100-mL shake flask. The flasks were sealed with rubber

stoppers equipped with needles for CO2 emitting and

placed in a shaking water bath (HS-SHWB-30, Hansol

Tech) for 24 h at 120 rpm. At selected time intervals, a

1.5 mL sample was withdrawn and immediately heated for

5 min in a boiling water bath to terminate the enzymatic

reaction. The hydrolysis mixture was centrifuged at 1300g

for 10 min to remove the solid matter. The supernatants

were used to monitor the total reducing sugar [29, 33], and

the degree of hydrolysis was calculated using following

Eq. [20]:

Hydrolysis yield ¼ TRS concentration ðg=LÞInitial concentration ðg/L) of substrate� 100 %

Release of soluble proteins during fermentation

Fermentation of the dissolved carbohydrate fraction (TRS)

released due to the pretreatments (sonication and enzy-

matic hydrolysis) was processed using Saccharomyces

cerevisiae YPH499 [Meyen ex E.C. Hansen (ATCC�

204679TM)]. Released protein fraction from the microalgal

biomass during the carbohydrate fermentation process was

monitored. S. cerevisiae was cultivated using yeast extract,

peptone, and dextrose (YPD) medium for 2–3 days. The

yeast biomass was harvested, washed and adjusted to 10 g/

L for inoculation in the fermentation rector. The pH of the

untreated, sonicated, and sonicated-hydrolyzed microalgal

cells was adjusted to 5 using 1 N HCl/KOH before yeast

inoculation. The fermentation experiments were performed

in triplicate using 250 mL serum bottles with a working

volume of 150 mL. The headspace of each bottle was

flushed with N2 gas for 15 min to provide an anaerobic

environment and then sealed tightly with a butyl rubber

stopper and aluminum crimp. The bottles were placed in a

shaking water bath (HS-SHWB-30, Hansol Tech) at 37 �Cand 120 rpm for 3 days. During carbohydrate fermentation,

the dissolved protein concentration was monitored. A

separate reactor containing yeast only was operated as a

control, and the protein concentration observed in the

control reactor was subtracted from the values obtained

from the reactor containing yeast ? algae to determine the

amount of proteins from algae only.

Scanning and transmission electron microscopic

examination

The structure of untreated, sonicated, and sonicated-hy-

drolyzed microalgal cells was observed with low-vacuum

scanning electron microscope (LV-SEM, S-3500N, Hita-

chi) and transmission electron microscope (TEM, Leo

912A 8B OMEGA EF-TEM, Carl Zeiss, Germany) at

120 keV electron energy emission. The microalgae cell

suspension was fixed using 4 % glutaraldehyde in 0.1 M

cacodylate buffer (pH 7.4), then post-fixed in 1 % OsO4

with 0.1 M cacodylate buffer for 1 h and rinsed with 0.1 M

cacodylate buffer. The cells were pelleted, fixed in glu-

taraldehyde, dehydrated in a series of Et-OH solutions and

embedded in EPON resin. The polymerized blocks were

anaerobically sectioned on a microtome and thin sections

were mounted on copper grids coated with Formvar and

carbon for TEM analysis [15, 16].

Statistical analysis

All experiments were conducted in triplicates and the data

were presented as the mean ± standard error mean (SEM).

One-way analysis of variance (ANOVA) followed by

Tukey’s multiple comparison test was used to examine the

difference among individual treatment and optimum con-

dition. GraphPad Prism version 5.0 for Windows (Graph-

Pad Software, Inc., USA) was used for all statistical

analysis and difference in the variables was considered

significant at the P\ 0.05 of confidence.

Results and discussion

Biochemical characterization

Carbohydrate quantification showed that C. mexicana

possessed the highest carbohydrate content of dry cell

weight (52.6 %) compared to T. aequale (47.72 %),

S. obliquus (41.4 %) and M. reisseri (20.23 %), and was

selected for further investigation. The carbohydrate frac-

tion of C. mexicana was divided into glucose (63.67 %),

mannose (24.33 %), galactose (8.66 %) and glucosamine

(3.34 %) (Fig. 1). Protein content of C. mexicana, T. ae-

quale, S. obliquus and M. reisseri was 37.40, 46.04, 25.50

and 43.72 % of dry biomass, respectively. Recently, pro-

teins have also gained much attention for generation of

higher alcohols. Huo et al. [34] modified the Escherichia

coli cells through metabolic engineering such that they can

de-aminate algal protein hydrolysates, enabling the cells to

convert proteins into C4 and C5 alcohols with 56 % the-

oretical yield. The target strain (C. mexicana) showed

37.4 % protein content which was further subjected to

Bioprocess Biosyst Eng

123

amino acid analysis. Leucine (Leu.), Asparagine (Asp.),

Glutamine (Gln.), Alanine (Ala.), Glycine (Gly.), Thre-

onine (Thr.) and Valine (Val.) accounted higher percentage

which represent *64 % of total amino acids (Fig. 1).

Threonine, glycine and valine have been reported to be the

major fermentable amino acids which can be converted

into (iso)butanol [35]. Protein and amino acid analysis will

give a preface for our future studies on protein

fermentation.

Optimization of sonication treatment

Sonication process as a pretreatment method for TRS

production was optimized with respect to temperature (30,

40 and 50 �C) and time (15, 30, 45 and 60 min). TRS

concentration increased with increasing sonication tem-

perature from 30 to 50 �C (Fig. 2), indicating a significant

influence of temperature on the TRS production. TRS

obtained at 50 �C were significantly different (*P\ 0.05)

than TRS produced at 30 and 40 �C. A slight increase in

TRS was observed from 15 to 60 min at 50 �C. Consid-ering the time and energy consumed, we selected 50 �Cand 15 min as optimum sonication conditions, which

released 14 % TRS of total carbohydrate. Jeon et al. [15]

reported that 32.4 % of dissolved carbohydrate was

released after sonication of microalgal biomass. Sonication

treatment involves the transmission of sonic waves through

the microalgal culture. The waves create a series of micro

bubbles cavitation, which impart kinetic energy into cell

surface, leading to the disintegration of the cell wall and

release of the intracellular carbohydrates into the exocel-

lular medium [14]. Sonication treatment in this study

enhanced the sugar solubility, but it showed a low effi-

ciency to release the carbohydrates residues. Pretreatment

of microalgae promotes the disintegration of polysaccha-

ride complexes which increases the availability of substrate

for enzymatic hydrolysis, facilitating the subsequent fer-

mentation process [36]. Therefore, enzyme hydrolysis of

the microalgal biomass was performed after sonication

treatment to enhance the TRS production as it offers higher

yield and selectivity, and mild operating conditions than

acid hydrolysis.

Optimization of enzymatic hydrolysis treatment

Effect of [E]:[S] ratio

Sonication treatment resulted in partial disintegration of

microalgal biomass, making the polysaccharides more

amenable to enzymatic hydrolysis. The optimization of

enzymatic hydrolysis (cellulase form T. reesei) conditions

with respect to [E]:[S] ratio, temperature and pH has been

described in Fig. 3. The TRS level increased along with

hydrolysis time up to 24 h producing higher simple fer-

mentable sugars (Fig. 3a). Comparative short duration

(24 h) for optimum release of fermentable sugars than

previous studies (50–80 h) [37–39] offers the advantages

of eliminating contamination, reducing inhibition effects

and making the process economically feasible. The TRS

yields after enzymatic hydrolysis of sonicated algal

Fig. 1 Amino acid profile and sugar composition of Chlamydomonas

mexicana. Results are expressed as percentage of each sugar/amino

acid [Leucine (Leu.), Asparagine (Asp.), Glutamine (Gln.), Alanine

(Ala.), Glycine (Gly.), Threonine (Thr.), Valine (Val.), Proline (Pro.),

Phenylalanine (Phe.), Lysine (Lys.), Arginine (Arg.), Serine (Ser.),

Isoleucine (Ile), Cystine (Cys.), Histidine (His.) and Methionine

(Met.)] and represent the real recovery of amino acid after analysis.

Concentration of Ammonia (NH3) corresponds to nitrogen recovery

from some free amino acids destroyed during acid hydrolysis

Fig. 2 Effect of sonication conditions (temperatures and time) on

TRS production. The amount of TRS obtained at 50 �C was

significantly different than TRS obtained at 30 and 40 �C (*P\ 0.05)

Bioprocess Biosyst Eng

123

biomass at various [E]:[S] ratios increased linearly with

increases in incubation period from 0 to 24 h showing a

maximum TRS yield (150 mg g-1) at the ratio [1]:[5]. TRS

produced at [1]:[5] was significantly different

(***P\ 0.0001) as compared to TRS produced at [1]:[10]

and [1]:[25]. The TRS concentration was decreased at

higher [E]:[S] ratios (1:10 and 1:25), which can be attrib-

uted to increased substrates’ viscosity, leading to inefficient

hydrolysis [24, 40]. High viscosity increases the contents of

insoluble materials, which hinders the efficiency of enzyme

to hydrolyze the substrates, and causes an end-product

inhibition and mass transfer limitations within the reaction

mixture, leading to a low TRS yield [41].

Effect of temperature

Temperature is a variable that has a significant effect on

enzyme activity. The hydrolysis of C. mexicana was car-

ried out at different temperatures ranging from 20 to 60 �Cwith the optimum [E]:[S] ratio (1:5) and pH 5. As illus-

trated in Fig. 3b, the TRS yield for 24 h ascended with an

increase in temperature from 20 to 50 �C and then des-

cended with a further increase in temperature to 60 �C. Theoptimum enzymatic hydrolysis temperature was 50 �C,reflecting the highest TRS yield of 280.5 mg g-1 after 24 h

of hydrolysis. The amount of TRS obtained at 50 �C was

significantly different (***P\ 0.0001) than TRS produced

at 20, 30, 40 and 60 �C. The optimum temperature for the

enzymatic hydrolysis of different cellulosic biomass has

also been reported to be 50 �C [19, 39, 42]. An increase in

the temperature affects the kinetic energy of enzymatic

reactions, which increases the frequency of collision

between the substrate and the active sites of an enzyme.

Such a thermal agitation may lead to denaturation of

enzymes, thereby reducing the availability of active sites

[43]. This explains the low TRS yield observed at high

temperature (60 �C) compared to the optimum temperature

(50 �C). Kim et al. [44] reported that over-dehydration of

TRS was incurred on exposure to high temperature,

resulting in the formation of by-products such as 5-Hy-

droxymethylfurfural, levulinic acid, formic acid and char.

Effect of pH

The effect of pHs (3–6) on the hydrolysis of C. mexicana

was investigated using optimum [E]:[S] ratio (1:5) and

temperature (50 �C) (Fig. 3c). The increase in medium pH

from 3 to 5 increased the TRS yield from 140 to

274 mg g-1 after 24 h of hydrolysis. The highest enzyme

activity was observed at pH 5, resulting in the release of

52.1 % TRS of total carbohydrates after 24 h of hydrolysis.

This finding was in agreement with the results reported by

Ingesson et al. [41], where the optimum glucose concen-

tration was observed at a pH ranging from 4.5 to 5.0. The

present results showed that TRS yield dropped drastically

with the subsequent increase in pH to 6. TRS obtained at

pH 5 were significantly different (***P\ 0.0001) than

TRS produced at pHs of 3, 4 and 6). Deflection of pH from

the optimum value affects the electrostatic bonding

between the enzyme and substrate during the hydrolysis

process [24]. The enzyme undergoes conformational

changes on disruption of their charge, making the active

site no longer suitable to catalyze the hydrolysis reactions

[43, 45].

Fig. 3 Effect of different enzyme [E]:substrate [S] ratios (condition:

pH 5 and 37 �C) (a), different temperatures (condition: ratio [1]:[5]

and pH 5) (b), and different pHs (condition: ratio [1]:[5] and

50 �C) (c), on TRS production by enzymatic hydrolysis. TRS

produced at [1]:[5], 50 �C and pH 5, were significantly different

than TRS obtained under other conditions (***P\ 0.0001)

Bioprocess Biosyst Eng

123

A comparison of the results reported in previous studies

with the results of this study for TRS production from algae

by enzymatic hydrolysis is shown in Table 1. Various

enzymes alone or in combination have been used to

hydrolyze the microalgae cells for maximizing the TRS

production. The TRS yield ranged from 120 to

327.2 mg g-1 biomass (Table 1). The variation might be

due to the differences in enzyme type and concentration,

pH value, temperature and microalgae species.

Release of protein during pretreatment

and subsequent fermentation

Utilization of the released proteins for higher alcohol

production will render the overall process cost effective by

increasing the recovery of bioenergy from microalgae

biomass. Dissolved protein concentration of C. mexicana

was monitored during optimization of sonication and

enzymatic hydrolysis treatments for maximizing the TRS

yield. Sonication treatment at 50 �C for 15 min liberated

2.3 mg protein g-1 biomass. Woods et al. [49] reported

that ultrasonication of the green filamentous algae (Cla-

dophora) resulted in 0.7 mg mL-1 protein yield. The

combined sonication and enzymatic hydrolysis treatment

increased the dissolved protein concentration up to

27.1 ± 0.9 mg g-1 biomass (Fig. 4). Carbohydrases attack

the carbohydrate components of the cell wall, decreasing

the cell wall integrity and increasing the liberation of the

intracellular protein pool [50].

Fermentation of microalgal carbohydrates for bioethanol

production leads to a simultaneous release of 56 %

(228.4 mg g-1) protein fraction under the combined

sonication and hydrolysis treatment. The amount of proteins

released during the fermentation of combined sonicated-

hydrolyzed biomass was significantly different as compared

to proteins liberated from untreated and sonicated alone

(***P\ 0.0001). The dissolved protein concentration was

increased up to 51.6, 108 and 228.5 mg g-1 during fer-

mentation of the untreated, sonicated, and sonicated-hy-

drolyzed microalgae biomass, respectively (Fig. 5).

Scanning and transmission electron microscopic

examinations

Scanning and transmission electron microscopes were used

to observe the cell integrity after the pretreatments. Scan-

ning electron microscopy (SEM) analysis revealed ultra-

Fig. 4 Concentration of dissolved protein released during combined

sonication and enzymatic hydrolysis

Table 1 Comparison of the results reported in previous studies with the results of this study for TRS production from algae by enzymatic

hydrolysis

Enzyme pH Temp �C Substrate Total reducing sugar (TRS)

yield

References

Pectinase (p4716) 4 35 Teraselmis suecica 400.0 mg (g biomass)-1 [46]

Cellulase (C1 U/mg) 4.8 40 Chlorococum humicola 64.2 % (w/w) [24]

Novozyme 188 (263 CBU/g) ? Celluclast 1.5 L

(798 EGU/g)

4.8 50 Eucheuma cottonii 327.2 mg (g biomass)-1 [20]

Cellulase (C1 U/mg) 5 50 Chlamydomonas

mexicana

280.5 mg (g biomass)-1 This study

Cellulase (22119) 4.8 45 Ulva fasciata 206.0 mg (g biomass)-1 [47]

Cellulase (10 U/g) ? b-glucosidase (5 U/g) 5 30 Parthenium

hysterophorus

187.4 mg (g biomass)-1 [23]

Novozyme 188 (263 CBU/g) ? Celluclast 1.5 L

(798 EGU/g)

4.5 55 Nannochloropsis

gaditana

129 mg (g biomass)-1 [1]

Cellulase (50 FPU/g) ? b-glucosidase (250 CBU/g) 4.8 50 Sargassum sp. 120 mg (g biomass)-1 [42]

Cellulase (20 FPU/g) ? b-glucosidase (60 U/g) 5 50 Gracilaria verrucosa 870 mg (g cellulose)-1 [39]

(Celluclast 1.5 L, Novoprime B957), ?

Amyloglucosidase (300L)

5.5 55 Dunaliella tertiolecta

LB999

42.0 % (w/w) [48]

Bioprocess Biosyst Eng

123

structural changes in C. mexicana during sonication and

enzymatic hydrolysis. As shown in Fig. 6a, the surface of

untreated sample was smooth and continuous. The soni-

cated samples showed partially ruptured cell wall (Fig. 6b),

while enzymatic hydrolysis increased the rupturing of cell

wall. The cell wall of sonicated-hydrolyzed cells appeared

to be thinner after enzymatic hydrolysis, indicating the

release of carbohydrate constituents of cell wall into the

medium (Fig. 6c).

Transmission electron microscope (TEM) images

showed that algae cells were lysed to a greater extent by

enzymatic hydrolysis compared to sonication (Fig. 6). The

nucleus materials in the nucleus membrane were clearly

visible and well defined for the untreated algae (Fig. 6d).

The nucleus materials in the sonicated microalgae (Fig. 6e)

were less spread throughout the cell interior compared to

enzymatic hydrolyzed microalgal cells where some of the

nucleus materials were spread outside the cell and were

accumulated within the algal periplasm because of the

complete lysis of the nucleus membrane. Enzymatic

hydrolysis altered the morphology of the microalgae cells,

which is in agreement with a previous study [48]. The TEM

micrographs of untreated C. mexicana showed well-defined

shapes with typical smooth surfaces and the outline of a

regular cell wall (Fig. 6d). The cell wall was disintegrated

after hydrolysis with the release of cell wall-associated

carbohydrates and proteins into the exo-cellular medium

(Fig. 6f). This finding confirmed the disintegration of the

samples’ cellulosic structure due to the enzymatic con-

version of cellulose to its constituent sugars [19].

Fig. 5 Effect of carbohydrate fermentation on protein release of

untreated, sonicated and combined sonicated-hydrolyzed microalgal

biomass. (Condition; 37 �C, 120 rpm for 3 days). Values are a mean

of three experiments ± SEM. The amount of proteins released during

the fermentation of sonicated-hydrolyzed biomass was significantly

different compared to untreated and sonicated treatment biomass

(***P\ 0.0001)

Fig. 6 SEM and TEM images showing the destruction of the cell

wall on the microalgae surfaces and in periplasm. SEM images of

untreated (a), SEM images of sonication treated (b), SEM images of

sonication and enzyme hydrolysis treated (c), TEM images of

untreated (d), TEM images of sonication treated (e), and TEM

images of sonication and enzyme hydrolysis treated (f), Chlamy-

domonas mexicana cells after 24 h of hydrolysis under optimized

conditions

Bioprocess Biosyst Eng

123

Conclusion

The combined sonication and enzymatic hydrolysis pre-

treatment of microalgae C. mexicana enhanced the release

of TRS and dissolved protein fractions up to 53 and 7 %

compared to sonication alone (14 and 2.3 %), respectively.

Fermentation of the TRS fraction enhanced the dissolved

protein fraction up to 56 %. SEM and TEM analyses

confirmed the complete cell distraction by combined son-

ication and enzyme hydrolysis treatment. These results

demonstrate that the combined sonication and enzymatic

hydrolysis pretreatment enhances the conversion of

microalgae biomass to soluble sugar, and subsequent fer-

mentation of the sugar residues significantly increases the

release of microalgal proteins. Such approach will render

the algae-based biofuel technology cost effective by

increasing the conversion of microalgae biomass to feed-

stocks (TRS and protein) for bioalcohol production.

Acknowledgments This work was supported by the Mid-career

Researchers Program (the National Research Foundation of Korea,

2013069183).

References

1. Hernandez D, Riano B, Coca M, Garcia-Gonzalez MC (2015)

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