) t?

ISS-CNS IOP Publishing

IOP Conf. Series: Earth and Environmental Science 31 (2016) 012034 doi: 10.1088/1755-1315/31/1/012034

Techno-economical evaluation of protein extraction for

microalgae biorefinery

Y W Sari12, J P M Sanders3, and M E Bruins 3

1
Bio based Chemistry and Technology, Wageningen University, P.O. Box 17 6700 AA
Wageningen, the Netherlands
2
Biophysics Division, Department of Physics, Bogor Agricultural University,
JI. Meranti, Gedung Wing S, Kampus IPB Darmaga, Bogor 16680, Jawa Barat,
Indonesia
3
Food and Biobased Research, P.O. Box 17, 6700 AA Wageningen, the Netherlands

E-mail : yessie.sari@apps.ipb.ac.id

Abstract. Due to scarcity of fossil feedstocks, there is an increasing demand for biobased
fuels. Microalgae are considered as promising biobased feedstocks. However, microalgae
based fuels are not yet produced at large scale at present. Applying biorefinery, not only for oil,
but also for other components, such as carbohydrates and protein, may lead to the sustainable
and economical microalgae-based fuels. This paper discusses two relatively mild conditions for
microalgal protein extraction, based on alkali and enzymes. Green microalgae ( Chlorella
fusca) with and without prior lipid removal were used as feedstocks. Under mild conditions,
more protein could be extracted using proteases, with the highest yields for microalgae meal
(without lipids). The data on protein extraction yields were used to calculate the costs for
producing 1 ton of microalgal protein. The processing cost for the alkaline method was 2448
/ton protein. Enzymatic method performed better from an economic point of view with 1367
/ton protein on processing costs. However, this is still far from industrially feasible . For both
extraction methods, biomass cost per ton of produced product were high. A higher protein
extraction yield can partially solve this problem, lowering processing cost to 620 and 1180
/ton protein product, using alkali and enzyme, respectively. Although alkaline method has
lower processing cost, optimization appears to be better achievable using enzymes. If the
enzymatic method can be optimized by lowering the amount of alkali added, leading to
processing cost of 633/ton protein product. Higher revenue can be generated when the
residue after protein extraction can be sold as fuel , or better as a highly digestible feed for
cattle.

1. Introduction
Owing to the scarcity of fossil feedstock, there is an increasing demand for biobased fuels.
Microalgae are considered one of the most promising biobased feedstocks, as their productivity in
converting carbon dioxide into lipids exceeds that of oilseed crops [l-3] . However, currently,
microalgae based fuels have not yet been produced at large scale. In depth works are being
conducted to enable microalgae-based fuel production in a sustainable and economical way.
Applying a biorefinery concept may aid here. Microalgae biorefinery uses the overall composition
of the algae, and next to lipids, including carbohydrates and proteins [3].
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ISS-CNS IOP Publishing
IOP Conf. Series: Earth and Environmental Science 31 (2016) 012034 doi: 10.1088/1755-1315/31/1/012034

Methods on microalgal protein extraction have been developed [4]. However, the yield is
still considered low, limiting commercial protein production from microalgae. This low protein
yield is due to the complexity of microalgae cell wall and microalgal protein properties. Extreme
extraction conditions can be used to obtain substantial cell wall degradation , but this may reduce
functional protein properties.
This paper discusses two relatively mild conditions for microalgae protein extraction. Green
microalgae (Chlarellafusca) with and without lipid are used as the feedstock. Two protein extraction
techniques were used; alkaline and enzyme assisted extraction. Data on the yield of extracted protein
were used to calculate cost for producing l ton of microalgae protein .

2. Materials and Methods

2.1 Materials
Microalgae meal (Chiarella fusca) and non-deoiled microalgae (microalgae) (Chiarella fus ca) were
obtained from Ingrepro B.V. , the Netherlands. Protex 40XL (activity: 52 MPU/ml) was obtained from
Genencor (Danisco) International Oy, Denmark.

2.2 Protein content analysis

Protein content in starting material and hydrolysate was determined using DUMAS analysis (FlashEA
1112 series, Thermo Scientific, Interscience). Methionine was used as a standard for the calibration
and a constant of 6.25 was used as a nitrogen-protein conversion factor to calculate the protein content
in the samples.

2.3 Protein extraction

Alkaline protein extraction was conducted as described in [5] while enzymatic assisted protein
extraction was conducted as described in [6] with some minor modifications.

2.3.1 Alkaline method

Protein extraction was conducted, according to [5], at three ascending temperatures : starting with a
one day incubation at 25C, followed by one hour at 60C, and ending with a one hour incubation at
120 C. Total protein yield obtained after three- step protein extraction was defmed as mass of
extracted protein relative to mass of protein in the starting material.

2.3.2 Protease assisted method

Protein extraction was conducted using Protex 40XL, a mixture of proteases, according to [6]. The
enzyme dosage in this study was 1 and 5% (volume enzyme per weight of protein). Total protein yield
obtained was defined as mass of extracted protein relative to mass of protein in the starting material.

2.4 Cost calculation

Cost analysis in this study focused on the main processing cost: biomass, NaOH, and heating. In the
case of the enzymatic method, enzyme costs were added. Biomass costs were calculated based on the
prices of microalgae meal and microalgae, which were 230 [7] and E 1650 [3] per ton dry mass.
NaOH cost were determined based the amount of NaOH required to extract protein per ton dry
biomass. The required NaOH mass was determined by lab scale experiments. Mass biomass used on
lab scale was 10 g. NaOH cost was determined as:

2
ISS -CNS IOP Publi shing
IOP Conf. Series: Earth and Environmental Science 31 (2016) 0 12034 doi: I0.1088/1755-1 3 15/3 111/012034

Jl;-.; ,,cm

~; l\)ll l<>t:...~!lr

m!.,to:nHS IJh.i(J.Je

;10H ( ~)
m:-.: ,on = mas .10H n'CJllll"L'd (ton)
P :-.:.1011 - pn " l\'.1 ( H _ +!. } -~ "Q
.:.ton ;: :.
1n 11 -~_;:::
. ["]

111"\ .iO H1..tn i .11,, =- m. 1ss I\,tOH nq111ncl dt1rJ11g l<lh sc;1lP txpt'rllJH'llt {g)
Ill i>iL~, 11 ,!".~ !.sbM ,1Jt. - 111 .1..;s dry !Jiomas'> d11rmg lr1li sc:ilp ('X(WrimP11t (g)
m hwr11 , ,.,., = mas~ dry hioma~s ( wn)

The energy costs w ere calculated in relation to the thennal energy required to heat up the mi-..;:ture
ofbiomass and sohent. Specific heat capacity for >-;at er is -l .2 J.g- 1.K and for biomass is assumed t o
be 1.3 J _g-:.K' (at _.5 :c i[9]. TI1eiatio ofbiomas.5 t o-..,atermedinthe cakulatiomis 1:9.

The themul energy required 'i";as calculated as :

The thennal energy in Joules is then come11ed to k\'\b nlue:;. L.!. J= 2.7 x 107 kV'l11) . 111e price
per k\\b used in the calculations: 0.0 -l 1 k\\b, is the price of natural gas for a mediurn -sized
indusuial comurner [10] .

.Assuming that~ O~ c ofheat inYohedin the extraction can be reco\'ered then the final energy cost is
defined as 50( x calculated energy cost .

The enz:.:me cos.ts used in this calculation >.<;ere based on the amount of enzyme that '-".'as used

Coo;;t 'l1i}1lJr -::- ll 1.11 1y111 r X ! \ . 11 !'111'

.~xm

Cos :u:m - co<;t '1li'y1111 { :)

J1l,. 11 z_~ ; 11 ,. = m ;.1 "5 Pnz~ftl{' nq11irPd (tnll)
..
11 .-n'"" '" '" - pncf f>fl7.Yl1H (11111) = 1000 ton enzyn1e solution.'
m n;:_ ml ib ... c,d ,- - lll.L " rn1ynw nqu1rid <innng l.!h s1,1h 'Xpt"filrn 11 (g)
m hi .. t:i ... :. l.;bM-.,1 ... = m .1..,-, dry b1tn11a~.., d11n11g l.!l.i "'L .1l1 t'Xp<'rtlllPllt (g)
m l>iomi "" = mass drylnnl11;-1$S (ton)

J t~xwl:x.~~W:;t;.t!&.~L~\~\ll." 0~,~J.,~~,...,;,,.L )J. .Gx.;. 2.;. :.Cc.rmuer21:tl pnce oi ell.-')"Ill.e is" GI kg a::tiYe en...;.we.
A:wJming that ,Px~!~~ ~ ' XL ha3 the ccncentntian p;, A;::l~:~ enzyme ~or : Cg L rnluticn , th2n the solution ::- m
a:rc . L r . U ' , ton.
I SS-CNS IOP Publishing
IOP Conf. Series: Earth and Environmental Science 31 (2016) 012034 doi : 10.1088/1755-1315/31/11012034

3. Results

3.1 Protein extraction

Microalgal protein has been extracted using alkali [5] and enzymes [6]. For both methods, microalgae
meal (with prior lipid removal) protein was more easily extracted as compared to protein from
microalgae (Fig l .). This fmding confirms the benefit of oil extraction prior to protein extraction [5].
The three-step alkaline extraction of microalgae meal resulted in 32.7% (w/w) protein extraction
yield. A higher protein yield was obtained when enzymes were used to aid in the extraction. As much
as 73.2% (w/w) of microalgal protein can be extracted from microalgae meal using 5% proteases (Fig.
l ). Less microalgal protein was extracted when less enzyme was used. The protein extraction yield
decreased to 62.7% w/w at 1% enzyme. Despite of this decrease, the 1% enzyme aided extraction still
showed higher yield than the alkaline method, indicating the technical superiority of the enzyme
assisted method in extracting microalgal protein.

80 M icroa lgae mea l

70 0 M icroa lgae
60
'i' 50
""3:
~ 40
-g
-a;
.> 30
c:
.Ci
20
0
Q.
10

0
1% 5%

IA!kali 11 e p ro tei n extracti on Enzyme assisted pro tei n extracti on

- -
Figure 1. Protein extraction yield from microalgae meal and microalgae using alkali (left) and
proteases at 1 and 5% (enzyme volume/protein weight).

3.2 Cost calculation

To gain insight in the cost effectiveness of alkaline and enzyme assisted protein extraction, economic
calculations were perfonned on part of the processing cost including cost of biomass, chemicals
(NaOH for alkaline protein extraction, NaOH and enzyme for enzymatic protein extraction), and
energy for heating. Biomass cost are the main contributor to the processing cost (Table 1). Figure 2
indicates that lipid removal has substantial effect on costs for production. It is therefore advisable to
de-oil microalgae prior to protein extraction.

4
ISS-CNS IOP Publishing
IOP Conf. Series: Earth and Environmental Science 31 (2016) 012034 doi: 10.1088/1755-1315/31/1/012034

40000 1\11icroalgae meal

35000 01\11i croa lga e

30000
c:
0 -c:
.;::; ~
25000
I.I ...
:::::J
"'C E
Q.
0
.... c: 20000
c. 0
t; -t'...
0 ~ 15000
u-
10000

5000

Alka line protein

JJ JJ 1% 5%

Enzyme ass isted protein extraction

Figure 2. Cost required to produce I ton protein from microalgae meal and microalgae using alkali
(left) and proteases at I and 5% (enzyme volume per weight of protein).

Table 1. Detailed processing cost calculation for producing 1 ton microalgal protein
Cost required (/ton protein)
Biomass Chemicals Heating Enzyme Total
Alkaline Microalgae meal 1948 IO 490 0 2448
Microalgae 33422 77 1173 0 34672
Enzymatic Microalgae meal 984 333 35 16 1367
(1%) Microalgae 11970 426 45 27 12468
Enzymatic Microalgae meal 858 308 30 70 1266
(5%) Microalgae 10141 338 38 115 10631

Due to the unavailability of a reliable industrial microalgae market price, the processing cost for
microalgae protein depicted in Fig. 2 will probably not reflect the actual possible industrial protein
production cost. The deviation on the predictive market price for microalgae is very high. In the
calculation we cite 1650/ton microalgae [3], which is already very high, while some also mentioned
values of microalgae as high as 25000/ton microalgae [7, 11].

4. Discussion
With the current low 17 .6% protein yield from the alkaline extraction from microalgae, industrial
microalgae protein production is far from being feasible . De-oiling increases protein yield to 32%
protein from microalgae meal (with alkaline method at elevated temperatures). Looking at the still low
yield, it seems that further optimization is required to extract microalgae protein. Protease-assisted-
protein extraction has been developed for extracting more protein from microalgae and microalgae
meal. With protease addition, the protein yie ld for microalgae and microalgae meals increased from
17.6 to 49 . 1% and from 31.7 to 73 .2%, respectively (using 5% enzyme dosage).
The enzymatic assisted method in general required lower cost as compared to alkaline method.
Without protease, protein production from microalgae requires 26 k/ton protein (using alkali, see Fig.
2). Addition of I% and 5% protease reduces the cost to 13 kE and 11 k/ton protein, respectively_. Yet,
this value is still too high for industrial production . The benefit of de-oiling microalgae brings p-rotein
ISS-CNS IOP Publishing
IOP Conf. Series: Earth and Environmental Science 31(2016)012034 doi: 10.1088/1755-1315/31/1/012034

production closer to industry. Here the addition of 1% and 5% protease to microalgae meal solution
reduces the cost to 1367 and 1266 /ton protein, respectively.
This makes the enzymatic method on microalgae meal economically preferred for microalgal
protein production. However, 1266 /ton protein is still much too high. In comparison, only 350 /ton
protein' is required to produce I ton soybean protein concentrate [12]. The details on the microalgal
protein cost calculation (Table l) points out that biomass cost contribute most to the processing cost
per ton of product A further optimized extraction method may contribute to higher extraction yield. If
protein extraction yield can be increased to 90%, biomass cost can be reduced to 555/ton protein (see
Table 2). Table 2 indicates total processing cost for producing 1 ton microalgae meal protein with
optimized method (90% yield).

Table 2. Processing cost for producing microalgae meal protein with optimized method (/ton protein)
Method Biomass Chemicals Heating Enzyme Total
Alkaline 555 15 50 0 620
Enzymatic 555 562 18 45 1180

For a theoretical 90% yield, the alkaline method is less costly than the enzymatic one (see Table 2).
However, in practice, it is rather difficult to increase the yield up to 90%. In this study, alkaline protein
extraction was conducted overnight with increased temperature up to 120 <C. Still, most protein
remained in the biomass.
Optimization of the enzymatic method appears more feasible . Cost minimization of the enzymatic
method should first focus on the chemical cost. The enzymatic method has higher chemical cost
compare to that of alkaline method due to use of a pH-stat during the extraction process. If the
extraction can be performed in the absence of pH-stat and optimized for a fixed, but lower amount of
alkali, then less chemicals will be required. Assuming as low a cost as 15/ton protein (similar to the
alkaline method without enzyme, see Table 2) the production cost may be reduced to 633 /ton
protein, using 5% enzyme on microalgae meal.
In addition to an improved extraction yield or lower chemicals usage, biorefinery of other
components from the microalgae is required to meet economic feasibility. In this case, the residual
microalgal fraction after protein and oil extraction can be used for fuel or for feed with increased
digestibility, due to the alkaline treatment. By refining microalgae into more than one or two products
a feasible process may be achieved.

5. Conclusion
We examined the technical and economic feasibility of protein extraction from microalgae meal
and microalgae, using alkali or proteases. For both methods, de-oiling microalgae prior to protein
extraction increased the protein extraction yield. Enzyme assisted extraction resulted in higher protein
yield as compared to alkaline method. Therefore, enzymatic extraction is preferred from an
economical point of view. A reduction in chemicals cost due to excessive buffering is however needed
to further reduce production cost. When combined with additional revenue from the residue after
protein extraction, biorefinery of microalgae may become a feasible process.

References
[l] Konur 0, 2011 Applied Energy 88(10) 3532-3540.
[2] Wijffels RH and M J Barbosa, 2010 Science 329(5993) 796-799
[3] Wijffels R H, M J Barbosa, and M H M Eppink, 2010 Biofuels, Bioproducts and Biorefining
4(3) 287-295
1
Biomass, chemicals, and power cost only.

6
ISS-CNS IOP Publishing
IOP Conf. Series : Earth and Environmental Science 31 (2016) 012034 doi: 10.108811755-1315/31/11012034

(4] Sari Y W, W J Mulders, J PM Sanders, and ME Bruins, 2015 Biotechnology Journal 10(8): p.
1138-1157
[5] Sari Y W, U Syafitri, J P M Sanders, and M E Bruins, 2015 Industrial Crops and Products 70
125-133
[6] Sari Y W, M E Bruins, and JP M Sanders, 2013 Industrial Crops and Products 43 78-83
[7] FAO, 20 l 0, Rome, Italy.
[8] Alibaba, NaOH price. [cited 2014 July 12] Available from: www.alibaba.com
[9] Dupont C. R Chiriac, G Gauthier, and F Toche, 2014 Fuel 115 644-651
[10] Eurostat 2014 Energy price statistics [cited 2014 October 27] Available from:
http://epp.eurostat.ec .europa.eu/statistics_ explained/index.php/Energy_price_statistics
[11] Pulz 0 and W Gross , 2004 Applied Microbiology and Biotechnology 65(6) 635-648
[12] Mustakas G C and VE Sohns, Soy process. equipment, capital, and processing cost. [cited
2014 August 27]; Available from : http ://naldc.nal.usda.gov/

7
 Industrial Enzymes Cost based on L. Hepner and Assoc.(1995)

Note: Manufacturing costs in the table do not include formulation and packaging cost, cost of
marketing and distribution, R&D, taxes, and general administration overhead

Chymosin Protease a-Amylase Lovastatin

Orqan ism Aspergillus sp Bacillus sp Bacillus sp Aspergilus sp
Fermentation yield (q/L) 5 10 20 15
Recovery yield (%) 83 83 85 85
Plant ou tput (kq a.i./yr) 2000 380,000 60 ,000 10,000
Formulated product (ton/yr) 2900 19,000 3,000 NA

Fermen tation cost ($/kg a. i.) 23 8 31 11

Purification cost ($/kq a.i .) 10 5 3 4
Other variable cost ($/kg a.i.) 88 20 11 64
(labor, depreciation, etc.)
Manufacturing cost ($/kg a.i.) 121 33 21 79

Comments :
1. Chymosin is formulated in coagulant preparations at 0. 7 g/L. Rec. chymosin is sold at
$ 6-9/L i. e. at $9-1 2/g.
2. Granulated protease product contains 2% a.i. and is sold at $5-7 /kg.
3. ex-Amylase formulated product contain 2% a.i. and is sold at $2-3 /kg.

Cost in 2011 used for QTI project - quotes from vendors given to QTI?
-==<.
Composition Indicative Price
Enzym e Category Indicated Application Dosage Guidelines Compatible Conditions (%Active Enzyme) per kg($)
<SOC (p H 3.S-6.S ), SO-
Opti flow RC 2.0 Cellu l ase Sta rch 1% wt/wt o n solids 65C (pH 4.0-5.5 ) 18-23% 10
<SO C (pH 3.S-6.S ), SO-
Opti mash XL Cel I ul ase/ xyl a nase Fermention alcoho l industry 1 % wt/wt on so lid s 6SC (pH 4.0-S.S) 18-23% 9
pH 3-6.5 (opt 4.2),
Cel lu l ase ( ~ ( 1,3 ) 1,4 - Fermention a lcoh ol industry, t emp up to 90C (opt
Opti mash TBG gl ucanase) st a rch pr ocessin g 1% wt/wton so lid s 80 C) , 7SC lon g ter m 10- 1S % w/w 12
Waste treatm ent, pr otein pH 7.5 -11 .0 (opt i mum

Pr ot ex P Al kaline pro tea se

proc essing for industria l end -
use 1% wt/wton solids
pH 8.5 ), Temp 50 -70C
{70C) 3-7%
K 20
pH 6 .5 -9.0, active pH 4-
4.5 at f erm entati o n
Spezyme Fan Substi l isins (p ro t eases) Fuel Alcoh ol prod uct i on 1% wt/ wt on so lid s t emp 1-5% 8
pH 3.5-5 .0 (opt pH 3.6 ),
Ferm gen Ac i d protease Etha no l produ ction 1% wt/ wt on so l i d s t emp up to 65 C 1-5% 12 .5

I
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