Protein separation and characterization from Neochloris Oleoabundans

17 
 
1.INTRODUCTION 
Since the last decades, many efforts have been spent in the direction of green fuels and 
reduction of CO
2
 emissions. A strong investment in researches in this field has been needed due 
to the limited amount of available petroleum in the oil fields and to the increasing temperature 
of the Earth. Anyway, research efforts may be not enough if the human and politic sensibility do 
not help the spread of results and methodology to improve production systems.  
Since a couple of decades the term “biofuels” has started its wide-spread among the world. The 
aim of the companies that produce these “biofuels” is to make economically affordable fuels 
from renewable resources.  The US Council in 2000 defined the term biomass as “organic matter 
that is available on a renewable or recurring basis (excluding old growth timber), including 
dedicated energy crops and trees, agricultural food and feed crop residues, aquatic plants, wood 
and wood residues, animal wastes, and other waste materials”. A significant improvement has 
taken place in biofuels field and that is why a clarification about their stages of progress is 
needed.  The first difference is about the utilization: primary biofuels consist on biomass (wood, 
pellets, vegetable wastes) that is burnt to produce heat. Instead of a direct utilization, secondary 
biofuels are obtained from chemical processes and they have a further classification in three 
different classes (Naik, Goud, Rout, & Dalai, 2010). The first generation of bio-fuels has been 
intended to produce fuels from corn starch for producing bio-ethanol but the economic value of 
the product is just slightly higher than all the production costs (Bounds, 2007). The first way to 
produce oil was by a thermochemical process called pyrolysis. In this process the biomass is 
heated at 500-800°C in absence of oxygen: in this way many products could be observed like 
acidic oils, that need further treatments, charcoal and sub-products such as CO
2
 and H
2
O. 
Pyrolysis has not been used for long time because the efficiency was around 50% and it was not 
profitable. Gasification is another thermochemical process, but it is not meant to produce oil 
from biomass. It has been largely used in syngas (mixture of CO and H
2
 and traces of CO
2
) 
production. The process consists in a high temperature treatment of biomass with air and water 
steam. The products are mainly CO and H
2
 that could be used directly in turbines or for the 
synthesis of chemicals such as methanol, dimethyl ether and Fischer Tropsch Diesel. Another 
example is the transesterification of vegetable oil to produce bio-diesel that consists on the 
mixture of esters produced by this chemical process. The best known process is FAME (Fatty 
Acid Methanol Esterification, Fig. 1.1). For this purpose rapeseeds and soy were used and 
chemically transterified to produce biodiesel. Many efforts were also spent to produce biodiesel 
by transesterification of cooking oil and waste of animal fats.  
 
Fig.1.1. The figure represents the FAME process (Chisti, 2007)
18 
 
In the second generation of biofuels, researches were focused on the conversion of the cellulose 
into glucose by enzymes and the further fermentation of the obtained sugars to ethanol. Many 
raw materials that were used for the production were lignocellulosic materials such as straw, 
wood and grass. Another route was gasification of agricultural residues to obtain syngas (mainly 
CO and H
2
) then used for Fischer Tropsch applications or methanol production, and pyrolysis of 
agricultural residues. In the third generation efforts have been focused on microalgae and 
especially on how to produce bioethanol, biodiesel and hydrogen from microalgae (Dragone & 
Fernandes, 2010). The third generation is represented by biofuels produced by microalgae. 
Microalgae are cell factories that convert CO
2
 to primitive compounds that can be processed to 
produce biofuels, foods, feed and other high value bioactives. Microalgae produce several 
biofuels such as methane obtained by anaerobic digestion of biomass and biodiesel from 
microalgal oil. Biodiesel is commercially produced from plant and animal oils, but the option of 
manufacturing it from microalgae is now taken into account more seriously due to the 
enhancing petroleum price (Chisti, 2007). 
1.1 Microalgae 
Microalgae are small plant-like organisms having a size of 1-50 micrometres in diameter. They 
make part of the aquatic biomass together with macroalgae and large aquatic plants. There are 
hundreds of thousands of existing microalgal species but just a few tens of thousands have been 
described in literature. They live both in freshwater and seawater and a single cell cannot be 
seen with naked eye, but usually when the concentration increases they turn the water colour to 
green, brown blue or orange (Fig. 1.2). Most species contain chlorophyll, so they can use sunlight 
to convert carbon dioxide into oxygen and biomass. Many products make part of the biomass, 
such as fatty acid, proteins, colorants, anti-oxidants and starch that can all be used in many 
everyday utilities (Pulz & Gross, 2004). 
 
 
 
Fig.1.2 On the left an example of microalgae cultivation. On the right some fed-batch reactors of different varieties of 
microalgae. 
Microalgae need a specific living setting to perform the highest productive condition. They 
should be exposed to light and the medium should provide the right compounds in the right
19 
 
quantity.  Nutrients amount can be estimated from the approximate molecular formula  for the 
microalgal biomass: 
  
    
 
    
 
    
 
    
 
presented by (Grobbelaar, 2004) even if other nutrients such as iron should be added. A 
dedicated analysis was made on Neochloris Oleoabundans by (Pruvost, Van Vooren, Cogne, & 
Legrand, 2009). They discovered that optimal growing conditions for this microalgal strain 
requires magnesium, sodium, copper, molybdenum, and calcium; all the salts required are 
reported in Table 1, page 5990 of the quoted scientific paper. 
Phosphorus should be added in excess because phosphates add complex with metal ions. 
Microalgae growth depends on many factors such as the size of the inoculum, the specific 
growth rate of the strain, and the capacity of medium and culturing conditions to support the 
growth. Main phases of growth are (Fig.1.3): lag, exponential, stationary, declining. Lag phase 
happens especially after inoculums in different growth conditions from the previous. Lag phase 
is usually proportional to the time that the inoculums spent in declining-death phase. 
Exponential phase is the measure of the growth of cells in the time and it depends on the culture 
parameters and the relation between medium, growth rate and size of the inoculum. Declining 
phase usually occurs when the biomass content becomes very high and the medium is running 
out of nutrient salts. Cells enter the stationary phase when the net growth is equal to zero and 
depending on the limiting nutrient, cells can undergo biochemical variations. Fig.1.3 shows the 
common behaviour of microalgae growth. 
 
Fig.1.3 Typical example of growth rate and growth phases of microorganisms. 
Microalgae oil content is various, and high quality fatty acids like omega-3 and omega-6 are even 
contained. Microalgae may become the source for omega-3, presently obtained from fishes 
(Wolkers, Barbosa, Kleinegris, Bosma, Wijffels, & Harmsen, 2011). From further analysis it was 
developed that the lipid mass in Dunaliella specie is comprised between 45% and 55% of the 
total organic mass and the most abundant fatty acids are palmitic (16 Carbon atoms), linoleic (18 
C) and palmitoleic (16 C) fatty acids (Sheelan, Dunahay, Benemann, & Roessler, 1998). The 
second important class of compounds found in microalgae are proteins. Most part of protein 
content estimations are based on so called crude proteins, mainly used in food and feed. The
20 
 
protein has to be declared safe for human consumption before being used in food applications. 
Tests have to be performed to gain the quality and safety certification and especially for 
microalgae, which are encountered in unconventional protein sources, several requirements 
have to be satisfied. Many tests have already been taken and serious anomalies were not found 
neither in short or long term experiments nor in analysis on chronic toxicity. None of the tests 
taken have revealed any restriction for using properly processed microalgae for human scopes. 
That is why from these results microalgae seem to be promising for the quality of proteins even 
higher than conventional plants proteins (Becker, 2007). The third most important compound-
class found in microalgae are carbohydrates. Unlike the most part of land plants, microalgae 
usually do not contain simple carbohydrates or easily hydrolysable polysaccharides. Linear 
carbohydrates can be found but they are often derivatized with acids or complexing groups like 
sulphate group. That means that industrial fermentation is not such an easy task because there 
are not known industrial yeast strains able in fermenting the most part of microalgae 
carbohydrates. The above mentioned are the most important compound-classes that can be 
observed in microalgae. So many products can be achieved from processing these 
microorganism that the efforts and researches performed in that field seem to be fully justified.  
1.2 Biofuels 
Microalgae represents the third generation of biofuels. At the beginning of the researches 
reduction of the cost was not the top priority, because the market of these products was small 
and the market price of the products very high. But in larger market of raw materials, such as 
fatty acids for producing biodiesel, the reduction of costs is of vital importance to make 
affordable the comparison between microalgal raw products and other sustainable raw 
materials. Many efforts have been spent to increase the productivity of this system because the 
production of biofuels does not pay-back  fixed and production investments. One alternative to 
such an economic situation is the profiting of more sub-products from microalgae instead of 
focusing the attention just on biofuels (Wolkers, Barbosa, Kleinegris, Bosma, Wijffels, & 
Harmsen, 2011) . It is estimated that a reduction of the growing costs down to 0.5€/kg biomass 
would make feasible the use of microalgae for biofuel production (Wolkers, Barbosa, Kleinegris, 
Bosma, Wijffels, & Harmsen, 2011) since the typical composition of the algal biomass is 40% 
lipids, 50% of proteins and 10% of sugars. With this assumption an approximate economic value 
of the biomass is possible and Fig.1.4 shows the distribution of the economic potential for every 
product that can be achieved. With the composition assumed the total economic value of the 
biomass would be 1.65 €/kg biomass. Biorefinery costs are not included in both estimations, but 
the range of income is wide and allows to affirm that with improvements in the productive 
chain, biofuels will be economically feasible.
21 
 
 
Fig. 1.4. The picture represents the distribution of various products from microalgae on a base scale of 100 kg of 
microalgal biomass (Wolkers, Barbosa, Kleinegris, Bosma, Wijffels, & Harmsen, 2011) 
The production of methane from microalgae has been proposed since the 50’s using wastewater 
as inoculums, however not much work has been done in this field. Recently the U.S. Department 
of Energy listed three main biobased fuels as the main ones from microalgae, namely: 
i. Production of methane gas via biological or thermal gasification 
ii. Production of ethanol via fermentation 
iii. Production of biodiesel 
A potential fourth option would be burning directly the biomass to produce steam and 
electricity. However, it would miss the transportation fuel purpose and its emphasis as an 
environmentally sustainable fuel, which is one of the most important issues for developed 
economies (i.e. USA ). (Sheelan, Dunahay, Benemann, & Roessler, 1998) 
Microalgae store organic matter through the photosynthesis. Microalgae find CO
2
 in atmosphere 
as well as anthropogenic gases like flue gases from fossil power plants. SunChem process was 
designed to produce methane from gasification of microalgae. It is mainly composed by 5 
different steps as shown in Fig. 1.5. The first step is represented by the production of the 
biomass in the PBR (photobioreactor). In this stage the fixation of CO
2
 via the photosynthesis 
leads to the synthesis of biomass. In the second step excess water is removed mechanically and 
recycled to the PBR. The algae concentration reached is approximately 20 wt% dry mass. The 
biomass is then liquefied by heating it up to 450°C at a pressure of 30 MPa . In the fourth step 
the organic stream is catalytically gasified under hydrothermal conditions to Bio-syngas, with 
methane as the main product. In the last step methane is separated from CO
2
, that is recycled to 
the PBR and methane is partially used to provide the heating needed in the process. (Haiduc, 
Brandenberger, S., F., Bernier-Latmani, & Ludwig, 2009).
22 
 
 
Fig.1.5. The figure shows the Block flow diagram of the SunChem process. (Wolkers, Barbosa, Kleinegris, Bosma, 
Wijffels, & Harmsen, 2011) 
Some species of algae, such as Chlorella vulgaris and Chlamydomonas perigranulata can produce 
ethanol and other alcohols through heterotrophic fermentation of starch. The microalgaes 
synthesizes starch by CO
2
  fixation (photosynthesis) with further anaerobic fermentation under 
dark condition. The process usually consists on closed photobioreactors, with metabolically 
enhanced microorganism producing alcohols while resisting to high temperature, high salinity 
and even high ethanol levels. One of the key parameters is the use of CO
2
 coming from power 
plant to feed microalgae and accelerate their growth. This technology is expected to produce up 
to 10,000 gallons per acre per year in the next years (Krassen, 2007). Also other alcohols, such as 
methanol and butanol can be produced in microalgae and the heavier alcohols have an energy 
density almost equal to gasolines but they are not produced for commercial purposes (Sheelan, 
Dunahay, Benemann, & Roessler, 1998) 
Microalgae can produce oils and every specie has its own capacity, as shown in Table 1.1 
(Becker, 2007). Microalgae can accumulate significant quantities of triacylglycerol’s (TAG) that 
are important fuel precursors indeed biodiesel can be achieved by a transesterification process, 
and FAME (Fatty Acid Methanol Transesterification), is the most used and is shown in Fig.1.1. 
Three moles of alcohol are required to esterify the triglyceride, but the reaction is reversible so 6 
alcohol moles per each mole of triglyceride are used on industrial scale to make the highest 
percentage possible of triglycerides react (Chisti, 2007). Chemical and enzymatic catalysts were 
studied to improve the process. (Fukuda, Kondo, & Noda, 2001) shows that alkali-catalysts can 
react with a 6:1 butanol:soybean ratio, despite of acid-catalysts that requires a 30:1 ratio. 
Furthermore (Chisti, 2007) proposed some scenarios about the 50% conversion of the USA 
transports from fossil fuels to bio-based fuels (Table 1.2). 
Table 1.2 shows that microalgae can provide the highest concentration of biofuels per land unit. 
Even considering the lowest concentration of oil in microalgae, investing 5% of the whole US 
cropping area would be enough to cover the 100% of US fuel demand with biofuel of 
microalgaes. These data show clearly that biofuels from microalgae require less land for growing
23 
 
and they does not exploit feed source, that would be a strong response to the ethical question 
about the investment of ground for producing fuels instead of food. It is acknowledged that 
fossil fuels cannot last forever and that is why researches about microalgae are required to 
improve the yield of fuel production. 
 
Table 1.1. General composition of different algae (% of dry matter)  (Becker, 2007) 
Alga Protein 
[% on dry matter] 
Carbohydrates 
[% on dry matter] 
Lipids 
[%on dry matter] 
Anabaena cylindrica 43-56 25-30 4-7 
Aphanizomenon flos-aquae 62 23 3 
Chlamydomonas rheinhardii 48 17 21 
Chlorella pyrenoidosa 57 26 2 
Chlorella vulgaris 51-58 12-17 14-22 
Dunaliella salina 57 32 6 
Euglena gracilis 39-61 14-18 14-20 
Porphyridium cruentum 28-39 40-57 9-14 
Scenedesmus obliquus 50-56 10-17 12-14 
Spirogyra sp. 6-20 33-64 11-21 
Arthrospira maxima 60-71 13-16 6-7 
Spirulina platensis 46-63 8-14 4-9 
Synechococcus sp. 63 15 11 
 
 
 
 
 
Table 1.2. Comparison of some sources of biodiesel (Chisti, 2007) 
Crop Oil yield (L/ha) Land area needed (Mha) 
Percent of existing US 
cropping area
a
 
Corn 172 1540 846 
Soybean 446 594 326 
Canola 1190 223 122 
Jatropha 1892 140 77 
Coconut 2689 99 54 
Oil palm 5950 45 24 
Microalgae
b
 136900 2 1.1 
Microalgae
c
 58700 4.5 2.5 
a For meeting 50% of all transport fuel needs of the US  b 70% oil (by wt) in biomass   c  30% oil (by wt) in biomass.
24 
 
1.3  Proteins 
 
Proteins are organic macromolecules made of amino acids which are molecules containing an 
amine group, a carboxylic group and a side-chain. The side-chain determines the nature of the 
amino acid. Key elements of amino acids are carbon, hydrogen, oxygen and nitrogen. Amino 
acids are bound with a peptide bond and that is why proteins are also called polypeptide. 
Peptide bond is generated with the formation of a molecule of water. Proteins are fundamental 
for organism life and they take part in several processes. Their structure is characterized at four 
different levels. The primary structure refers to amino acid sequence. Amino acids are bound 
together by peptide bonds. The ends of the polypeptide chain have a carboxyl group and an 
amine group. The secondary structure is referred to local sub-structures. Main types of 
secondary structures are alpha helix and beta sheets and they present a regular structure. The 
tertiary structure is referred to the 3-dimensional structure of the proteins. It is referred to the 
arrangement among secondary structures. The structure is stable only when specific tertiary 
interactions take place such as salt bridges and disulfide bonds. The quaternary structure is the 
gathering of multiple protein chains (polypeptides) and its stabilized by the same interactions of 
the tertiary structure. Tertiary and quaternary structures makes a protein functional, while the 
secondary structures itself do not. 
Protein are thermo-sensitive molecules, and that is why protein samples should be kept in a 
fridge or in a freezer. According to (Kampinga, 1995) proteins start to denaturate. This change in 
protein structure can be due to thermal vibrations and collision between molecules. Protein 
solubility is pH-depending. (Schwenzfeier, Wierenga, & Gruppen, 2011) showed that water 
soluble proteins from Tetraselmis present the lowest solubility value between the pH values of 3 
and 4. Fig.1.7 presents their results changing the pH and the salinity of the solution with 
different kinds of microalgal protein solution.  This is a consequence of the Isoelectric Point, that 
changes from protein to protein and it is a pH value at which the protein gains a neutral electric 
charge. Fig.1.6 shows the behaviour of Zeta potential that is depending by the electric charge 
while changing the pH. The solubility is strictly connected to the charge, because if the protein 
does not have any electric charge on it, intermolecular interaction are not possible. 
 
 
Fig.1.6 The picture represents the behaviour of  
Zeta potential at pH-variations
25 
 
 
Fig.1.7 Protein solubility as a function of pH for algae juice (A),  
crude algae protein isolate (B) and final algae protein isolate (C)  
at different ionic strengths (I=0.03M (▪), 0.2M ( ) I=0.5M (□).  
100% =soluble protein concentration at pH 7.6.  
Dashed lines indicates pH 5.5. Error bars According to standard                                                                                                 
deviation (Schwenzfeier, Wierenga, & Gruppen, 2011) 
 
 
 
 
 
 
 
 
 
1.3.1 Protein quantification method 
Concentration of total proteins was determined by Lowry method that was published by Oliver 
H. Lowry in 1951. The method is based on the reactions of copper ions with the peptide bonds 
under alkaline conditions. The Lowry method is based on the reaction of Cu
+
 ion produced by the 
oxidation of peptide bonds, with Folin-Ciocalteu reagent (a mixture of phosphotungstic acid and 
phosphomolybdic acid in the Folin-Ciocalteu reaction). This method is recommended for this 
type of analysis because it is 10 to 20 times more sensible than ultraviolet (280nm) absorbance 
reading, it is less disturbed by turbidity, and it is 100 times more sensible than Biuret reaction. 
The list of all interfering substances with the Folin-Ciculteau’s reagent is elsewhere reported 
(Lowry, 1951). The main disadvantages of this method are that the amount of color can change 
with different proteins and that the optical density is not strictly correlated to the protein 
concentration. Despite of this disadvantages the Lowry assay is still the most recommended way 
to measure low protein concentration (Lowry, 1951). For this assay the recommended protein 
concentration range, according to (Lowry, 1951) is [0.1-1 g/L] to avoid proteins concentration 
values on the hysteresis range of the calibration line. The experiment is conducted under 
alkaline condition by adding NaOH to the samples that enables peptide bonds to become 
negative charged. It is even noticed that in presence of copper, working in alkaline conditions 
increase several times the color of the samples. Samples are then incubated at 95°C to make the 
proteins more linear and to increase the rate of the copper reaction. After the addition of Folin’s 
reagents the samples has to be placed into the darkness and then the absorbance can be read at 
750nm. In Fig.1.8 (Lowry, 1951) is shown that the highest value of the absorbance is reached 
after 30 minutes of adding the Folin reagents.