104 Marine Macro- and Microalgae: An Overview
investigated: viscozyme, driselase, crude papain, lipase from Rhizomucor miehei, and proteinase K. These
enzymes were selected due to their specific mode of action. Viscozyme contains, among others, a wide
range of carbohydratases (Latif and Anwar 2011) acting on branched pectin-like substances found in plant
cell walls. Driselase contains (among others) cellulase, pectinase, β-xylanase, and β-mannanase. Crude
papain contains among others cysteine endopeptidases papain, chymopapain, glycyl endopeptidase, and
caricain. The lipase from R. miehei catalyses hydrolysis of TAG molecules while proteinase K is an
alkaline serine protease with broad substrate activity. The microalgae investigated were P. tricornutum,
T. pseudonana, and N. oculata and the efficiency of the treatment was evaluated on the basis of lipid
release. For P. tricornutum the most effective enzymes were viscozyme, proteinase K, and papain
crude extract. For N. oculata the most efficient enzymes were viscozyme and proteinase K and for
T. pseudonana only driselase was efficient. This study also confirmed that the enzymatic activity is very
specific to each algal species and for instance papain had little or no effect on T. pseudonana while it had
a very promising activity on P. tricornutum, despite the fact that both algae are diatoms. This difference
lies in the structure of the silica shell. Inhibitory studies of the enzymatic activity showed that the cysteine
protease activity of papain was crucial to separate the lower and upper parts of the diatom shell. As
enzymatic digestion processes can be pretty costly, this study also investigated the enzyme quantity and
residence time in order to minimize them, and found that 2.5 mg enzyme per 200 mg dry weight and a
holding time of 2 hr were acceptable.
Similarly, Liang et al. (2012) screened various enzymes for disrupting cell membranes of Scenedesmus
dimorphus, and Nannochloropsis sp. The enzymes tested were cellulase, snailase (a mixture of more than
30 enzymes), neutral protease, alkaline protease, and trypsine. Higher lipid recovery was obtained with
a pretreatment by snailase and trypsine. Enzyme dosage and reaction time were also investigated. By
the time 4% enzyme dosage was reached, the lipid recovery approached maximum with a duration of
treatment of 12 hr.
A wide array of enzymes was tested on the marine microalgae P. tricornutum (CCMP 632),
Nannochloropsis sp., Franceia sp., and Ankistrodesmus falcatus (Gerken et al. 2013). The two latter
strains were resistant to almost all the enzymes tested. P. tricornutum was sensitive to the enzymes
chitinase, β-glucuronidase, hyaluronidase, and pectinase. Nannochloropsis sp. showed significant
sensitivity to a number of enzymes: chitinase, chitosanase, β-glucuronidase, β-glucosidase, hyaluronidase,
lysozyme, lyticase, pectinase, sulphatase, trypsin, and zymolyase. The enzymes inhibited the growth
of the microalgae most probably due to the degradation of cell walls. This could be demonstrated by
showing cell permeability to a DNA staining dye, SYTOX Green, which normally cannot pass the cell
wall/membrane of living cells. Nannochloropsis was sensitive to lysozyme, but the effect was even more
drastic when it was combined with one of the following enzymes: sulfatase, trypsin, or lyticase.
Production of bioethanol from biomass requires a preliminary saccharification process step from
which fermentable sugars such as glucose and mannose are released (Harun and Danquah 2011). These
authors carried out a study using cellulase from Trichoderma reesei, ATCC26921, to perform the
enzymatic hydrolysis of the marine alga Chlorococcum sp. and showed that the highest glucose yield
of 64.2% (w/w) was obtained at a temperature of 40ºC, pH 4.8 and a substrate concentration of 10 g/L
of microalgal biomass. This enzymatic process was evaluated as an effective mechanism to enhance the
saccharification process.
Acid pretreatment
Talukder et al. (2012) used sulfuric acid to perform cell lysis. The dried microalgae biomass of N. salina
(1 g) was treated with sulfuric acid (5% w/v first shaken with the algal biomass at 30ºC and 150 rpm for
10 min, followed by hydrolysis at 120ºC for 1 h). The algal lipids were then extracted using hexane. For
comparison purposes, the same extraction was carried out without acid pretreatment. Lipid yield increased
from 48.7% to 85.6% when applying the sulfuric acid lysis pretreatment. This improvement was not only
attributed to the disruption or deformation of the microalgae cell wall but also to the acid hydrolysis of
fatty acids from polar lipids such as phospholipids and glycolipids. Additionally, in that study, the acidic
pretreatment contributed to the release of a maximum of 64.3% of sugar available (as glucose and xylose)
investigated: viscozyme, driselase, crude papain, lipase from Rhizomucor miehei, and proteinase K. These
enzymes were selected due to their specific mode of action. Viscozyme contains, among others, a wide
range of carbohydratases (Latif and Anwar 2011) acting on branched pectin-like substances found in plant
cell walls. Driselase contains (among others) cellulase, pectinase, β-xylanase, and β-mannanase. Crude
papain contains among others cysteine endopeptidases papain, chymopapain, glycyl endopeptidase, and
caricain. The lipase from R. miehei catalyses hydrolysis of TAG molecules while proteinase K is an
alkaline serine protease with broad substrate activity. The microalgae investigated were P. tricornutum,
T. pseudonana, and N. oculata and the efficiency of the treatment was evaluated on the basis of lipid
release. For P. tricornutum the most effective enzymes were viscozyme, proteinase K, and papain
crude extract. For N. oculata the most efficient enzymes were viscozyme and proteinase K and for
T. pseudonana only driselase was efficient. This study also confirmed that the enzymatic activity is very
specific to each algal species and for instance papain had little or no effect on T. pseudonana while it had
a very promising activity on P. tricornutum, despite the fact that both algae are diatoms. This difference
lies in the structure of the silica shell. Inhibitory studies of the enzymatic activity showed that the cysteine
protease activity of papain was crucial to separate the lower and upper parts of the diatom shell. As
enzymatic digestion processes can be pretty costly, this study also investigated the enzyme quantity and
residence time in order to minimize them, and found that 2.5 mg enzyme per 200 mg dry weight and a
holding time of 2 hr were acceptable.
Similarly, Liang et al. (2012) screened various enzymes for disrupting cell membranes of Scenedesmus
dimorphus, and Nannochloropsis sp. The enzymes tested were cellulase, snailase (a mixture of more than
30 enzymes), neutral protease, alkaline protease, and trypsine. Higher lipid recovery was obtained with
a pretreatment by snailase and trypsine. Enzyme dosage and reaction time were also investigated. By
the time 4% enzyme dosage was reached, the lipid recovery approached maximum with a duration of
treatment of 12 hr.
A wide array of enzymes was tested on the marine microalgae P. tricornutum (CCMP 632),
Nannochloropsis sp., Franceia sp., and Ankistrodesmus falcatus (Gerken et al. 2013). The two latter
strains were resistant to almost all the enzymes tested. P. tricornutum was sensitive to the enzymes
chitinase, β-glucuronidase, hyaluronidase, and pectinase. Nannochloropsis sp. showed significant
sensitivity to a number of enzymes: chitinase, chitosanase, β-glucuronidase, β-glucosidase, hyaluronidase,
lysozyme, lyticase, pectinase, sulphatase, trypsin, and zymolyase. The enzymes inhibited the growth
of the microalgae most probably due to the degradation of cell walls. This could be demonstrated by
showing cell permeability to a DNA staining dye, SYTOX Green, which normally cannot pass the cell
wall/membrane of living cells. Nannochloropsis was sensitive to lysozyme, but the effect was even more
drastic when it was combined with one of the following enzymes: sulfatase, trypsin, or lyticase.
Production of bioethanol from biomass requires a preliminary saccharification process step from
which fermentable sugars such as glucose and mannose are released (Harun and Danquah 2011). These
authors carried out a study using cellulase from Trichoderma reesei, ATCC26921, to perform the
enzymatic hydrolysis of the marine alga Chlorococcum sp. and showed that the highest glucose yield
of 64.2% (w/w) was obtained at a temperature of 40ºC, pH 4.8 and a substrate concentration of 10 g/L
of microalgal biomass. This enzymatic process was evaluated as an effective mechanism to enhance the
saccharification process.
Acid pretreatment
Talukder et al. (2012) used sulfuric acid to perform cell lysis. The dried microalgae biomass of N. salina
(1 g) was treated with sulfuric acid (5% w/v first shaken with the algal biomass at 30ºC and 150 rpm for
10 min, followed by hydrolysis at 120ºC for 1 h). The algal lipids were then extracted using hexane. For
comparison purposes, the same extraction was carried out without acid pretreatment. Lipid yield increased
from 48.7% to 85.6% when applying the sulfuric acid lysis pretreatment. This improvement was not only
attributed to the disruption or deformation of the microalgae cell wall but also to the acid hydrolysis of
fatty acids from polar lipids such as phospholipids and glycolipids. Additionally, in that study, the acidic
pretreatment contributed to the release of a maximum of 64.3% of sugar available (as glucose and xylose)