Pentoses and light intensity increase the growth and carbohydrate production and alter the protein profile of Chlorella minutissima

Accepted Manuscript Pentoses and light intensity increase the growth and carbohydrate production and alter the protein profile of Chlorella minutissima B.C.B. Freitas, A.P.A. Cassuriaga, M.G. Morais, J.A.V. Costa PII: DOI: Reference:

S0960-8524(17)30512-6 http://dx.doi.org/10.1016/j.biortech.2017.04.031 BITE 17926

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

18 February 2017 6 April 2017 7 April 2017

Please cite this article as: Freitas, B.C.B., Cassuriaga, A.P.A., Morais, M.G., Costa, J.A.V., Pentoses and light intensity increase the growth and carbohydrate production and alter the protein profile of Chlorella minutissima, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.04.031

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Pentoses and light intensity increase the growth and carbohydrate production and alter the protein profile of Chlorella minutissima 1

Freitas, B. C. B.; 1Cassuriaga, A. P. A.; 2Morais, M. G.; 1*Costa, J. A. V.

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College of Chemistry and Food Engineering, Federal University of Rio Grande,

Laboratory of Biochemical Engineering, Rio Grande, RS, Brazil 2

College of Chemistry and Food Engineering, Federal University of Rio Grande,

Laboratory of Microbiology and Biochemistry, Rio Grande, RS, Brazil

*Corresponding Author: Prof. Dr. Jorge Alberto Vieira Costa Laboratory of Biochemical Engineering College of Chemistry and Food Engineering Federal University of Rio Grande P.O. Box 474, 96203-900 Av. Itália, km 8 Rio Grande, RS Brazil Phone: +55 53 32935373 Fax: +55 53 32336968 E-mail: [email protected]

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ABSTRACT High concentrations of carbon, which is considered a necessary element, are required for microalgal growth. Therefore, the identification of alternative carbon sources available in large quantities is increasingly important. This study evaluated the effects of light variation and pentose addition on the carbohydrate content and protein profile of Chlorella minutissima grown in a raceway photobioreactor. The kinetic parameters, carbohydrate content, and protein profile of Chlorella minutissima and its theoretical potential for ethanol production were estimated. The highest cellular concentrations were obtained with a light intensity of 33.75 µmol.m-2.s-1. Arabinose addition combined with a light intensity of 33.75 µmol.m-2.s-1 increased the carbohydrate content by 53.8% and theoretically produced 39.1 mL.100 g-1 ethanol. All of the assays showed that a lower light availability altered the protein profile. The luminous intensity affects xylose and arabinose assimilation and augments the carbohydrate content in C. minutissima, making this microalga appropriate for bioethanol production.

Key-words: Carbohydrate, Chlorella, luminosity, pentose, Raceway

1. Introduction The generation of industrial residues is a concerning environmental problem, and lignocellulosic material constitutes a high proportion of these wastes. When hydrolyzed, this material releases large amounts of pentoses, such as xylose and arabinose (Soccol et al., 2010). Pentose has no relevant applications as a source of carbon in microalgae culture, but recently, some wild species that are capable of

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metabolizing these sugars, such as Chlorella sorokiniana (Zheng et al., 2014), Chlorella minutissima (Freitas et al., 2016) and indigenous strains of Chlorella sp. (Leite et al., 2015), have been isolated. Carbon represents a large share of the nutrients required for microalgal growth and could be replaced by residues to minimize the costs of cultivation and the environmental impact of industrial waste generation. In Brazil, pentoses (C5), which represent 25% of the sugars available in sugar cane bagasse (Mariano et al., 2013), can be considered a potential source of carbon. Researchers have recently started to study the utilization of D-xylose by microalgae, and the first report of the metabolic absorption of this pentose by microalgae (Chlorella sorokiniana cells) was published by Zheng et al. (2014). In addition, the effects of D-xylose on some enzymes involved in photosynthesis, such as ribulose-1,5-biophosphate-carboxylase/oxygenase (Rubisco) and adenosine triphosphate (ATP), were first determined by Freitas et al. (2016). These studies have encouraged interest in the incorporation of pentoses into microalgal cultivation. Many biocompounds and fuels, such as biodiesel, bioethanol, biohydrogen and biogas, can be produced by microalgae (Brennan and Owende, 2010). The composition of the resulting biomass depends directly on the cultivated species and the physical and nutritional conditions to which the strains are exposed (Kim et al., 2014; Skorupskaite et al., 2014). One of the most important open-system limitations for microalgal culture is the absorption of light because light gradients and shading can arise in high-density cellular systems. To avoid these problems, the luminosity and agitation conditions must be improved to favor the photosynthetic process. Raceway reactors have been

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used in the industrial production of microalgae, specifically for Spirulina, Chlorella and Dunaliella cultures, since the 1950s (Chisti, 2007). These reactors allow high biomass production, but care must be taken to control the effects of evaporation, temperature fluctuations, and cellular and nutritional dilution by rain (Norsker et al., 2011; Richmond and Hu, 2013). Therefore, due to the high availability of lignocellulosic residues, this work aimed to study the effects of luminous intensity variation and the addition of pentoses on the growth, carbohydrate production and protein profile of Chlorella minutissima cultivated in raceway photobioreactors and assess the potential of this biomass for biofuel production.

2. Materials and Methods 2.1 Microalgae and culture conditions A strain of C. minutissima from the Collection of the Laboratory of Biochemical Engineering of the Federal University of Rio Grande (FURG), Rio Grande do Sul, Brazil, was used for the assays. The microalga was exposed to different luminosity intensities, reduced levels of nitrogenous compounds and the addition of D-xylose and/or L-arabinose (Vetec Quimica, Sigma-Aldrich Corporation). The strain was cultivated in liquid BMM medium (Watanabe, 1960), which had the following composition: 0.250/0.125 g.L-1 KNO3, 0.01 g.L-1 CaCl2, 0.075 g.L-1 MgSO4.7H2O, 0.075 g.L-1 K2HPO4, 0.175 g.L-1 KH2PO4, 0.025 g.L-1 NaCl, 0.02 g.L-1 FeSO4.7H2O, and 1 mL of solution A5 (2.86 g.L-1 H3BO3, 1.81 g.L-1 MnCl2.4H2O, 0.222 g.L-1 ZnSO4.7H2O, 0.079 g.L-1 CuSO4.5H2O, and g.L-1 0.015 NaMoO4). The

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cultivations were conducted in raceway photobioreactors with a utilization volume of 3.5 L. 2.2 Light intensity variation and addition of pentoses To assess the effects of varying light intensity, the cultures were exposed to light intensities of 33.75, 16.88 and 8.44 μmol.m-2.s-1 using 40-W fluorescent light bulbs. The assays were conducted in a thermostated greenhouse with a temperature of 30 °C, a 12-h light/dark photoperiod and agitation via aeration pumps until a potential stationary phase was reached. The conditions utilized in this study are listed in Table 1. The addition of pentoses (C5) was achieved using various synthetic broths composed of xylose and/or arabinose. The broth composed of both xylose and arabinose contained 5% pentoses, which is equivalent to the concentration present in hydrolyzed sugar cane bagasse broth (obtained through steam explosion treatment), which was utilized as the control. The broths containing only xylose or arabinose were prepared as described previously (Freitas et al., 2016), and each broth contained 20-mg.L-1 pentose.

2.3 Biomass concentration The cell concentration was monitored daily by measuring the optical density of the cultures with a spectrophotometer (QUIMIS Q798DRM, Diadema - SP - Brasil) at 670 nm and comparing it to a standard curve established using C. minutissima to determine the corresponding dry weight (Costa et al., 2002).

2.4 Biomass volumetric productivity

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The maximum biomass productivity (Pmax, g.L-1.d-1) was obtained using Equation 1, where Xt is the biomass concentration (g.L-1) at time t (d) and X0 is the biomass concentration (g.L-1) at time t0 (d). ma

(1)

-

2.5 Maximum specific growth rate The ma imum specific grow h μmax, d-1) was determined by applying exponential regression to the logarithmic growth phase. 2.6 Biomass pentose conversion factor The substratum conversion factor (xylose and arabinose) for the biomass (YX/S, mg.mg-1) was determined with Equation (2), where X0 and S0 represent the biomass concentration and the substratum concentration at the beginning of the culture, respectively, Xmax is the maximum biomass concentration, and Sf is the final substratum concentration. The total pentose consumption (Sf=0) was determined by applying the methodology described by Somogyi (1952) to the supernatant. ma

- f

(2)

2.7 Carbohydrate and protein contents The biomass of Chlorella minutissima obtained in the different trials was characterized in terms of the carbohydrate and protein compositions and the theoretical conversion of ethanol. The total carbohydrate content in the C. minutissima biomass was determined by the phenol-sulfuric method using a standard glucose curve (DuBois et al., 1956). The calculations performed to estimate the conversion of biomass carbohydrates into

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ethanol were based on 100 g of biomass and a theoretical stoichiometric conversion value for glucose into ethanol of 0.511. The total protein content in C. minutissima at the end of each assay was determined through the colorimetric method proposed by Lowry et al. (1951) based on the thermal and alkaline properties of the pre-treated biomass. The characteristics of the biomass obtained from the cultures with pentose (CC5) were compared with those of the biomass control cultures (CCs) as proposed by Deamici et al. (2016). This relationship is described by Equation 3, where R corresponds to the difference between the results obtained with and without added pentoses. -

1

(3)

2.8 Extraction and protein profile The proteins were extracted by adding sample buffer (4× sample buffer: 80-mM Tris/HCl, pH 6.8, 2% [w/v] sodium dodecyl sulfate [SDS], 15% [v/v] glycerol, 0.006% [w/v] m-purple cresol, and 0.1-M 2- mercaptoethanol), heating for 5 min at 98 °C, and centrifuging at 10000×g for 1 min. The samples were then subjected to discontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970) using a 5% acrylamide stacking gel and a 12.5% acrylamide resolving gel. The gel wells were loaded with 1 mg of freeze-dried biomass.

2.9 Assessment of bacterial contamination and raceway reactor asepsis Because the strains used were not necessarily axenic and the assays with pentose addition were mixotrophic, it was necessary evaluate the degree of bacterial contamination. The contamination assessment was performed as proposed by Freitas et

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al. (2016). To ensure raceway reactor asepsis, the reactors were exposed to a solution containing chlorine for 24 h and then rinsed with sterile water.

2.10 Statistical analyses The differences between the average values obtained from each assay were assessed through analysis of variance followed by Tukey’s test with a confidence level of 95%.

3. Results and Discussion At the highest luminous intensity (33.75 µmol.m-2.s-1), the control cultures (C1) presented the highest cell concentration (1.34 g.L-1). This luminous intensity also yielded the highest productivity and specific growth rate, and the values did not differ significantly between the control cultures and those subjected to pentose addition (X1, A1 and P1; Table 2). The use of an adequate luminosity intensity might be solely responsible for the optimization of mixotrophic cultures because photosynthetic microorganisms depend on their own photosynthetic systems to supply their metabolic needs (Barsanti and Gualtieri, 2006; Costa and Morais, 2011). C. minutissima consumed all of the available pentoses (20 mg.L-1), as confirmed by the fact that after 2 d of cultivation, no pentoses were detected in the supernatant (Sf=0). According to Zheng et al. (2014), the improvement in the utilization of D-xylose could be attributed to the additional chemical energy derived from light-dependent reactions, which justifies the better conversion factors for pentose into biomass observed (Table 2; YX/S=0.04 mg.mg-1). The same results were found for the X1, A1 and P1 cultures. Gautam et al. (2013) assessed the effects of four-carbon (succinate [4C]) and threecarbon (glycerol [3C]) structures and found that these substances increased C.

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minutissima growth during the first 15 d of cultivation. In this study, as observed previously for 4C and 3C structures, the introduction of pentoses (5C) favored the growth of C. minutissima cells, and this effect was more evident in assays in which only xylose or arabinose was added (X1 and A1; Fig. 1-a). The use of xylose in C. minutissima cultures contributed to the C/N balance, as shown in a previous study (Freitas et al., 2016) in which higher cellular densities were obtained by reducing the nitrogen source level by 50% (0.125 g.L-1 KNO3) and adding xylose to cultures in Erlenmeyer flasks. The addition of C5 also contributed to maintaining the growth capacity when the luminous intensity was reduced by 50% (16.88 µmol.m-2.s-1; Fig. 1-b). With the exception of the control cultures (C2 and C3), exposure to the lowest luminous intensity tested (8.44 µmol.m-2.s-1) altered the growth of C. minutissima. The observed changes are attributable to the lower resulting cell concentrations (Table 2) and the relatively rapid achievement of a potential stationary phase (Fig. 1-c; X3: 10 d; A3: 9 d; and P3: 8 d). Mixotrophic cultures usually utilize high amounts of the carbon source. For example, with a luminous intensity of 40.5 µmol.m-2.s-1, Prakash Rai et al. (2013) found maximum cellular concentrations of 0.77 g.L-1 and 0.87 g.L-1 in the presence of 5- and 10-g.L-1 acetate, respectively, and 0.78 g.L-1 with 2.5-g.L-1 acetate and 0.25% glycerol. These values are lower than those obtained with 20-mg.L-1 pentose in this study (X1, A1 and P1; Table 2). Given the small amounts of pentoses used in this work, the obtained kinetic parameters (Table 2) demonstrate the potential application of these sugars for microalgal biomass production under adequate luminosity conditions. The cultures were maintained until a potential stationary phase was achieved because the carbohydrate content might be relatively high during this phase (Renaud et al.,

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1999). With the exception of the control assays, higher carbohydrate contents (X1=52.8%, A1=60.3% and P1=53.4%) were obtained with the highest tested irradiance (33.75 µmol.m-2.s-1). This finding confirms that increasing the luminous intensity stimulates the production of CO2 and other carbon compounds from organic substances (Chang et al., 2016). Margarites and Costa (2014) cultured C. minutissima in BMM medium with 50% KNO3 (0.125 g.L-1) in Erlenmeyer flasks (2 L) and found a final polysaccharide concentration of approximately 44.6%. In raceway reactors, the addition of arabinose resulted in a carbohydrate production of 60.3%, which demonstrates that the type of photobioreactor and carbon source used directly affect the bioproduct production. The highest carbohydrate production observed in raceway reactors was accompanied by a decrease in the protein concentration of C. minutissima biomass (Table 3; positive and negative effects). This phenomenon explains the metabolism deviation caused by the addition of pentoses and the reduction of nitrogen. Additionally, the addition of xylose to C. minutissima cultures significantly reduced (by 25.1%) the neutral lipid content (Freitas et al., 2016). The greatest increase in the carbohydrate content (53.8%; Table 3) was found in the assays with arabinose, followed by those with xylose and arabinose (36.2%) and those with xylose alone (34.7%) (all at 33.75 µmol.m-2.s-1). Thus, the increase in the polysaccharide content detected in this strain was related to the addition of pentoses and the provision of an adequate luminous intensity (Table 3). Application of the lowest tested luminous intensity (8.44 µmol.m-2.s-1) resulted in a decreased carbohydrate concentration, and the most marked effect (-28.3%) was found with A3 (Table 3).

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As a result of decreasing oil reserves and the environmental consequences of burning fossil fuels, the development of renewable fuels has recently attracted increased attention. Microalgae are considered a promising alternative to the conventional raw materials used for the production of biofuels (Suganya et al., 2016). Because some microalgae have high carbohydrate contents in terms of starch and cellulose, they are likely excellent substrata for bioethanol production. In addition, the sugars of microalgae are more easily converted into ethanol than lignocellulosic materials (Ho et al., 2013). In general, the C. minutissima biomass obtained using raceway photobioreactors showed elevated carbohydrate contents, and the theoretical ethanol production values are shown in Table 3. For cultures with added pentoses, the theoretical ethanol production values were estimated to equal X1=34.2 mL.100 g-1 and P1=34.6 mL.100 g1

under a high irradiance level (33.75 µmol.m-2.s-1).

Because the growth rates of microalgae are at least 5- to 10-fold higher than those of higher plants (Lee et al., 2015), the production of ethanol from microalgal biomass will likely be strategically important in the biofuel industry. Because of the high carbohydrate content (60.3% m.m-1) produced by C. minutissima cultured with arabinose in raceway reactors, 39.1 mL of ethanol could be generated from each 100 g of biomass (Table 3). Other studies have investigated the potential application of microalgae in ethanol production. For example, Rosa et al. (2015) studied the production of macromolecules by Spirulina sp. LEB 18 in the presence of monoethanolamine (MEA) and CO2 and determined a theoretical ethanol efficiency of 12.8 mL for each 100 g of Spirulina biomass cultured with MEA. Furthermore, by culturing Spirulina platensis LEB 52 with ultra- and nanofiltration whey protein

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residues, Vieira Salla et al. (2016) achieved a carbohydrate content of 58.2%, which corresponds to a theoretical ethanol production rate of 37.6 mL.100 g-1. Similar to other Chlorella species, C. minutissima has a thick and complex cellular wall, which hinders cell disruption (Safi et al., 2015; Zheng et al., 2011). Thus, studying its metabolic responses in terms of the proteins present in different parts of the cell is difficult. Freitas et al. (2016) recently reported the first results regarding the detection of proteins (Rubisco and AtpB) in C. minutissima. In addition to the increased Rubisco and AtpB levels detected in C. minutissima cells cultured under reduced-nitrogen conditions, Freitas et al. (2016) confirmed that the addition of xylose directly affects the Rubisco levels. The protein profiles observed by SDS-PAGE in this study (Fig. 2) revealed that the protein levels increased as the luminous intensity decreased to 16.88 and 8.44 µmol.m2 -1

.s in both control assays (without the addition of pentoses: wells 5 and 9) and those

with added pentoses (wells 6, 7, 8, 10, 11 and 12). The cultures exposed to the lowest tested luminous intensity (8.44 µmol.m-2.s-1; wells 9, 10, 11 and 12) resulted in higher band intensities. The proteins themselves were directly affected by exposure to 33.75 µmol.m-2.s-1. Higher Rubisco (56 kDa) degradation was detected in the cultures exposed to 33.75 µmol.m-2.s-1 compared with the cultures subjected to lower luminous intensities. Furthermore, Rubisco exhibited less degradation in the cultures supplemented with arabinose (well 7; 16.88 µmol.m-2.s-1), xylose (well 10; 8.44 µmol.m-2.s-1) and both xylose and arabinose (well 12; 8.44 µmol.m-2.s-1). The enzymes D1 (38.9 kDa) and D2 (39.5 kDa) constitute the photosystem II (PSII) reaction center, which is a key complex in the electron transport chain. PSII,

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particularly the D1 subunit, is sensitive to light-exposure damage (Melis et al., 2000). This sensitivity explains the low degradation of the D1 and D2 subunits observed as the luminous intensity was reduced to 16.88 and 8.44 µmol.m-2.s-1 (wells 5-12). In addition to the effect observed as the luminous intensity was reduced, D2 was positively affected by the addition of xylose and arabinose, as confirmed by the higher band intensities observed for wells 5 and 7.

4. Conclusion The incorporation of pentoses and the application of different luminous intensities alter the growth and biomass composition of C. minutissima grown in raceway reactors. The higher carbohydrate content (60.3% m.m-1) achieved in cultures supplemented with arabinose alone demonstrates the potential application of this microalga for bioethanol production. Indeed, the theoretical ethanol production was calculated to equal 39.1 mL.100 g-1. The influence of culture conditions on enzymes, such as Rubisco, D1 and D2, was determined. This study is among the first to demonstrate that reducing the luminous intensity and adding pentoses reduces degradation of the protein profile of C. minutissima.

5. Acknowledgements The authors would like to thank the Coordination for the Improvement of Higher Education Personnel (CAPES), the National Council for Scientific and Technological Development (CNPq) and the Ministry of Science, Technology, Innovations and Communication (MCTIC) for the financial support provided.

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29. Suganya, T., Varman, M., Masjuki, H.H., Renganathan, S., 2016. Macroalgae and microalgae as a potential source for commercial applications along with biofuels production: A biorefinery approach. Renew. Sustain. Energy Rev. 55, 909–941. doi:10.1016/j.rser.2015.11.026 30. Vieira Salla, A.C., Margarites, A.C., Seibel, F.I., Holz, L.C., Brião, V.B., Bertolin, T.E., Colla, L.M., Costa, J.A.V., 2016. Increase in the carbohydrate content of the microalgae Spirulina in culture by nutrient starvation and the addition of residues of whey protein concentrate. Bioresour. Technol. 209, 133–141. doi:10.1016/j.biortech.2016.02.069 31. Watanabe, A., 1960. List of Algal Strains in Collection At the Institute of Applied Microbiology, University of Tokyo. J. Gen. Appl. Microbiol. 6, 283– 292. 32. Zheng, H., Yin, J., Gao, Z., Huang, H., Ji, X., Dou, C., 2011. Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: A comparison of grinding, ultrasonication, bead Milling, enzymatic lysis, and microwaves. Appl. Biochem. Biotechnol. 164, 1215–1224. doi:10.1007/s12010-011-9207-1 33. Zheng, Y., Yu, X., Li, T., Xiong, X., Chen, S., 2014. Induction of D-xylose uptake and expression of NAD(P)H-linked xylose reductase and NADP + linked xylitol dehydrogenase in the oleaginous microalga Chlorella sorokiniana. Biotechnol. Biofuels 7, 125. doi:10.1186/s13068-014-0125-7

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Figure captions Figure 1: C. minutissima growth in raceway photobioreactors: () C1, () X1, () A1 and () P1 exposed to (a) 33.75 µmol.m-2.s-1; () C2, () X2, () A2 and () P2 exposed to (b) 16.88 µmol.m-2.s-1; and () C3, () X3, () A3 and () P3 exposed to (c) 8.44 µmol.m-2.s-1.

Figure 2: Protein profiles of different C. minutissima cultures grown in raceway photobioreactors. The protein profiles were determined by SDS-PAGE. PS, protein standards; Well 1, C1; Well 2, X1; Well 3, A1; Well 4, P1; Well 5, C2; Well 6, X2; Well 7, A2; Well 8, P2; Well 9, C3; Well 10, X3; Well 11, A3; and Well 12, P3.

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Tables and Figures Table 1: Assays performed to evaluate the effects of varying the light intensity on C. minutissima grown in raceway photobioreactors. Light intensity

Nitrogen concentration

Pentose concentration

(µmol.m-2.s-1)

(g.L-1)

(mg.L-1)

33.75 16.88

0.250

8.44

-

C1

-

C2

-

C3

33.75 16.88

Assay

X1 0.125

D-xylose: 20

X2

8.44

X3

33.75

A1

16.88

0.125

L-arabinose: 20

8.44

A2 A3

33.75

P1 D-xylose: 19.16

16.88

0.125

P2 L-arabinose: 0.89

8.44

P3

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Table 2: Maximum biomass concentration (Xmax, g.L-1), maximum productivity (Pmax, g.L-1.d-1), maximum specific growth rate (μmax, d-1) and biomass pentose conversion factor (YX/S mg.mg-1) of C. minutissima cultures in raceway photobioreactors. Assays

Xmax

Pmax

µmax

YX/S

(g.L-1)

(g.L-1.d-1)

(d-1)

(mg.mg-1)

C1

1.34±0.14h

0.10±0.01c

0.41±0.02d

-

C2

0.69±0.01a,d,e

0.04±0.01b,d

0.16±0.01a,b

-

C3

0.42±0.01b,c

0.03±0.01b

0.21±0.01a,b

-

X1

0.88±0.08e,f,g

0.09±0.01a,c

0.33±0.03c,d

0.04±0.01a

X2

0.81±0.04a,e,f

0.07±0.01a,d

0.17±0.02a,b

0.03±0.01a,d

X3

0.51±0.02b,c,d

0.03±0.01b

0.21±0.01a,b

0.02±0.01b,e

A1

1.01±0.07g

0.08±0.03a,c

0.31±0.03c,e

0.04±0.01a

A2

0.74±0.07a,e,f

0.07±0.01a,d

0.25±0.01b,e

0.03±0.01c,d

A3

0.54±0.05a,b,c,d

0.04±0.01b

0.16±0.02a

0.02±0.01b,c

P1

0.96±0.16f,g

0.09±0.01a,c

0.34±0.04c,d

0.04±0.01a

P2

0.66±0.03a,c,d

0.05±0.01a,c,d

0.17±0.02a,b

0.02±0.01b,c,d

P3

0.29±0.01a

0.03±0.01b

0.19±0.01a,b

0.01±0.01e

The same superscript letter in the same column indicates that the means are not significantly different at a 95% confidence level (p > 0.05).

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Table 3: Carbohydrate (%w.w-1) and protein (%w.w-1) concentrations (mean ± standard deviation) of C. minutissima cultivated in raceway photobioreactors and differences between the carbohydrate and protein contents of the control cultures and those with added pentoses (R %). Assays

Carbohydrates

R (%)

(%w.w-1)

Proteins

R (%)

(%w.w-1)

Theoretical ethanol production (mL.100 g-1 biomass)

C1

39.2±3.55b,e

**

19.6±0.76a,b,c,d

**

25.4±2.30

C2

47.5±1.19a,b,c,d

**

20.4±2.81a,b,c,d

**

30.8±0.78

C3

43.5±3.41a,b,c,e

**

23.5±0.19c,d

**

28.2±2.21

X1

52.8±1.72a,d,f

(+) 34.7

14.8±1.73a,b

(-)24.5

34.2±1.11

X2

50.4±0.72a,c,d,f

(+) 6.1

13.8±2.12a,b

(-)32.6

32.6±0.47

X3

b,c,e

21.3±0.71

(-)9.4

26.2±0.97

40.5±1.50

(-) 6.9

a,b,d

A1

60.3±0.40f

(+) 53.8

15.6±0.44a,b

(-)20.4

39.1±0.26

A2

51.7±0.93a,d,f

(+) 8.8

21.5±3.64a,b,d

(+)5.4

33.5±0.60

A3

31.2±2.06e

(-) 28.3

26.0±1.99d

(+)10.6

20.6±0.46

P1

53.4±2.45d,f

(+) 36.2

16.7±1.29a,b,c

(-)14.8

34.6±1.59

P2

45.9±0.49a,b,c,d

(-) 3.4

12.5±0.06b

(-)38.7

29.7±0.32

P3

43.3±0.26a,b,c,e

(-) 0.5

23.2±1.01c,d

(-)1.3

28.0±0.16

The same superscript letter in the same column indicates that the means are not significantly different at a 95% confidence level (p > 0.05).

23

Figure 1

24

Figure 2:

25

Highlights 

Xylose and arabinose increase the carbohydrate content of Chlorella minutissima.



A low luminous intensity and the presence of pentoses affect the protein profile of Chlorella.



Higher luminosities promote Chlorella growth in raceway reactors.



Xylose and arabinose can be used as carbon sources for microalgal cultures.