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A cost-effective strategy for the bio-prospecting of mixed microalgae with highcarbohydrate content: diversity fluctuations in different growth media

Glenda Cea-Barcia, Germán Buitrón, Gloria Moreno, Gopalakrishnan Kumar

PII: S0960-8524(14)00619-1DOI: http://dx.doi.org/10.1016/j.biortech.2014.04.079Reference: BITE 13375

To appear in: Bioresource Technology

Received Date: 18 January 2014Revised Date: 15 April 2014Accepted Date: 21 April 2014

Please cite this article as: Cea-Barcia, G., Buitrón, G., Moreno, G., Kumar, G., A cost-effective strategy for the bio-prospecting of mixed microalgae with high carbohydrate content: diversity fluctuations in different growth media,Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.04.079

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A cost-effective strategy for the bio-prospecting of mixed microalgae with

high carbohydrate content: diversity fluctuations in different growth media

Glenda Cea-Barcia, Germán Buitrón*, Gloria Moreno and Gopalakrishnan Kumar Laboratory for Research on Advanced Processes for Water Treatment, Instituto de Ingeniería,

Unidad Académica Juriquilla, Universidad Nacional Autónoma de México, Blvd.

Juriquilla,3001, Queretaro 76230, Mexico.

*Corresponding Author GbuitronM@ii.unam.mx

Abstract

In recent years, widespread efforts have been directed towards decreasing the costs associated

with microalgae culture systems for the production of biofuels. In this study, a simple and

inexpensive strategy to bio-prospect and cultivate mixed indigenous chlorophytes with a high

carbohydrate content for biomethane and biohydrogen production was developed. Mixed

microalgae were collected from four different water-bodies in Queretaro, Mexico, and were

grown in Bold’s basal mineral medium and secondary effluent from a wastewater treatment plant

using inexpensive photo-bioreactors. The results showed large fluctuations in microalgal genera

diversity based on different culture media and nitrogen sources. In secondary effluent,

Golenkinia sp and Scenedesmus sp. proliferated. The carbohydrate content, for secondary

effluent, varied between 12% and 57%, and the highest volumetric and areal productivity were

61 mg L-1 d-1 and 4.6 g m-2 d-1, respectively. These results indicate that mixed microalgae are a

good feedstock for biomethane and biohydrogen production.

Keywords: bio-prospecting; biofuel; microalgae; carbohydrates; hydrogen; methane

1. Introduction

Microalgae have recently demonstrated various advantages due to their ease of growth, high

accumulation of lipids and carbohydrates during starvation conditions and higher productivity

(Bahadar and Bilal Khan, 2013; González-Fernández and Ballesteros, 2012). Biofuels produced

from microalgae, such as bioethanol, biodiesel, biohydrogen and biomethane, have been gaining

more attention in recent years for use as fuel sources in lieu of fossil fuels, which are being

depleted daily due to overextraction (Bahadar and Bilal Khan, 2013; Chisti, 2013). To date,

research efforts have been primarily focused on the limitations for the commercialization of

microalgal biofuels, such as inexpensive and energy efficient biomass production methods, the

supply of N and P nutrients, water constraints and the availability of concentrated carbon

dioxide. Moreover, microalgal biotechnology provides new opportunities for wastewater

treatment (Chisti, 2013). The bio-prospecting of unique microalgal strains could lead the way to

finding suitable microalgal strains for the production of biofuels from biomass (Mutanda, 2011).

However, using pure cultures or strains of microalgae presents difficulties in industrial

applications due to contamination issues. The exploitation of mixed indigenous microalgae

cultures may be a possible solution and has the capacity for commercialization. A previous

report on the potential of mixed microalgae for biodiesel production provided information about

lipid content (Venkata Mohan et al, 2011). However, it was lacking data from different reactor

types for growth and carbohydrate content analyses. The carbohydrate content is important for

production of gaseous fuels such as biomethane and biohydrogen (Liu et al., 2012). Thus, the

aim of this study was to bio-prospect for mixed indigenous microalgae cultures with high

carbohydrate contents that were able to grow efficiently in secondary effluent and without CO2

enrichment by assessing various inexpensive photo-bioreactors for biomethane and biohydrogen

production.

2. Methods

2.1. Collection of mixed microalgae

Mixed microalgae were collected from four different aquatic environments from Queretaro State

in central México, which are denoted here as A, B, C and D. The samples of phytoplankton were

collected using a mesh net (20 µm mesh) and then stored in vials prior to inoculation. Sample A

was collected from a freshwater streamlet at a depth of 60 cm, and the following conditions were

observed: pH 8.4, temperature 12 °C, total dissolved solids (TDS) 615 mg/L, conductivity 1226

µS cm-1 and dissolved oxygen (DO) 7.1 mg/L. Sample B was collected from a freshwater lake at

a depth of 1 m, and the following conditions were recorded: pH 8.3, temperature 17 °C, TDS 530

mg/L, conductivity 1070 µS cm-1 and DO 6.8 mg/L. Sample C was collected from a water dam

receiving treated wastewater at 1 m depth under the following conditions: pH 8.9, temperature 16

°C, TDS 1840 mg/L, conductivity 3680 µS cm-1 and DO 6.6 mg/L. Sample D was collected from

a water dam that provides drinking water under the following conditions: pH 8.4, temperature 12

°C, TDS 300 mg/L, conductivity 609 µS cm-1 and DO 7.0 mg/L. Finally, all collected samples

were sieved with a 250 µm mesh to remove impurities and insect larvae.

2.2. Enrichment and growth of mixed microalgae in various photo-reactors

To select mixed microalgae belonging to the class chlorophyceae, the freshwater phytoplankton

(A, B, C and D) were grown in Bold’s basal medium, which is a nutrient medium widely used

for freshwater microalgae of the classes chlorophyceae, xanthophyceae, chrysophyceae, and

cyanophyceae (Wehr and Sheath, 2003). All the inocula were cultivated in Erlenmeyer flasks

with magnetic stirring at room temperature (23 °C). The reactors were illuminated with a

continuous light supply from by a 54 W daylight neon lamp with an intensity of 100 µmol m-2 s-1

(LT 300 Extech Instruments, USA). Afterwards, the cultures were successively grown in

different sized microalgal reactors of 2 L (glass), 5 L (plastic bottle) and 20 L (plastic bag). The

reactors were aerated at a flow rate of 1 L/min and kept under the same culture conditions. The

inoculum for each reactor was 10% (v/v) and was taken from a previously grown culture. The

biomass growth was measured using a relationship between optical density (UV/visible;

Shimadzu spectrophotometer, Japan) and total suspended solids (SS). The maximal absorbance

was set at 685 nm for all cultures. The specific growth rate (µ) was calculated according to the

equation: lnXt = µt + lnX0, where X and X0 are the SS (g L-1) at time (t) and initial time (0),

respectively.

2.3. Mixed microalgae growth in secondary effluent

To evaluate nutrient removal, the mixed cultures of microalgae were grown in secondary effluent

collected from a secondary settler of a municipal wastewater treatment plant in Queretaro.

Microalgae previously grown in Bold’s medium were used for this experiment. The culture

conditions and reactor types were used as described previously. SS, total chemical oxygen

demand (COD), ammoniacal nitrogen (N-NH3), nitrate nitrogen (N-NO3-) and soluble total

phosphorus (TP) were measured.

2.4. Mixed microalgae identification

The four non-axenic microalgal cultures were characterized regarding the types of genera present

in the initial environmental samples, in Bold’s medium cultures and in the secondary effluent

cultures. The microalgae genera were identified according to Wehr and Sheath (2003). Samples

were taken periodically from the microalgae reactors to identify the genera growing in the

cultures. An optical microscope (Leica DM500, Japan) with Leica LAS EZ 2.0.0 software was

used for microalgae identification. Photomicrographs were taken to document the genera present

and were then used for comparison with micrograph catalogs of known genera. The cell number

was determined by direct counting with a 0.1 mm improved Neubauer counting chamber. In this

study, a simple and cost-effective method for microalgae characterization is presented; thus, at

this stage, the molecular biology analysis was not considered.

2.5. Analytical methods

Total volatile solids (VS), SS, NH3-N, NO3--N and COD concentrations were determined

according to standard methods (APHA, 2005). The TP was measured in a TNPC-4110 C

analyzer (Shimadzu, Japan). The total carbohydrate concentration was determined by the phenol

sulfuric acid method, using glucose as a standard according to Dubois et al (1956). All

experimental runs were performed in triplicate. The mean and standard deviation values were

reported.

3. Results and discussion

3.1. Diversity variance of mixed microalgae during growth

The initial samples of phytoplankton showed a large diversity of microorganisms, primarily

different microalgal genera, diatoms, bacteria and protozoa, because they were collected from

bodies of open water. Table 1 shows the microalgae diversity and the genus changes from the

initial four inocula. The initial inocula were analyzed immediately after collection, after six

months of successive cultures in Bold’s medium and after three consecutive cultures in

secondary effluent. Due to the selection pressure applied by the Bold’s medium, there was an

enrichment of chlorophytes, which were the predominant microalgae in all cultures. In the

cultures grown in secondary effluent, chlorophytes also predominated and genus variation was

observed. All of the microalgae cultures became significantly varied. Major changes between

Bold’s medium and the secondary effluent (nearly 50% variation) were observed in culture D,

mainly due to the differences in the amount and type of nitrogen source. The Bold’s medium

contained a higher amount of the nitrogen content as nitrite (41 mg N-NO3. L-1) than the

secondary effluent, which had principally ammonium (22 ± 14 mg N-NH3 L-1). Wu et al (2013)

studied the effects of different nitrogen sources in a culture of Monoraphidium sp SB2 and

showed that potassium nitrate is the best nitrogen source in an artificial medium. The biomass

concentration of Monoraphidium sp SB2 in media with ammonium chloride and ammonium

nitrate as the nitrogen source decreased by 74 and 83%, respectively. In fact, this effect was also

observed in culture D, where Monoraphidium sp represented 73% of the genera in Bold´s

medium but decreased to 7% in secondary effluent (Table 1). In the four microalgae cultures, the

genera that proliferated successfully with ammonium nitrogen were Scenedesmus sp,

Keratococcus sp, Golenkinia sp, and filamentous microalgae such as Oscillatoria sp and Ulotrix

sp. (Table 1).

3.2. Growth kinetics of mixed microalgae in Bold’s medium

At the beginning of the enrichment process, the biomass concentrations of the cultures were 76 ±

16, 36 ± 6, 32 ± 4 and 76 ± 9 mg L-1 for cultures A, B, C and D, respectively, in a working

volume of 650 mL. To increase the biomass concentration, a supply of air was introduced into

the liquid phase of the Erlenmeyer reactors with a working volume of 1.6 L, and the growth

kinetics were observed over a 14 day period. The growth kinetics and the specific growth rates of

each mixed microalgae culture are shown in Fig. 1. After 14 days of cultivation, cultures A and

D had the highest biomass concentrations of 830 and 850 mg L-1, respectively, and they also

showed similar growth rates of 0.25 d-1 and 0.22 d-1, respectively. In contrast, cultures B and C

achieved the lower final biomass concentrations of 470 and 220 mg SS L-1, respectively.

Cultures A and D are composed primarily of the genera Scenedesmus sp. and Monoraphidium sp,

respectively (Table 1). Previous reports (Li et al., 2010; Yu et al., 2012;) showed that

Scenedesmus sp has a specific growth rate of 0.34 ± 0.19 d-1 at 25 °C, whereas Monoraphidium

sp showed 0.15 ± 0.05 d-1, suggesting that the data are consistent with these reports under similar

conditions. The differences in the final biomass concentrations observed in this study are

possibly due to the microalgae diversity present in the different cultures. Unlike cultures A and

D, culture B contained a wider range of microalgae genera; however, a predominant genus was

absent (Table 1). This makes it difficult to compare growth rates with a specific microalgae

genus. Culture C contained a higher percentage of filamentous microalgae, which tend to form

flocs that encapsulate the dispersed microalgae, limiting their growth and therefore resulting in

lower productivities.

3.3. Mixed microalgae growth in secondary effluent and carbohydrate composition

Microalgae cultures were also grown in domestic secondary effluent to evaluate the potential of

the mixed microalgae for nutrient removal and whether wastewater could be used for growth

rather than Bold's medium. The initial chemical composition of the secondary effluent was 99 ±

48 mg total COD L-1, 39 ± 24 mg SS L-1, 22 ± 14 mg N-NH3 L-1, 0 mg N-NO3

- L-1, 5.7 ± 0.2 mg

TP L-1 and pH 8.2 ± 0.4. After the growth of microalgae, the removal percentages of N-NH3, TP

and total COD were 100%, 70%, and 10%, respectively. Moreover, the pH increased to 8.9 ±

0.18. This result indicated that microalgae grown in wastewater is a good alternative for the

removal of inorganic nutrients. Table 2 shows the productivities achieved in various aerated

microalgal reactors in Bold’s medium and secondary effluent. In both culture media, the plastic

bags were identified as the most efficient microalgal reactors tested. The productivities obtained

in Bold’s medium and secondary effluent in plastic bags showed conflicting results, as the

productivities in the secondary effluent decreased by 27, 16 and 40% for cultures A, B and D,

respectively. According to the microscopy analysis, the dominant genera Scenedesmus sp and

Keratococcus sp that were present in culture A during growth in Bold’s medium remained while

growing in the secondary effluent. In contrast, the genus Dictyosphaerium sp, present in culture

B in Bold’s medium (30%), was replaced by the genera Scenedesmus sp and Keratococcus sp

when grown in secondary effluent (Table 1). This result may explain the similar productivities

achieved by the growth of cultures A and B in secondary effluent (Table 2). Some authors report

that the genus Scenedesmus sp has the ability to grow in domestic secondary effluent and can

efficiently remove inorganic nutrients (Li et al., 2010), which is consistent with the results

presented in this study. The significant decrease in the biomass productivity of culture D may be

due to the great variation in the predominant genus observed in secondary effluent. The genera

Golenkinia sp (42%) and Keratococcus sp (29%) significantly displaced the genus

Monoraphidium sp (73%), present mostly in the Bold’s medium culture (Table 1). Regarding the

genera in secondary effluent, Soler et al. (1991) reported the proliferation of the genus

Golenkinia sp in a wastewater self-depuration lagoon, but insufficient information exists about

culturing the genus Keratococcus sp. in wastewater. Comparing the productivities of culture C in

Bold’s medium versus secondary effluent, no significant differences were observed (section 3.1).

Culture C was not grown in plastic bags due to technical difficulties. The abundant filamentous

microalgae adhered to the walls of the reactors and ultimately obstructed the passage of light

through the reactor. Ruiz et al. (2013) reported productivities for Scenedesmus sp, growth in

secondary effluent and using 5% CO2-enriched air, ranging from 5.7 to 10.1 g m-2 d-1. Maximal

productivities obtained in the present study, in both Bold´s and secondary effluent were lower

than the previous report because of the lower concentration of CO2 contained in ambient air used

for the microalgae cultivation (Table 2).

The carbohydrate content of all mixed microalgae cultures grown in secondary effluent was

determined. Table 2 shows the carbohydrate content of all the mixed cultures expressed in g

glucose equivalent/g VS. Cultures A and D have the highest quantities of carbohydrates, with 37

± 0.5% and 57 ± 1%, respectively. These values are higher than different axenic microalgal

cultures in which the average carbohydrate content is approximately 20 ± 6% of dry matter

(Becker, 2007). This is the first report regarding the carbohydrate content of mixed microalgae

and provides useful information for biohydrogen and biomethane production.

4. Conclusions

It was demonstrated that it is feasible to develop a cost-effective strategy to bio-prospect and

cultivate mixed indigenous microalgae with high carbohydrate content. A large variation in

genus diversity was observed due to the growth medium and nitrogen source. In Bold’s medium,

Scenedesmus sp and Monoraphidium sp were the predominant genera, whereas in secondary

effluent, Golenkinia sp and Scenedesmus sp. proliferated. The carbohydrate content of

microalgae grown in secondary effluent varied between 12% and 57%, and the highest

volumetric and areal productivity were 61 mg L-1 d-1 and 4.6 g m-2 d-1, respectively, indicating

that mixed microalgae are a good feedstock for biomethane and biohydrogen production.

Acknowledgments

The authors gratefully acknowledge the financial support of the project FOMIX-CONACYT-

Queretaro (grant 192341). Enrique Cantoral and Jaime Perez Trevilla are acknowledged for their

technical assistance and DGAPA-UNAM for the postdoctoral fellowship to G. Cea.

Supplementary data

Supplementary data associated with this article can be found in the online version.

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newly isolated freshwater microalga, Scenedesmus sp LX1, growing in secondary effluent.

New Biotechnol. 27, 59-63.

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hydrogen production by Clostridium butyricum CGS5 using carbohydrate-rich microalgal

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9. Mutanda, T., Ramesh, D., Karthikeyan, S., Kumari, S., Anandraj, A., Fux, B., 2011.

Bioprospecting for hyper lipid producing microalgal strains for sustainable biofuel production.

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10. Ruiz, J., Álvarez-Díaz, P.D., Arbib, Z., Garrido-Pérez, C., Barragán, J., Perales, J.A., 2013.

Performance of a flat panel reactor in the continuous culture of microalgae in urban

wastewater: Prediction from a batch experiment. Bioresour Technol. 127, 456-463.

11. Soler, A., Sáez, J., Lloréns, M., Martinez I., Torrella F., Berná, M., 1991. Changes in

physico-chemical parameters and photosynthetic microorganisms in a deep wastewater self-

depuration lagoon. Wat Res. 25 (6), 689-695.

12. Venkata Mohan, S., Prathima devi, M., Mohankrishna, G., Amarnath, N., Lenin, Babu, M.,

Sarma, P.N., 2011. Potential of mixed microalgae to harness biodiesel from ecological

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15. Yu, X., Zhao, P., He, C., Li, J., Tang, X., Zhou, J., Huang, Z., 2012. Isolation of a novel

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Figure Legend

Fig. 1. Growth kinetics of four mixed microalgae cultures in Erlenmeyer reactors in Bold’s

medium at 23 °C and 100 µmol m-2 s-1 (culture A: µ = 0.25 d-1; culture B: µ = 0.34 d-1; culture C:

µ = 0.15 d-1; culture D: µ = 0.22 d-1).

Table 1. Diversity variance during growth of mixed microalgae cultures

Culture 1Environmental sample (collection site)

Bold’s medium Secondary effluent

Culture A Scenedesmus, Pandorina,

Pediastrum, Caloxtrix and undetermined (<1%)

Scenedesmus (79%), Keratococcus (19%), Oscillatoria and undetermined (<2%)

Scenedesmus (98%), Keratococcus (1%) and undetermined (<1%)

Culture B Scenedesmus,

Dictyosphaerium, Pediastrum,

Oscillatoria and undetermined

(<1%)

Scenedesmus (35%), Monoraphidium (8%), Dictyosphaerium (30%), Nitzchia (2%), Keratococcus

(15%), Oscillatoria and undetermined (<10%)

Scenedesmus (95%), Keratococcus (4%), Closterium and

undetermined (<1%)

Culture C1 Chlorella, Ulotrix, Anabaena,

Oscillatoria and undetermined (<1%)

Scenedesmus, Ulotrix,

Anabaena, Oscillatoria,

Oocystis and undetermined (<1%)

Scenedesmus, Ulotrix,

Anabaena, Oscillatoria,

Oocystis and undetermined (<1%)

Culture D Scenedesmus, Closterium,

Phacus, Coelastrum, Oocystis,

Euglena, Chlorella,

Oscillatoria, Diploneis,

Aphanocapsa, Anabaena,

Nitzchia and undetermined (<1%)

Scenedesmus (14%), Monoraphidium (73%), Closterium (2%), Chlorella

(3%), Oscillatoria and undetermined (<8%)

Scenedesmus (21%), Keratococcus (29%), Golenkinia (42%), Monoraphidium (7%), Oscillatoria and

undetermined (<1%)

1Percentages not determined.

Table 2. Summary of the biomass productivities achieved from different microalgal reactors and carbohydrate content

N.M.: Not measured; 11.6 L working volume;

24 L working volume;

38 L working volume;

4Based on facility area. The

standard deviation of the productivities corresponds to 3 consecutive growths.

Culture Productivities in Bold’s medium Productivities in secondary effluent

Reactor Erlenmeyer

1

(mg L-1

d-1

) Plastic bottles

2

(mg L-1

d-1

) Plastic bags

3

(mg L-1

d-1

)

Plastic bags4

(g m-2

d-1

) Plastic bottles

2

(mg L-1

d-1

) Plastic bags

3

(mg L-1

d-1

)

Plastic bags4

(g m-2

d-1

) % Carbohydrate

(g glucose/gVS)

A 55 ± 4 62 ± 4 83 ± 3 6.2 ± 0.2 32 ± 5 61 ± 4 4.6 ± 0.3 37 ± 0.5

B 68 ± 9 42 ± 13 75 ± 3 5.6 ± 0.2 34 ± 16 63 ± 3 4.7 ± 0.2 19 ± 1

C 12 ± 11 31 ± 16 N.M. N.M. 30 ± 6 N.M. N.M. 12 ± 1

D 60 ± 6 59 ± 8 70 ± 4 5.2 ± 0.3 32 ± 9 42 ± 10 3.1 ± 0.7 57 ± 1

15

Highlights:

• A cost-effective strategy for cultivation of indigenous microalgae was proposed.

• Diversity decreased when microalgae were grown in secondary effluent.

• Scenedesmus sp, Keratococcus sp and Golenkinia sp were the predominant genera.

• High carbohydrate content was observed for microalgae growth in secondary effluent.