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Short Communication

Coupling algal biomass production and anaerobicdigestion: Production assessment of some nativetemperate and tropical microalgae

Eric Fouilland a,b,*, Christophe Vasseur a,b, Christophe Leboulanger a,b,Emilie Le Floc'h a,b,c, Claire Carr�e a, Bruno Marty d, Jean-Philippe Steyer e,Bruno Sialve e

a Ecologie des Syst�emes Marins cotiers UMR 5119 ECOSYM (Universit�e Montpellier 2, CNRS, IRD, IFREMER,

Universit�e Montpellier 1), Universit�e Montpellier 2, Place E. Bataillon, CC093, 34095 Montpellier Cedex 5, Franceb Ecologie des Syst�emes Marins cotiers UMR 5119 ECOSYM (Universit�e Montpellier 2, CNRS, IRD, IFREMER,

Universit�e Montpellier 1), Station M�editerran�eenne de l'Environnement Littoral, 2 Rue des Chantiers, 34200 S�ete,

Francec Centre d'�ecologie marine exp�erimentale MEDIMEER (Mediterranean Center for Marine Ecosystem Experimental

Research) UMS 3301 (Universit�e Montpellier 2, CNRS), Station M�editerran�eenne de l'Environnement Littoral, 2 Rue

des Chantiers, 34200 S�ete, Franced Naskeo Environnement, Avenue des Etangs, Narbonne F-11100, Francee INRA, UR050, Laboratoire de Biotechnologie de l'Environnement, Avenue des Etangs, Narbonne F-11100, France

a r t i c l e i n f o

Article history:

Received 10 January 2014

Received in revised form

19 June 2014

Accepted 22 August 2014

Available online xxx

Keywords:

Microalgae

Digestates

Wastewaters

Extreme natural environments

Chlorophyta

Cyanobacteria

* Corresponding author. Ecologie des Syst�emUniversit�e Montpellier 1), Station M�editerran

E-mail address: eric.fouilland@univ-mon

Please cite this article in press as: Fouillaassessment of some native temperatej.biombioe.2014.08.027

http://dx.doi.org/10.1016/j.biombioe.2014.08.0961-9534/© 2014 Elsevier Ltd. All rights rese

a b s t r a c t

Coupling algal biomass production and anaerobic digestion is one of the most promising

bioprocesses for economically viable algal production. This study assesses the production

rates of some native microalgae growing in media supplemented with algal digestate,

urban wastewater or digested sludge. Native microalgal populations isolated from

temperate freshwaters (Scenedesmus spp.) and marine ecosystems (Nannochloris spp.) had

the highest potential production rates (about 100 mg DW L�1 d�1) with algal digestate at

about 20% loading ratio. However, no growth was measured for Nannochloris spp., when the

ammonium concentration exceeded 100 mg L�1 although Scenedesmus spp. appeared to be

tolerant to higher NH4þ concentrations. Very low production rates, or no growth, were

measured when microalgae isolated from high salinity waters (Dunaliella salina, Lyngbya

aestuarii) were used, suggesting that populations well adapted to extreme environmental

conditions are not suitable candidates for growing on wastewater or anaerobic digestate.

© 2014 Elsevier Ltd. All rights reserved.

es Marins cotiers UMR 5�eenne de l'Environnemetp2.fr (E. Fouilland).

nd E, et al., Coupling aland tropical microalga

027rved.

119 ECOSYM (Universit�e Montpellier 2, CNRS, IRD, IFREMER,nt Littoral, 2 Rue des Chantiers, 34200 S�ete, France.

gal biomass production and anaerobic digestion: Productione, Biomass and Bioenergy (2014), http://dx.doi.org/10.1016/

b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 4 ) 1e62

1. Introduction

Since thepublicationof thepaperbyOswaldandGotaas [1], the

combination ofwastewater treatment andmicroalgal biomass

productionhasbeen tested successfully inmanycountries and

shown to be an efficient means of removing nitrogen (N),

phosphorus (P) and toxic metallic or organic micropollutants

[2]. Processing wastewater grown algal biomass by lipid

transesterification, carbohydrate fermentation or anaerobic

digestion was recently proposed for sustainable production of

biofuels [3e5]. Coupling algal biomass production and anaer-

obic digestion could be the most promising bioprocess as

anaerobic digestion of a wide variety of microalgae seems

achievable [6,7]. Anaerobic digestion can convert the whole of

themicroalgae biomass or organic residue into energy (biogas)

and fertilizer (digestate). This process can, therefore, provide a

sustainable source for the energy required from the cultivation

step through to extraction as well as the nutrients required for

growth [8]. At lab and pilot scales, the amount of microalgal

biomass used to feed the digester (loading rate) has been re-

ported from 0.28 to 22 g VS per liter and awide range of species

and operating conditions has been used successfully for

anaerobic digestionwithmethane yields from70 to 587mlCH4

g VS�1 [9]. The methane produced can be used directly for en-

ergy production or indirectly for innovative chemical applica-

tions (e.g. graphene synthesis, [10]).

A range of valuable microalgae (e.g. Scenedesmus sp.,

Botryococcus sp., Spirulina sp.) has been reported to growwell in

urban, industrial and agricultural wastewaters [4,5]. The use

of native species from both freshwater and marine ecosys-

tems has recently been tested for local bioresource production

[11], including the production of lipids [12,13], basedmainly on

the adaptation of indigenous organisms to local biotic and

abiotic conditions to limit potential invasive or noxious

Fig. 1 e Production rates of microalgae growing on urban,

industrial or agricultural wastewaters in raceways

reported in the literature.

Please cite this article in press as: Fouilland E, et al., Coupling aassessment of some native temperate and tropical microalgaj.biombioe.2014.08.027

behavior. It can reasonably be assumed that native species,

selected from highly productive natural systems, would also

be good candidates for developing a bioprocess requiring a

high biomass production rate with nutrient loading from

treated or untreated wastewater. This is supported by the

production rates of mixtures of isolated native microalgae,

being higher than the rates measured for commonly culti-

vated microalgae species [4] in open ponds (Fig. 1).

In order to validate this assumption, we tested several

consortia from temperate and tropical systems considered to

be naturally very productive (up to 0.6 g L�1 of biomass in dry

weight). Each consortiumwas dominated by a population of a

single microalgal species or genus. These microalgae were all

supplied with wastewater and digestate containing different

concentrations of ammonium (NH4þ) and phosphate (PO4

3�).The growth rate, biomass production and production rate

were measured in batch lab cultures for different loading ra-

tios for each of the wastewater and digestate sources.

2. Methods

2.1. Selection of microalgal populations

The microalgal populations studied were obtained from

samples from diverse ecosystems and were acclimated to

laboratory culture conditions. The acclimation process led to

the dominance of a single homogeneous population. We refer

to i) a group of species (spp.) when no microscopic discrimi-

nation between species within one genus was possible, and ii)

a true species when only one taxon was identified in the

acclimated cultures.

The population of the freshwater microalgae Scenedesmus

spp. (Fig. 2A) was originally isolated from a wastewater

treatment lagoon system near M�eze, southern France

(43�2503400N, 3�3602700E). Prior to the experiment, Scenedesmus

spp. was cultivated at 20 �C under an average photosynthetic

photon flux density (PPFD) of 261 ± 20 mE m�2 s�1 with a 12:12

light:dark cycle in batch cultures using a modified Z8 medium

where ammonia (supplied asNH4Cl) replaced nitrate (supplied

as NaNO3) as the sole source of nitrogen and NaH2PO4 was

added as a source of phosphorus.

The population of the marine microalgae Nannochloris spp.

(Fig. 2B) was isolated from Thau Lagoon, southern France

(43�2405300N, 3�4101600E), one of the most intensive oyster

farming sites in France. Prior to the experiment, Nannochloris

spp. was cultivated under the same temperature and light

conditions as those for Scenedesmus spp. in batch cultures in

enriched seawater using a modified Conway medium where

ammonia (supplied as NH4Cl) replaced nitrate (supplied as

NaNO3) as the sole source of nitrogen and NaH2PO4 was added

as a source of phosphorus.

The population of halophile microalgae Dunaliella salina

(Fig. 2C) was isolated from temperate salterns in Gruissan,

southern France (43�0602700N, 3�0501100E). Prior to the experi-

ment, D. salina was cultivated using the same modified Con-

way medium and conditions as for Nannochloris spp.

The saline cyanobacteria Lyngbya aestuarii (Fig. 2D) was

cultivated from a tropical saline lake (Dziani Dzaha, Mayotte,

France) in the Indian Ocean (12�500 S, 45�100 E) where primary

lgal biomass production and anaerobic digestion: Productione, Biomass and Bioenergy (2014), http://dx.doi.org/10.1016/

b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 4 ) 1e6 3

production reached its highest natural levels (0.8 g Chloro-

phyll a L�1, 20 g O2 m�2 d�1). This population was acclimated

at 35 �C under an average PPFD of 261 ± 20 mE m�2 s�1 with a

12:12 light:dark cycle during a few days prior to the

experiment.

2.2. Growth and production rates of microalgae usingdifferent types of wastewater and digestate supplements

Urban wastewater was collected from the wastewater treat-

ment plant in Narbonne (southern France) a few days before

the experiment and contained 106mgNH4þ L�1 and 19mg PO4

3�

L�1. The concentrations of NH4þ and PO4

3� were determined by

ionic chromatography (ICS 3000, Dionex, USA). The secondary

activated sludge was collected from the same wastewater

treatment plant and digested (1 m3 continuous digester under

mesophilic conditions with a loading rate of 1 g L�1 d�1).

The freshwater microalgae Scenedesmus spp. isolated from

the wastewater treatment lagoon system near M�eze was

cultivated in batch conditions using the modified Z8 medium

described above in a 56 m2 outdoor open pond at pilot scale.

The biomass was harvested by settling when the concentra-

tion reached 0.5 gDW L�1. The concentrated biomass of Sce-

nedesmus spp. was digested using a 2.5 L digester inmesophilic

conditions with a loading rate of 1 g L�1 d�1, prior to the

experiment presented here. The methane yield was about 143

(±20) mL CH4 gVS�1. The liquid phase of both digested sludge

and algal digestates was separated by centrifugation (5 min,

18,600 G, 5 �C).The liquid digested sludge contained 1360 mg NH4

þ L�1 and

400 mg PO43� L�1, while the liquid algal digestates contained

between 630 and 960 mg NH4þ L�1 and 160 mg PO4

3� L�1.

Fig. 2 e Microalgae populations isolated from (A) wastewater tre

waters (Nanochloris spp), (C) Mediterranean salterns (Dunaliella

aestuarii).

Please cite this article in press as: Fouilland E, et al., Coupling alassessment of some native temperate and tropical microalgaj.biombioe.2014.08.027

Each of the four microalgal populations was inoculated

into a 96 well microplate using 30 mL of parent culture (10% of

total well volume) and incubated with different volumes of

wastewater and digestates (from 0% to 50% of the total well

volume: loading ratios) in 6 replicates and topped up with

deionized water. For D. salina only, the experiment was also

performed with 35 gNaCl L�1 in deionized water. The micro-

plates were incubated for 7 days at a fixed temperature (21 �Cfor temperate populations and 35 �C for the tropical popula-

tion) and the PPFD was maintained at an average of

261 ± 20 mE m�2 s�1 with a 12:12 light:dark cycle using OSRAM

L18W/954 daylight fluorescent tubes. The absorbance of the

cultures was chosen as a non-destructive proxy for the

microalgal biomass. The chlorophyll a þ b peak absorption

was targeted in order to reduce the interference caused by

adding the substrate and to improve the signal:noise ratio. A

wavelength of 650 nm (±10 nm bandpass filter) was chosen,

according to previous studies [14]. The linearity of the rela-

tionship between OD650 and the dry weight was checked. For

each algal population collected in the parent culture just

before the inoculation into the microplate, the microalgal

biomass was diluted with the culture media using from 6 to 8

different dilution rates ranging from 0 to 100% to determine

the relationship between OD650 and the dry weight of the

diluted microalgal biomass. During the microplate incubation

period, OD650 was measured daily using a Chameleon micro-

plate reader (Hidex, Turku, Finland) and corrected for the

absorbance of the digestate measured immediately after

microplate inoculation. The daily values of the corrected

OD650 were then converted into dry weight (mg DW L�1) using

the linear relationship determined above. The Verlhurst

equation was fitted to the biomass measured for the different

atment lagoon system (Scenedesmus spp), (B) marine coastal

salina) and (D) tropical saline Dziani Dzaha lake (Lyngbya

gal biomass production and anaerobic digestion: Productione, Biomass and Bioenergy (2014), http://dx.doi.org/10.1016/

Table 1 e Mean (avg) and standard deviation (std) of the maximum growth (d¡1) and production (mg DW L¡1) estimated from 6 replicates and derived production rates(mg DW L¡1 d¡1) for the four species with different loading ratios (0e50%) of untreated wastewater, organic waste sludge digestate and digestate from Scenedesmus spp.The values for Dunaliella salina in this table were obtained using salted (35 g NaCl L¡1) waste and digestates, as no growth was reported when using unsalted waste anddigestates. Significant differences between growth and production values measured with loading ratio of 0% of and those measured with a loading ratio greater than 0%are shaded (t-student test, p ≤ 0.05). NG: No Growth, NP: Not Performed.

Dilution(%)

Untreated wastewaters Digested sludges Digestates from Scenedesmus spp

Max growthrate (d�1)

Max biomass(mgDW L�1)

Max productionrate (mgDW L�1 d�1)

Max growthrate (d�1)

Max biomass(mgDW L�1)

Max productionrate (mgDW L�1 d�1)

Max growthrate (d�1)

Max biomass(mgDW L�1)

Maxproduction rate(mgDW L�1 d�1)avg std avg std avg std avg std avg std avg std

Scenedesmus spp 0 1.84 0.31 33 1 65 1.56 0.05 22 0 51 1.50 0.13 78 1 66

2.5 1.11 0.02 101 4 55 0.89 0.05 101 1 45 1.07 0.01 123 2 58

5 1.78 0.31 44 1 68 0.71 0.03 146 2 43 0.83 0.06 169 3 55

7.5 1.17 0.20 101 4 60 0.77 0.04 112 2 40 0.77 0.05 236 5 61

17.5 0.66 0.08 112 4 34 NG NG NG 0.73 0.03 485 9 99

25 0.93 0.21 55 2 36 NG NG NG 0.61 0.02 723 23 115

37.5 0.58 0.10 101 8 29 NG NG NG 0.54 0.03 508 27 76

50 0.79 0.098 180 9 52 NG NG NG 0.42 0.03 282 27 37

Nannochloris spp 0 0.49 0.05 159 10 27 0.69 0.10 28 1 20 0.51 0.10 49 3 16

2.5 0.59 0.05 231 9 42 1.01 0.10 193 4 78 0.78 0.05 426 10 88

5 0.68 0.08 226 9 48 NG NG NG 0.69 0.05 618 21 107

7.5 0.60 0.07 332 20 56 NG NG NG NG NG NG

17.5 0.77 0.04 537 13 105 NG NG NG NG NG NG

25 0.88 0.07 509 27 115 NG NG NG NG NG NG

37.5 NG NG NG NG NG NG NG NG NG

50 NG NG NG NG NG NG NG NG NG

Dunaliella salina 0 0.80 0.11 62 2 15 1.36 0.26 56 2 22 1.30 0.23 109 35 35

2.5 0.67 0.07 111 3 19 NG NG NG 0.92 0.03 382 4 88

5 NG NG NG NG NG NG 0.94 0.37 230 29 54

7.5 NG NG NG NG NG NG NG NG NG

17.5 NG NG NG NG NG NG NG NG NG

25 NG NG NG NG NG NG NG NG NG

37.5 NG NG NG NG NG NG NG NG NG

50 NG NG NG NG NG NG NG NG NG

Lyngbya aestuarii 0 NP NP NP NG NG NG NG NG NG

1 NP NP NP 0.36 0.01 286 49 18 1.16 0.24 81 3 23

5 NP NP NP NG NG NG 0.74 0.22 78 8 14

10 NP NP NP NG NG NG 0.84 0.10 113 6 24

20 NP NP NP NG NG NG 1.93 0.24 34 1 16

33 NP NP NP NG NG NG NG NG NG

40 NP NP NP NG NG NG NG NG NG

50 NP NP NP NG NG NG NG NG NG

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b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 4 ) 1e6 5

loading ratios of the wastewater and digestates, using the

regression tools in SigmaPlot 10.1. The algal growth was

expressed as a sigmoid curve using equation (1):

fðtÞ ¼ K1

1þ ae�rt(1)

where r is the maximum growth rate (d�1) and K is the

maximum achievable production or biomass (i.e. carrying

capacity of the culture system in mg DW L�1). The first de-

rivative was calculated from each fitted equation to estimate

the maximum production rate (mg DW L�1 d�1).

Fig. 3 e Maximum production rates measured for each

population incubated at different loading ratios as a

function of the NH4þ concentration at the start of

incubation. The values for Dunaliella salina in this figure

were obtained using salted (35 g NaCl L¡1) waste and

digestates, as no growth was reported when using

unsalted waste and digestates.

3. Results and discussion

The temperate freshwater microalgae Scenedesmus spp. had

significant growth rates (between 0.4 and 1.8 d�1) with all the

unsalted wastewater and digestate supplements at all

loading ratios (Table 1), except for digested sludge at a

loading ratio greater than 7.5%. The highest maximum

growth rates for this population were measured at low

loading ratios, while the highest maximum production rates

(99e115 mg DW L�1 d�1) were measured when the micro-

algae was growing on its own digestate at a loading ratio of

about 20% (Table 1). Similar maximum production rates were

measured for the marine microalgae Nannochloris spp. with

unsalted wastewater (17.5% and 25% loading ratios) and

digestate of Scenedesmus spp. (5% and 17.5% of loading ratios).

The temperate freshwater and marine populations tested in

this study were isolated from Mediterranean ecosystems

known to have highly variable climatic (temperature, light)

and hydrological (flood) conditions. The populations of Nan-

nochloris spp. and Scenedesmus spp. isolated from these eco-

systems made efficient use of the algal digestate nutrients

and the marine population Nannochloris spp. also tolerated

considerable salinity variations. This supports the feasibility

of bioenergy production by coupling anaerobic digestion and

microalgal biomass production. The study clearly showed

that not only microalgal species from freshwaters but also

microalgae isolated from coastal marine waters could

potentially achieve high biomass production rates using un-

salted digestates.

However, no growth was measured for the three marine

populations (Nannochloris spp., D. salina and L. aestuarii) with

unsalted or salted water (only D. salina was tested with

salted water) and digested sludge at a loading ratio of more

than 2.5% (Table 1). The highest initial NH4þ concentration

was measured in the digested sludge (>1 g L�1). The very

high NH4þ concentration may inhibit microalgae growth,

especially in populations growing naturally under lower

NH4þ concentrations. Scenedesmus spp., isolated from a

wastewater treatment lagoon system, seemed to be more

tolerant of NH4þ as shown by the high rates measured at

NH4þ concentrations greater than 100 mg L�1 (Fig. 3). In

addition to high NH4þ concentrations, the potential presence

of concentrated toxic compounds in the digested sludge [15]

or a lack of essential growth micronutrients, e.g. Mg [16],

may also explain the reduction or absence of microalgal

growth.

Please cite this article in press as: Fouilland E, et al., Coupling alassessment of some native temperate and tropical microalgaj.biombioe.2014.08.027

D. salina isolated from Mediterranean salterns only grew

with wastewater and digestates when salt (NaCl) was added

and the maximum production rate rate (88 mg DW L�1 d�1)

was reachedwith very low loading ratios (2.5%) of Scenedesmus

spp. digestate. Neither L. aestuarii, which had the lowest

maximumproduction rate rates (14e24 mg DW L�1 d�1) nor D.

salina appeared to grow well when wastewater or digestates

were added. These populations were isolated from ecosys-

tems with specific, extreme environmental characteristics

(salinity of over 50 PSU) resulting in selection in the natural

environment. This specialization appeared to have led to

reduced phenotypic plasticity and reduced ability to grow in

other conditions (e.g. low salinity, high NH4þ concentrations)

and may not be suitable for producing biomass using waste-

water or digestates. This supports the suggestion that many

algal species living in extreme environments appear to be

highly specialized organisms, adapted to a narrow set of

environmental conditions [17].

4. Conclusion

Theseresultsshowthat temperatemicroalgaefrombothmarine

and freshwaters ecosystems have great potential as candidates

for coupled anaerobic digestion and algal biomass production

whereas microalgae isolated from extreme environmental eco-

systems appear to be unsuitable for such coupling.

Acknowledgments

This work was supported by the French National Research

Agency, under the Symbiose (ANR-08-BIOE-11) project, and by

gal biomass production and anaerobic digestion: Productione, Biomass and Bioenergy (2014), http://dx.doi.org/10.1016/

b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 4 ) 1e66

the Scientific Council of University Montpellier II (METTRO

project, 2010). This work benefited from the facilities provided

the Centre d'�ecologie marine exp�erimentale MEDIMEER (UMS

3301). C�ecile Roques (UMR 5119 ECOSYM) and Myriam Le

Chevanton (IFREMER PBA Nantes) provided some of the pic-

tures of microalgal population used in this work.

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