**4. Microalgae growth in anaerobic digestates**

#### **4.1. Physico-chemical characterization of digestates**

**Microalga Co-substrate Conditions Improvement in** 

Laboratory scale, continuously stirred tank reactor, at mesophilic temperature

temperature

temperature

temperature

temperature

temperature

temperature

temperature

temperature

temperature

temperature

temperature

temperature

temperature

semi-continuous

thermophilic temperature

Sewage sludge Batch at mesophilic and

Batch at mesophilic temperature

Batch at mesophilic temperature

Batch experiments 77 (compared to

Batch experiments No positive effect [83]

Glycerol C/N = 12.44

*Chlorella* sp. (4%) WAS (96%) Batch at mesophilic

(80%)

(20%)

(88%)

(75%)

*C. sorokiniana (25%)* WAS (75%) Batch at mesophilic

*C. vulgaris (80%)* Manure (20%) Batch at mesophilic

*Scenedesmus* sp. (15%) Pig manure (85%) Batch at mesophilic

*Scenedesmus* sp. (20%) WAS (80%) Batch at mesophilic

*D. salina (25%)* OMSW (75%) Batch at mesophilic

*N. salina (16.6%)* Corn silage (83.4%) Batch at mesophilic

Lipid-spent *B. braunii* WAS and glycerol Batch at mesophilic

*Micractinium* sp. (79%) WAS (21%) Batch at mesophilic

*A. platensis* (85%) Barley straw (15%) Batch at mesophilic

*A. platensis* (45%) Beet silage (55%) Batch at mesophilic

*A. platensis* (15%) *L. digitata* (85%) Batch at mesophilic

*O. tenuis* (66.6%) Pig manure (33.3%) Batch at mesophilic

*A. platensis* (33.3%) WAS (66.6%) Two stages

*Chlorella* 1067 (20%) Chicken manure

Pretreated *Chlorella* sp. (80%) Chicken manure

*Chlorella* sp. (12%) Wastewater sludge

*Scenedesmus* sp. (25%) *O. maxima* cladodes

Lipid-extracted *Chlorella*

76 Microalgal Biotechnology

biomass

*I. galbana* and *S. capricornutum*

**methane yield (%)**

>50 (compared to microalga)

73–79 (compared to microalga)

microalga)

39 (compared to microalga)

12 (compared to single substrate)

3.8 (compared to microalga)

66.4 (compared to microalga)

50.3 (compared to microalga)

39.5 (compared to microalga)

3 (compared to single substrate)

6 (compared to microalga)

10 (compared to microalga)

76.7 (compared to single substrate)

1.1 (compared to microalga)

1.6 (compared to single substrate)

32.5 (compared to microalga)

\* [73]

No positive effect [92]

No positive effect [93]

**Reference**

[80]

[81]

[82]

[84]

[85]

[86]

[87]

[88]

[22]

[36]

[90]

[27]

[95]

[95]

[95]

[97]

The anaerobic digestate studied by Solé-bundó et al. [100] presented low dry matter content (~3%), and these digestates can therefore be treated as liquids that could be directly spread onto soil as fertilizer. A problem arises when transportation is required and moisture reduction could be necessary. Anaerobic digestate from microalgae co-digestion was observed to present better water release than the digestate from single microalga digestion.

Other parameters that could have a negative impact on soil (pH, electrical conductivity, and volatile fatty acids) were lower in the co-digestion digestates, indicating that microalgae codigestion resulted in a more stable digestate.

In general, among the bibliography, anaerobic digestates from agro-food industries presented higher organic contents than those from microalgae digestion [101], which could be explained due to organic matter mineralization during anaerobic digestion processes. The use of microalgae as co-substrate in the digester reduces the VS/TS ratio when compared to microalga alone (from 53 to 54–47%) due to the better biodegradability of the organic compounds of the co-substrate.

In order to evaluate the feasibility of these anaerobic digestates as fertilizers, some elemental nutrients were evaluated. The total nitrogen content was higher in the non-co-digested microalgae (80 g/kg TS and 56 g/kg TS), although the N-NH<sup>4</sup> + /TKN ratio, which represents the soluble mineral nitrogen fraction, only varied from 30.9 to 33.8% among all digestates. Moreover, the C/N ratio was low across the board, which means that in each case the nitrogen content is too high for its use as fertilizers, although it could be used as soil amendment. This problem could be sorted out by using a high-carbon content co-substrate like OMSW or corn silage. Phosphorous and potassium were found slightly higher in the digestates from non-codigestion, although in each case the content was relatively low and similar to other anaerobic digestates reported in the literature. Calcium, magnesium, and sodium were also analyzed, and no difference was observed among the different digestates [100].

On the whole, the anaerobic digestate from microalgae co-digestion presented better suitability for nutrient supply in soil due to its low C/N ratio, which could be enhanced by using a co-substrate with a higher carbon content.

#### **4.2. Microalgae growth in digestates**

The anaerobic digestion of biomass produces a high-nutrient digestate, which is usually used as crop fertilizer, and also could be used as a nutrient supply for microalgae growth in order to reduce the use of external sources of nitrogen and phosphorous [102]. Moreover, wastewaters and other biomass present a reduction in suspended solids and color, better degradability, a more stable pH, and a reduction in pathogens after the anaerobic digestion process, which could enhance microalgae growth when compared to the non-digested biomass.

Moreover, according to the nutrient removal, the nitrogen depletion (up to 100%) and the phosphorous reduction (93.4%) were higher when the anaerobic digestate was sterilized and diluted by up to 2%. Nevertheless, the maximum COD removal (33.3%) was achieved with the non-sterilized anaerobic digestate and a higher dilution (10%). Regarding the fatty acid accumulation, after 25 days of growth, the concentration observed (31.1% dry weight) was

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79

De la Noüe et al. [103] studied the growth of different microalgae in swine manure anaerobic digestate diluted with tap water (0.6–3.0%). The results showed that *Scenedesmus obliquus* presented a response to high temperature, which could be a problem for outdoor work. This microalga was able to reduce the COD content of the anaerobic digestate by up to 85% with a microalga concentration of 57 mg dry wt/L·d at 20°C and with a manure concentration of

In a different study, *S. obliquus* was cultivated in the abovementioned conditions [107]. Under the best conditions (2% dilution), *S. obliquus* presented a biomass yield of 231 mg/L, regardless of the use of sterilized or non-sterilized anaerobic digestate. Moreover, according to the nutrient removal, the nitrogen depletion was higher (up to 100%) when the anaerobic digestate was sterilized and diluted by up to 2%. Nevertheless, the phosphorous reduction was higher (92.5%) when the anaerobic digestate was not sterilized, and the maximum COD removal (53.7%) was achieved with the non-sterilized anaerobic digestate and a higher dilution (10%). The fatty acid accumulation (26.6% dry weight) was higher after 25 days of growth than in the

Different anaerobic digestates from microalgae biomass co-digestion with swine and cow manure and vegetable wastes were selected for the growth of *Scenedesmus* sp. AMDD at

phosphorous concentrations were evaluated. Moreover, digestates were filtered to reduce the bacterial load. This study showed that the use of an anaerobic digestate from the codigestion of microalgae biomass presented a good microalga growth rate. Animal manure digestate without co-digestion did not produce a complete nitrogen removal, which was improved when Mg+2 was added in the media growth. This element was indicated as a key nutrient for microalgae growth, and it was concluded that 0.03 ± 0.02 mM was adequate for

*M. pusillum* was grown in a cheese factory anaerobic digestate at 20°C and proven to present a satisfactory microalga growth rate. After 4 days, the pH reached 8.5, and the ammonia depletion was complete, although, according to the high pH, it could be due to the stripping

137 ± 21 mg dry wt/L·d. Moreover, it was observed that the presence of suspended organic matter caused cell clogging and the adhesion of *M. pusillum* to the walls of the culture vessels [105].


3− removal reached 33%, and the biomass yield was

higher than in the control essay (19.6% dry weight).

*4.2.1.3. Scenedesmus genus*

control essay (24.5% dry weight).

22°C [102]. Nitrogen was adjusted to 1.5 mM (NH<sup>3</sup>

2% after 15 days.

optimal growth.

*4.2.1.4. Micractinium pusillum*

of ammonia or bacterial activity. P-PO<sup>4</sup>

The main factors that could affect the microalgae growth in anaerobic digestates are the nitrogen and phosphorous contents as well as the pH profile. pH could be increased due to active photosynthesis or insufficient CO<sup>2</sup> supply, which could provoke a N-NH<sup>4</sup> + disappearance through gas stripping and a P-PO<sup>4</sup> 3− precipitation when the medium presents a high concentration of Ca2+ [103]. Thus, when the pH of the medium is increased due to microalgae activity, nitrogen and phosphorous depletion do not necessarily mean an increase in biomass. Moreover, it has been reported that an ammonia concentration higher than 2 mM, when pH exceeds 8.1, presented a toxic effect on algae growth [104]. Regarding phosphorous content, it has been reported that 5 mg P/L was sufficient for adequate algae growth when the N/P ratio was around 15, although other studies suggested that N should be the limiting factor [103].

On the other hand, the organic load in these anaerobic digestates is reduced after microalgae cultivation. Nitrogen and phosphorous could be completely removed when the conditions are optimum and COD reduction could reach 44–85% depending on culture conditions and microalgae species [103].

#### *4.2.1. Chlorophytes*

#### *4.2.1.1. Chlorella genus*

An early study used different microalgae cultivated in swine manure anaerobic digestate diluted with tap water (0.6–3.0%) in order to evaluate its effect on microalgae growth. *Chlorella* sp. was the only species that presented pH stability (pH = 8.5 during 8 days), which indicated that the nitrogen removal was directly related to biomass production. Regarding temperature conditions, *Chlorella* sp. did not show any difference in biomass yield when the temperature was raised from 10 to 20°C. COD reduction in the anaerobic digestate reached 60%. The best conditions for the highest concentration (41 mg dry wt/L·d) were 20°C and a manure concentration of 2% [103].

#### *4.2.1.2. Parachlorella kessleri*

*P. kessleri* was cultivated (12 days; 25°C; air flow: 0.5–1 L/min; illumination: 200 μmol/m<sup>2</sup> ·s) in the anaerobic digestate derived from the co-digestion of end-of-life dairy products with a given mixture of agro-industrial wastes [107]. Prior to the growth of algae, the anaerobic digestate was filtered, diluted (2–10%), and then split into two different samples, one sterilized and the other not. Under the best conditions (2% dilution), *P. kessleri* presented a biomass yield of 270 mg/L, regardless of the use of sterilized or non-sterilized anaerobic digestate. Moreover, according to the nutrient removal, the nitrogen depletion (up to 100%) and the phosphorous reduction (93.4%) were higher when the anaerobic digestate was sterilized and diluted by up to 2%. Nevertheless, the maximum COD removal (33.3%) was achieved with the non-sterilized anaerobic digestate and a higher dilution (10%). Regarding the fatty acid accumulation, after 25 days of growth, the concentration observed (31.1% dry weight) was higher than in the control essay (19.6% dry weight).

#### *4.2.1.3. Scenedesmus genus*

**4.2. Microalgae growth in digestates**

78 Microalgal Biotechnology

photosynthesis or insufficient CO<sup>2</sup>

through gas stripping and a P-PO<sup>4</sup>

microalgae species [103].

*4.2.1. Chlorophytes*

*4.2.1.1. Chlorella genus*

tration of 2% [103].

*4.2.1.2. Parachlorella kessleri*

The anaerobic digestion of biomass produces a high-nutrient digestate, which is usually used as crop fertilizer, and also could be used as a nutrient supply for microalgae growth in order to reduce the use of external sources of nitrogen and phosphorous [102]. Moreover, wastewaters and other biomass present a reduction in suspended solids and color, better degradability, a more stable pH, and a reduction in pathogens after the anaerobic digestion process, which could enhance microalgae growth when compared to the non-digested biomass.

The main factors that could affect the microalgae growth in anaerobic digestates are the nitrogen and phosphorous contents as well as the pH profile. pH could be increased due to active

centration of Ca2+ [103]. Thus, when the pH of the medium is increased due to microalgae activity, nitrogen and phosphorous depletion do not necessarily mean an increase in biomass. Moreover, it has been reported that an ammonia concentration higher than 2 mM, when pH exceeds 8.1, presented a toxic effect on algae growth [104]. Regarding phosphorous content, it has been reported that 5 mg P/L was sufficient for adequate algae growth when the N/P ratio was around 15, although other studies suggested that N should be the limiting factor [103].

On the other hand, the organic load in these anaerobic digestates is reduced after microalgae cultivation. Nitrogen and phosphorous could be completely removed when the conditions are optimum and COD reduction could reach 44–85% depending on culture conditions and

An early study used different microalgae cultivated in swine manure anaerobic digestate diluted with tap water (0.6–3.0%) in order to evaluate its effect on microalgae growth. *Chlorella* sp. was the only species that presented pH stability (pH = 8.5 during 8 days), which indicated that the nitrogen removal was directly related to biomass production. Regarding temperature conditions, *Chlorella* sp. did not show any difference in biomass yield when the temperature was raised from 10 to 20°C. COD reduction in the anaerobic digestate reached 60%. The best conditions for the highest concentration (41 mg dry wt/L·d) were 20°C and a manure concen-

*P. kessleri* was cultivated (12 days; 25°C; air flow: 0.5–1 L/min; illumination: 200 μmol/m<sup>2</sup>

in the anaerobic digestate derived from the co-digestion of end-of-life dairy products with a given mixture of agro-industrial wastes [107]. Prior to the growth of algae, the anaerobic digestate was filtered, diluted (2–10%), and then split into two different samples, one sterilized and the other not. Under the best conditions (2% dilution), *P. kessleri* presented a biomass yield of 270 mg/L, regardless of the use of sterilized or non-sterilized anaerobic digestate.

supply, which could provoke a N-NH<sup>4</sup>

3− precipitation when the medium presents a high con-

+

disappearance

·s)

De la Noüe et al. [103] studied the growth of different microalgae in swine manure anaerobic digestate diluted with tap water (0.6–3.0%). The results showed that *Scenedesmus obliquus* presented a response to high temperature, which could be a problem for outdoor work. This microalga was able to reduce the COD content of the anaerobic digestate by up to 85% with a microalga concentration of 57 mg dry wt/L·d at 20°C and with a manure concentration of 2% after 15 days.

In a different study, *S. obliquus* was cultivated in the abovementioned conditions [107]. Under the best conditions (2% dilution), *S. obliquus* presented a biomass yield of 231 mg/L, regardless of the use of sterilized or non-sterilized anaerobic digestate. Moreover, according to the nutrient removal, the nitrogen depletion was higher (up to 100%) when the anaerobic digestate was sterilized and diluted by up to 2%. Nevertheless, the phosphorous reduction was higher (92.5%) when the anaerobic digestate was not sterilized, and the maximum COD removal (53.7%) was achieved with the non-sterilized anaerobic digestate and a higher dilution (10%). The fatty acid accumulation (26.6% dry weight) was higher after 25 days of growth than in the control essay (24.5% dry weight).

Different anaerobic digestates from microalgae biomass co-digestion with swine and cow manure and vegetable wastes were selected for the growth of *Scenedesmus* sp. AMDD at 22°C [102]. Nitrogen was adjusted to 1.5 mM (NH<sup>3</sup> -N) with deionized water, and different phosphorous concentrations were evaluated. Moreover, digestates were filtered to reduce the bacterial load. This study showed that the use of an anaerobic digestate from the codigestion of microalgae biomass presented a good microalga growth rate. Animal manure digestate without co-digestion did not produce a complete nitrogen removal, which was improved when Mg+2 was added in the media growth. This element was indicated as a key nutrient for microalgae growth, and it was concluded that 0.03 ± 0.02 mM was adequate for optimal growth.

#### *4.2.1.4. Micractinium pusillum*

*M. pusillum* was grown in a cheese factory anaerobic digestate at 20°C and proven to present a satisfactory microalga growth rate. After 4 days, the pH reached 8.5, and the ammonia depletion was complete, although, according to the high pH, it could be due to the stripping of ammonia or bacterial activity. P-PO<sup>4</sup> 3− removal reached 33%, and the biomass yield was 137 ± 21 mg dry wt/L·d. Moreover, it was observed that the presence of suspended organic matter caused cell clogging and the adhesion of *M. pusillum* to the walls of the culture vessels [105].

#### *4.2.2. Cyanobacteria*

## *4.2.2.1. Phormidium bohneri*

De la Noüe et al. [103] also studied the growth of *P. bohneri.* The nitrogen toxic effect for *P. bohneri* was observed at 3.2 mM N-NH<sup>4</sup> + , which indicated that *P. bohneri* presented a higher nitrogen resistance than other common cyanobacteria. Moreover, an increase in temperature (from 10 to 35°C) produced an increase in biomass production. It was observed that a concentration of 0.1–0.5 mg Cu2+/L showed a toxic effect on *P. bohneri.* Seventy-five percent of COD removal from the anaerobic digestate was achieved. The higher concentration of *P. bohneri* (32 mg dry wt/L·d) was reached with a 2% swine manure dilution at 20°C.

pure species by favoring positive synergetic effects. Further studies will be needed in order to

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81

The authors wish to express their gratitude to the regional government of Andalucía "Junta de Andalucía" (project RNM-1970) for providing financial support. Dr. Rincón wishes to thank the "Ramón y Cajal" Program (RYC-2011-08783 contract) from the Spanish Ministry of

Bárbara Rincón\*, María José Fernández-Rodríguez, David de la Lama-Calvente and

Instituto de la Grasa, Consejo Superior de Investigaciones Científicas (CSIC), Pablo de

Science and Biotechnology. 2016;**15**:243-264. DOI: 10.1007/s11157-016-9392-z

[1] Santos-Ballardo DU, Rossi S, Reyes-Moreno C, Valdez-Ortiz A. Microalgae potential as a biogas source: Current status, restraints and future trends. Reviews in Environmental

[2] Najafi G, Ghobadian B, Tavakoli T, Yusaf T. Potential of bioethanol pro-duction from agricultural wastes in Iran. Renewable and Sustainable Energy Reviews. 2009;**13**(6-7):

[3] MacIntyre HL, Kana TM, Anning T, Geider RJ. Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in microalgae and cyanobacteria.

[4] Laws EA, Taguchi S, Hirata J, Pang L. Optimization of microalgal production in a shallow outdoor flume. Biotechnology and Bioengineering. 1988;**32**(2):140-147. DOI: 10.1002/

[5] Geider RJ. Light and temperature dependence of the carbon to chlorophyll a ratio in microalgae and cyanobacteria: Implications for physiology and growth of phytoplankton. The New Phytologist. 1987;**106**:1-34. DOI: 10.1111/j.1469-8137.1987.tb04788.x [6] Jankowska E, Sahu AK, Oleskowicz-Popiel P. Biogas from microalgae: Review on microalgae's cultivation, harvesting and pretreatment for anaerobic digestion. Renewable and

Journal of Phycology. 2002;**38**(1):17-38. DOI: 10.1046/j.1529-8817.2002.00094.x

Sustainable Energy Reviews. 2017;**75**:692-709. DOI: 10.1016/j.rser.2016.11.045

Economy and Competitiveness for providing financial support.

\*Address all correspondence to: brlloren@cica.es

1418-1427. DOI: 10.1016/j.rser.2008.08.010

Olavide University Campus, Sevilla, Spain

obtain a proper mixture culture.

**Acknowledgements**

**Author details**

Rafael Borja

**References**

bit.260320204

When *P. bohneri* was cultivated in a cheese factory anaerobic digestate at 20°C, a rapid increase in pH was observed after 4 days (from 8.4 to 10.9). No significant amount of NH<sup>4</sup> + was observed after the process, although, according to the high pH, it could be due to the stripping of ammonia or bacterial activity. P-PO<sup>4</sup> 3− removal reached 69% with a biomass yield of 329 ± 24 mg dry wt/L·d [105].

#### *4.2.2.2. Spirulina maxima*

In an early study, *S. maxima* was observed to need a high concentration of bicarbonate ions for optimal growth [106]. When it was cultivated in swine manure anaerobic digestate diluted with seawater, an increase in the microalga growth rate was observed with CO<sup>2</sup> supplementation. After 15 days, the anaerobic digestate presented a complete N-NH<sup>4</sup> + reduction, phosphate removal of 99.3%, nitrogen depletion of 76%, and a reduction in volatile solids of 28%.
