**3. Use of microalgae for biogas production through anaerobic digestion**

Anaerobic digestion is a series of biological processes in which microorganisms break down biodegradable material in the absence of oxygen. The end-products of anaerobic digestion are biogas and a digestate. Recently, algal biomass has been identified and developed as a renewable fuel source, and the growth of algal biomass for methane production has been increased.

The first study concerning the anaerobic digestion of microalga was carried out by Goluke et al. [30]. *Scenedesmus* sp. and *Chlorella* sp. were used as substrates for anaerobic digestion under different conditions. The authors finally concluded that microalgae have a relatively low digestibility due to the slowly biodegradable cell wall. Recently, one of the first studies about using algal biomass in anaerobic digestion was carried out by De Schamphelaire et al. [75]. This work consisted of designing a closed loop where algal biomass was used to obtain biogas. The maximum methane yield reached was 65 mL/day. More recently, in 2013, Torres et al. [35] defined the ideal microalgae for anaerobic digestion as a large cell microalga with a very thin cell wall or lacking it, with a high-growth rate in non-sterile medium and great resistance against natural pollutants. In one of the latest studies on the anaerobic digestion of microalgae, the authors pointed out the main limitations during the anaerobic digestion of microalgae, noting the low degradability of the cell wall, ammonium toxicity, and salinity as the main inhibitors of anaerobic digestion [76].

However, the use of microalgae as co-substrate is an approach to dilute complex compounds and balance the C/N ratio. Co-digestion has several advantages such as adjusting the C/N ratio, nutrients, and inhibitor compounds [34]. Ajeej et al. [77] also reported the increased activity of methanogenic microorganisms, a decreased anaerobic digestion inhibition by ammonium, and even increased cellulose activity when carbon-rich materials were added. Taking into account that the C/N ratio of the microalgal biomass is around 10:1 [78], the microalgae biomass can be considered as a suitable feedstock for carbon-rich substrates [79].

The main microalgae used for co-digestion have been described in the following paragraphs.

#### **3.1. Chlorophytes**

different ways: binary culture can use wastewater as a nutrients source without sterilization unlike in single systems; microalgae observe a low level of contamination in binary culture because bacteria protect those invading pathogens; microalgae with increased growth rate would decrease the cultivation time and reduce the overall cost; binary culture also aids in

Species selection is crucial for the success of microalgae cultivation in wastewater. Combining different species with varying metabolic potential would provide robustness to fluctuations in environmental factors and wastewater compositions, thereby giving more stability to the system. For instance, the potential application of microalgae consortia (*Chlorella* sp., *Scenedesmus* sp., and *C. zofingiensis*) compared to monoculture (*Chlorella* sp.) for the treatment of dairy wastewater was evaluated by Qin et al. [74]. They reported a significantly higher COD removal (57–62%) and phosphorous removal (91–96%) by microalgae consortia when compared to the monoculture of *Chlorella* sp. Furthermore, FAME profiles indicated that lipids produced from the microalgae consortia cultivation system were more suitable for biodiesel production [74]. In a very recent study [8], a mixed microalgae consortium (highly dominated by *Chlorella* species and small portions of *Scenedesmus* sp.) was cultivated using digestate (D), animal manure (AM), and textile wastewater (TW) as growth medium providing mainly N (nitrogen) and P (phosphorous) sources without any extra nutrient addition. After a cultivation period of 13 days, P was completely removed (100%); however, N was still remaining, and the removal rates of 70.1, 72.3, and 16.7% for TW, AM, and D, respectively, were achieved. The peak growth rate and biomass production of 0.419 d−1 and 0.4 g/L (in terms of volatile solids,

bioflocculation and lipid induction; and so on [38].

VSs) were achieved using TW as growth medium [8].

**digestion**

70 Microalgal Biotechnology

**3. Use of microalgae for biogas production through anaerobic** 

Anaerobic digestion is a series of biological processes in which microorganisms break down biodegradable material in the absence of oxygen. The end-products of anaerobic digestion are biogas and a digestate. Recently, algal biomass has been identified and developed as a renewable fuel source, and the growth of algal biomass for methane production has been increased. The first study concerning the anaerobic digestion of microalga was carried out by Goluke et al. [30]. *Scenedesmus* sp. and *Chlorella* sp. were used as substrates for anaerobic digestion under different conditions. The authors finally concluded that microalgae have a relatively low digestibility due to the slowly biodegradable cell wall. Recently, one of the first studies about using algal biomass in anaerobic digestion was carried out by De Schamphelaire et al. [75]. This work consisted of designing a closed loop where algal biomass was used to obtain biogas. The maximum methane yield reached was 65 mL/day. More recently, in 2013, Torres et al. [35] defined the ideal microalgae for anaerobic digestion as a large cell microalga with a very thin cell wall or lacking it, with a high-growth rate in non-sterile medium and great resistance against natural pollutants. In one of the latest studies on the anaerobic digestion of microalgae, the authors pointed out the main limitations during the anaerobic digestion of

#### *3.1.1. Chlorella genus*

Ehimen et al. [80] added lipid-extracted *Chlorella* biomass resulting from microalga diesel production to glycerol (main by-product formed during the transesterification process) and observed an increase in the methane yield of 50% when compared to the digestion of residual biomass alone.

Wang et al. [81] used the biomass of microalga *Chlorella* sp. grown in laboratory culture for co-digestion with WAS. The batch experiments were carried out under mesophilic conditions with a working volume of 100 mL. Different volumes of algae and WAS were added to the digester. They experimentally proved that the addition of WAS improved the anaerobic digestion of the microalga *Chlorella*, producing 73–79% more methane than single microalga digestion. Similar results were obtained by Li et al. [82], who co-digested *Chlorella* sp. with chicken manure in batch experiments. The co-digestion enhanced the methane production obtained during the single digestion of chicken manure and *Chlorella* sp. by 14.20 and 76.86%, respectively. By contrast, Retfalvi et al. [83], using the same C/N ratio, but pretreating the microalga, did not observe any positive effects on methane production.

Beltran et al. [84] assessed the co-digestion of *C. sorokiniana* with WAS. Different co-digestion mixtures were tested in biochemical methane potential (BMP) tests under mesophilic conditions. The highest methane yield obtained was 442 mL CH<sup>4</sup> /g VS for the mixture 75% WAS and 25% microalga. This value was 22 and 39% higher than that obtained in the anaerobic digestion of the sole substrates, WAS and microalga, respectively. This mixture clearly improved anaerobic digestion by ensuring its viability, suitability, and efficiency.

Rusten and Sahu [85] co-digested *Chlorella* sp. biomass and wastewater sludge (pretreated sludge liquor).The specific methane gas production (mL CH<sup>4</sup> /g VS fed) was not increased when compared to the anaerobic digestion of wastewater sludge alone. The co-digestion process achieved between 65 and 90% of specific methane gas production for sludge liquor depending on the HRT, temperature of incubation, and pretreatment of algae biomass. However, this study indicated that tested microalga could be cultivated in reject water to remove nitrogen and phosphorus from the sludge liquor.

In a recent study, Mahdy et al. [86] investigated the anaerobic co-digestion of *C. vulgaris* and manure. They used five different mixtures in a batch mesophilic experiment. The percentage 80:20 microalga:manure produced 431 mL CH<sup>4</sup> /g VS, while the methane yield of the single microalga produced 415 mL CH<sup>4</sup> /g VS. Despite the high-ammonium levels (3.7–4.2 g NH<sup>4</sup> + - N/L), using ammonia tolerant inoculums resulted in a relatively high-methane yield.

132 mL CH<sup>4</sup>

*3.1.3. Dunaliella salina*

/g VS. The results showed neither positive nor negative synergies between sub-

The Influence of Microalgae Addition as Co-Substrate in Anaerobic Digestion Processes

/g VS, and the highest

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73

strates, meaning that co-digestion did not improve microalga anaerobic biodegradability [22].

According to Fernández-Rodríguez et al. [36], the addition of olive mill solid waste (OMSW) to *D. salina* biomass resulted in the improvement in methane yield and biodegradability of OMSW when compared to the anaerobic digestion of the sole substrates. The experiment was carried out in batch, and different percentages of OMSW and *D. salina* biomass were tested. The highest biodegradability was found for the co-digestion mixture of 50% OMSW and 50%

methane production rate were obtained for the co-digestion mixture of 75% OMSW and 25%

Another approach to enhance biogas production from microalga through co-digestion was assessed by Schwede et al. [90]. Corn silage is one is the most common waste products produced around all over the world. Corn silage is characterized as being a lignocellulosic residue and very difficult to digest by anaerobic digestion [91]. The experiment carried out by Schwede et al. [90] reached a high-methane yield using *N. salina* as a co-substrate of corn silage. The mixture balanced the nutrient composition due to the corn silage providing mainly carbon and the microalga providing nitrogen, which helped to balance the C/N ratio from 65 (*N. salina*) or 32.6 (corn silage) to 21.2 (Mixture *N. salina*/corn silage). This mixture, C/N = 21.2, reached 9% more methane than that obtained in the anaerobic digestion of the

Neumann et al. [92] reported that the anaerobic co-digestion of lipid-spent *B. braunii* (LSBB) with WAS and glycerol showed no significant increase in BMP when mixing these substrates. However, the kinetic constant of the mixture 25% WAS-75% LSBB was much higher than those obtained for WAS and LSBB alone. The mixture of 10% glycerol and 90% LSBB did not show a higher kinetic constant or methane production. The authors concluded that the application of different cultivation procedures, lipid extraction methods, and anaerobic conditions will result in different microalga biomass compositions and characteristics, which affect the

Wang et al. [27] applied WAS to the digestion of microalga biomass consisting of *Micractinium* sp. The algae biomass was grown in high-nitrogen wastewater (mixture of sludge centrate and primary effluent wastewater). The microalga showed a good ability for nutrient removal throughout the growth. The co-digestion of microalga biomass and WAS improved the solubilization efficiency and the biodegradability of the microalgae. The methane yield obtained

*D. salina*. Nevertheless, the maximum methane production, 330 mL CH<sup>4</sup>

*D. salina*, keeping a C/N ratio close to 26.7.

*3.1.4. Nannochloropsis salina*

corn silage alone.

*3.1.5. Botryococcus braunii*

*3.1.6. Micractinium genus*

productivity of microalgal methane.

According to Li et al. [82], *Chlorella* 1067 was cultivated in a chicken manure-based digestate, and the resulting algae biomass was used as co-substrate with chicken manure in anaerobic co-digestion. The growth of microalga in manure-based digestate recycled about 91% of the total nitrogen and 86% of the soluble organic phosphorous. During co-digestion, the highest methane production was 238.71 mL CH<sup>4</sup> /g VS, obtained at the mixing ratio of 8:2 (chicken manure to *Chlorella* 1067 according to the VS).

#### *3.1.2. Scenedesmus genus*

Ramos-Suarez et al. [87] described *Scenedesmus* sp. biomass as a non-suitable substrate for anaerobic digestion due to its low degradability and low methane production. In contrast, during their investigations, they used the biomass of microalga as co-substrate with *Opuntia maxima* cladodes. Bioreactors were used to grow *Scenedesmus* sp., and the biomass was codigested with different percentages of cladodes of 1 or 2 years of age in order to avoid an increase in lignocelluloses. C/N ratios from 6.0 to 51.3 were used, proving that co-digestion improved methane yield and kinetics when compared to the mono-digestion of both substrates. The best mixture turned out to be the C/N ratio of 15.6. The methane yield for this mixture was 233.6 ± 16.4 mL CH<sup>4</sup> /g VS and was increased by 66.4 and 63.9% when compared to *Scenedesmus* sp. biomass and *O. maxima*, respectively, when digested alone.

Astals et al. [88] assessed the co-digestion of pig manure and *Scenedesmus* sp. with and without the extraction of intracellular algal co-products. Proteins and/or lipids were extracted from *Scenedesmus* sp. This process increased methane yield by 29–37% when compared to raw microalga biomass. Co-digestion experiments showed a synergy effect between pig manure and raw microalga that increased raw algae methane yield from 163 to 245 mL CH<sup>4</sup> /g VS. A similar synergy effect was not observed when algal residues were co-digested with pig manure.

Arias et al. [22] used microalga digestate and secondary effluent to grow microalga in a tertiary wastewater treatment, and then the microalga biomass was co-digested for biogas generation. The algal biomass was mainly composed of *Scenedesmus* sp. The algae biomass and the WAS were pretreated by autohydrolysis reaching 11.4 and 25.7% of solubilization, respectively. The solubilization of *Scenedesmus* biomass was lower than the solubilization of WAS after pretreatment, and *Scenedesmus* has been reported to have a complex multilayer cell wall [89]. After pretreatment both substrates were co-digested in different proportions. The maximum methane yield obtained was 204 mL CH<sup>4</sup> /g VS for the anaerobic digestion of 100% WAS. On the other hand, the methane yield of the anaerobic digestion of 100% microalga exhibited a 64% lower methane production and reached 134 mL CH<sup>4</sup> /g VS. The mixture of 20% microalga and 80% WAS produced 187 mL CH<sup>4</sup> /g VS, while the mixture of 50% microalgae and 50% WAS produced 162 mL CH<sup>4</sup> /g VS, and the mixture of 80% microalga and 20% WAS produced 132 mL CH<sup>4</sup> /g VS. The results showed neither positive nor negative synergies between substrates, meaning that co-digestion did not improve microalga anaerobic biodegradability [22].

#### *3.1.3. Dunaliella salina*

In a recent study, Mahdy et al. [86] investigated the anaerobic co-digestion of *C. vulgaris* and manure. They used five different mixtures in a batch mesophilic experiment. The percentage

According to Li et al. [82], *Chlorella* 1067 was cultivated in a chicken manure-based digestate, and the resulting algae biomass was used as co-substrate with chicken manure in anaerobic co-digestion. The growth of microalga in manure-based digestate recycled about 91% of the total nitrogen and 86% of the soluble organic phosphorous. During co-digestion, the highest

Ramos-Suarez et al. [87] described *Scenedesmus* sp. biomass as a non-suitable substrate for anaerobic digestion due to its low degradability and low methane production. In contrast, during their investigations, they used the biomass of microalga as co-substrate with *Opuntia maxima* cladodes. Bioreactors were used to grow *Scenedesmus* sp., and the biomass was codigested with different percentages of cladodes of 1 or 2 years of age in order to avoid an increase in lignocelluloses. C/N ratios from 6.0 to 51.3 were used, proving that co-digestion improved methane yield and kinetics when compared to the mono-digestion of both substrates. The best mixture turned out to be the C/N ratio of 15.6. The methane yield for this

Astals et al. [88] assessed the co-digestion of pig manure and *Scenedesmus* sp. with and without the extraction of intracellular algal co-products. Proteins and/or lipids were extracted from *Scenedesmus* sp. This process increased methane yield by 29–37% when compared to raw microalga biomass. Co-digestion experiments showed a synergy effect between pig manure and raw microalga that increased raw algae methane yield from 163 to 245 mL CH<sup>4</sup>

VS. A similar synergy effect was not observed when algal residues were co-digested with pig

Arias et al. [22] used microalga digestate and secondary effluent to grow microalga in a tertiary wastewater treatment, and then the microalga biomass was co-digested for biogas generation. The algal biomass was mainly composed of *Scenedesmus* sp. The algae biomass and the WAS were pretreated by autohydrolysis reaching 11.4 and 25.7% of solubilization, respectively. The solubilization of *Scenedesmus* biomass was lower than the solubilization of WAS after pretreatment, and *Scenedesmus* has been reported to have a complex multilayer cell wall [89]. After pretreatment both substrates were co-digested in different proportions. The maximum

the other hand, the methane yield of the anaerobic digestion of 100% microalga exhibited a

to *Scenedesmus* sp. biomass and *O. maxima*, respectively, when digested alone.

N/L), using ammonia tolerant inoculums resulted in a relatively high-methane yield.

/g VS, while the methane yield of the single

/g VS, obtained at the mixing ratio of 8:2 (chicken

/g VS and was increased by 66.4 and 63.9% when compared

/g VS for the anaerobic digestion of 100% WAS. On

/g VS, while the mixture of 50% microalgae and 50%

/g VS, and the mixture of 80% microalga and 20% WAS produced

/g VS. The mixture of 20% microalga

+ -

/g

/g VS. Despite the high-ammonium levels (3.7–4.2 g NH<sup>4</sup>

80:20 microalga:manure produced 431 mL CH<sup>4</sup>

methane production was 238.71 mL CH<sup>4</sup>

*3.1.2. Scenedesmus genus*

mixture was 233.6 ± 16.4 mL CH<sup>4</sup>

methane yield obtained was 204 mL CH<sup>4</sup>

and 80% WAS produced 187 mL CH<sup>4</sup>

WAS produced 162 mL CH<sup>4</sup>

64% lower methane production and reached 134 mL CH<sup>4</sup>

manure.

manure to *Chlorella* 1067 according to the VS).

microalga produced 415 mL CH<sup>4</sup>

72 Microalgal Biotechnology

According to Fernández-Rodríguez et al. [36], the addition of olive mill solid waste (OMSW) to *D. salina* biomass resulted in the improvement in methane yield and biodegradability of OMSW when compared to the anaerobic digestion of the sole substrates. The experiment was carried out in batch, and different percentages of OMSW and *D. salina* biomass were tested. The highest biodegradability was found for the co-digestion mixture of 50% OMSW and 50% *D. salina*. Nevertheless, the maximum methane production, 330 mL CH<sup>4</sup> /g VS, and the highest methane production rate were obtained for the co-digestion mixture of 75% OMSW and 25% *D. salina*, keeping a C/N ratio close to 26.7.

#### *3.1.4. Nannochloropsis salina*

Another approach to enhance biogas production from microalga through co-digestion was assessed by Schwede et al. [90]. Corn silage is one is the most common waste products produced around all over the world. Corn silage is characterized as being a lignocellulosic residue and very difficult to digest by anaerobic digestion [91]. The experiment carried out by Schwede et al. [90] reached a high-methane yield using *N. salina* as a co-substrate of corn silage. The mixture balanced the nutrient composition due to the corn silage providing mainly carbon and the microalga providing nitrogen, which helped to balance the C/N ratio from 65 (*N. salina*) or 32.6 (corn silage) to 21.2 (Mixture *N. salina*/corn silage). This mixture, C/N = 21.2, reached 9% more methane than that obtained in the anaerobic digestion of the corn silage alone.

#### *3.1.5. Botryococcus braunii*

Neumann et al. [92] reported that the anaerobic co-digestion of lipid-spent *B. braunii* (LSBB) with WAS and glycerol showed no significant increase in BMP when mixing these substrates. However, the kinetic constant of the mixture 25% WAS-75% LSBB was much higher than those obtained for WAS and LSBB alone. The mixture of 10% glycerol and 90% LSBB did not show a higher kinetic constant or methane production. The authors concluded that the application of different cultivation procedures, lipid extraction methods, and anaerobic conditions will result in different microalga biomass compositions and characteristics, which affect the productivity of microalgal methane.

#### *3.1.6. Micractinium genus*

Wang et al. [27] applied WAS to the digestion of microalga biomass consisting of *Micractinium* sp. The algae biomass was grown in high-nitrogen wastewater (mixture of sludge centrate and primary effluent wastewater). The microalga showed a good ability for nutrient removal throughout the growth. The co-digestion of microalga biomass and WAS improved the solubilization efficiency and the biodegradability of the microalgae. The methane yield obtained for the microalga was 209 mL/g VS. The co-digestion of algae with WAS improved the volatile solid reduction, the solubilization efficiency of the algae, and their biogas yield. However, the methane production of the WAS alone showed no improvement.

*A. platensis* was co-digested with WAS in batch and in semi-continuous systems [97]. During the batch tests, the system reached 89–93% volatile solid reduction. The biogas

The Influence of Microalgae Addition as Co-Substrate in Anaerobic Digestion Processes

anaerobic digestion system was investigated. The system achieved 60% of volatile solid reduction with 525 mL biogas/gVS·d. The co-digestion of *A. platensis* and sewage sludge improved biogas production and volatile solid reduction. The best mixture was 66.6% WAS and 33.3% *A. platensis* based on volatile solids. The maximum methane production was 640 mL biogas/g VS·d with a 62.5% reduction in volatile solids. The methane content in the

Cheng et al. [73] carried out batch experiments to investigate the performance of *O. tenuis* to remove nitrogen, phosphorus, and COD and from the secondary effluents of municipal domestic wastewater. The potential of biogas production was also investigated by applying the co-digestion of *O. tenuis* with pig manure. *O. tenuis* had a good biomass productivity, which ranged from 104 to 150 mg/L·d, and was beneficial for the subsequent anaerobic diges-

Zhen et al. [98] used a mixed microalgae culture of *Scenedesmus* sp. and *Chlorella* sp., which were co-digested with food waste in a batch system under mesophilic conditions. The results showed that supplementing food waste with microalga significantly improved the performance of microalga digestion. The highest methane yield achieved was 639.8 ± 1.3 mL/g VS, which was reached at a microalga/food waste ratio of 0.2:0.8, obtaining a 4.99-fold increase

Solé-Bundó et al. [99] grew microalgae biomass in wastewater, and subsequently, the algaebacteria biomass was co-digested with wheat straw. Batch systems were used for testing different substrate percentages (20–80%, 50–50% and 80–20%, microalgae and wheat straw, respectively, on a volatile solid basis). The highest synergies in degradation rates were observed by adding at least 50% wheat straw. Therefore, the co-digestion of 50% microalgae biomass and 50% wheat straw was further investigated in mesophilic semi-continuous labscale reactors. The results showed that the methane yield was increased by 77% in the co-

**Table 1** summarizes the different microalgae and co-substrates tested in anaerobic co-digestion

/g VS. In the continuous studies, a two-phase

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75

/g VS was achieved through co-digestion of

production was between 210 and 260 mL CH<sup>4</sup>

tion. A maximum methane yield of 191 mL CH<sup>4</sup>

*3.3.1. Scenedesmus genus and Chlorella genus*

this microalga with pig manure at a mixing ratio of 2.0.

with respect to microalgae alone (106.9 ± 3.2 mL/g VS).

digestion when compared to microalgae biomass mono-digestion.

processes including the improvement in the methane yields observed.

biogas was 77%.

*3.2.2. Oscillatoria tenuis*

**3.3. Binary culture system**

*3.3.2. Microalgae and bacteria*

### *3.1.7. Selenastrum capricornutum (Chlorophyta) and Isochrysis galbana (Haptophyta)*

*I. galbana* and *S. capricornutum* were co-digested with sewage sludge under mesophilic (33°C) and thermophilic (55°C) conditions [93]. Under mesophilic conditions, the anaerobic digestion of sewage sludge produced 451 ± 12 mL biogas/g VS. The microalga *I. galbana* produced 439 mL biogas/g VS, and *S. capricornutum* produced 271 mL biogas/g VS. When a substrate mixture was fed, biogas production showed quite similar values for all experiments, regardless of the sludge to microalga ratio in the mixture. The average biogas production was 440 ± 25 mL biogas/g VS. So, microalga and sewage sludge co-digestion did not improve biogas yield in comparison with individual digestions of both substrates under mesophilic conditions. Under thermophilic conditions, the biogas production of *I. galbana* was 261 ± 11 mL biogas/g VS, and the production of *S. capricornutum* was 185 ± 7 mL biogas/g VS. The amount of methane decreased by 40.5 and 31.7% for *I. galbana* and *S. capricornutum*, respectively, when compared to their biogas production at 33°C. The increase in temperature had a negative influence on microalga digestion. However, temperature had a huge beneficial effect on sewage sludge. The production of biogas reached 566 ± 5 mL biogas/g VS, indicating that 25.5% more biogas was produced by increasing temperature. The experiment presented similar tendencies, the higher the volatile solid, the lower the biogas production.

#### **3.2. Cyanobacteria**

#### *3.2.1. Arthrospira platensis*

*A. platensis* was characterized as having a high level of protein and, therefore, a high-nitrogen content [94]. Biomass with a high-nitrogen content could be used as co-substrate with highcarbon content substrates [95]. This study investigated the co-digestion of *A. platensis* with barley straw, beet silage, and brown seaweed at a C/N ratio of 25, the optimal ratio for anaerobic digestion [96]. The experiments were carried out in batch and semi-continuous systems. The C/N ratios of the substrates were 4.3, 145.5, 41.7, and 28.7 for *A. platensis*, barley straw, beet silage, and seaweed *Laminaria digitata*, respectively. The methane productions during the batch experiments were 357.1, 196.8, 393.5, and 306.5 mLN/gVS for *A. platensis*, barley straw, beet silage, and seaweed *L. digitata*, respectively. The co-digestion of 45% *A. platensis* and 55% beet silage produced 360.9 mLN/gVS. The co-digestion of 85% *A. platensis* and 15% barley straw produced 347.8mLN/gVS, and the best co-digestion mixture of *A. platensis* and *L. digitata* (15–85%) produced 311.5 mLN/gVS. Mono-digestion of *A. platensis* led to high-methane yields in the semicontinuous mode but only at low-organic rates of 1.0 g VS/L·d. Co-digestion with carbon-rich substrates had a positive effect on process stability. The highest biogas production occurred during co-digestion of microalga with beet silage. The best process stability was found at an organic loading of 4.0 g VS/L·d during co-digestion with the seaweed *L. digitata* [95].

*A. platensis* was co-digested with WAS in batch and in semi-continuous systems [97]. During the batch tests, the system reached 89–93% volatile solid reduction. The biogas production was between 210 and 260 mL CH<sup>4</sup> /g VS. In the continuous studies, a two-phase anaerobic digestion system was investigated. The system achieved 60% of volatile solid reduction with 525 mL biogas/gVS·d. The co-digestion of *A. platensis* and sewage sludge improved biogas production and volatile solid reduction. The best mixture was 66.6% WAS and 33.3% *A. platensis* based on volatile solids. The maximum methane production was 640 mL biogas/g VS·d with a 62.5% reduction in volatile solids. The methane content in the biogas was 77%.

#### *3.2.2. Oscillatoria tenuis*

for the microalga was 209 mL/g VS. The co-digestion of algae with WAS improved the volatile solid reduction, the solubilization efficiency of the algae, and their biogas yield. However, the

*I. galbana* and *S. capricornutum* were co-digested with sewage sludge under mesophilic (33°C) and thermophilic (55°C) conditions [93]. Under mesophilic conditions, the anaerobic digestion of sewage sludge produced 451 ± 12 mL biogas/g VS. The microalga *I. galbana* produced 439 mL biogas/g VS, and *S. capricornutum* produced 271 mL biogas/g VS. When a substrate mixture was fed, biogas production showed quite similar values for all experiments, regardless of the sludge to microalga ratio in the mixture. The average biogas production was 440 ± 25 mL biogas/g VS. So, microalga and sewage sludge co-digestion did not improve biogas yield in comparison with individual digestions of both substrates under mesophilic conditions. Under thermophilic conditions, the biogas production of *I. galbana* was 261 ± 11 mL biogas/g VS, and the production of *S. capricornutum* was 185 ± 7 mL biogas/g VS. The amount of methane decreased by 40.5 and 31.7% for *I. galbana* and *S. capricornutum*, respectively, when compared to their biogas production at 33°C. The increase in temperature had a negative influence on microalga digestion. However, temperature had a huge beneficial effect on sewage sludge. The production of biogas reached 566 ± 5 mL biogas/g VS, indicating that 25.5% more biogas was produced by increasing temperature. The experiment presented similar tendencies, the higher the volatile solid, the lower the biogas production.

*A. platensis* was characterized as having a high level of protein and, therefore, a high-nitrogen content [94]. Biomass with a high-nitrogen content could be used as co-substrate with highcarbon content substrates [95]. This study investigated the co-digestion of *A. platensis* with barley straw, beet silage, and brown seaweed at a C/N ratio of 25, the optimal ratio for anaerobic digestion [96]. The experiments were carried out in batch and semi-continuous systems. The C/N ratios of the substrates were 4.3, 145.5, 41.7, and 28.7 for *A. platensis*, barley straw, beet silage, and seaweed *Laminaria digitata*, respectively. The methane productions during the batch experiments were 357.1, 196.8, 393.5, and 306.5 mLN/gVS for *A. platensis*, barley straw, beet silage, and seaweed *L. digitata*, respectively. The co-digestion of 45% *A. platensis* and 55% beet silage produced 360.9 mLN/gVS. The co-digestion of 85% *A. platensis* and 15% barley straw produced 347.8mLN/gVS, and the best co-digestion mixture of *A. platensis* and *L. digitata* (15–85%) produced 311.5 mLN/gVS. Mono-digestion of *A. platensis* led to high-methane yields in the semicontinuous mode but only at low-organic rates of 1.0 g VS/L·d. Co-digestion with carbon-rich substrates had a positive effect on process stability. The highest biogas production occurred during co-digestion of microalga with beet silage. The best process stability was found at an

organic loading of 4.0 g VS/L·d during co-digestion with the seaweed *L. digitata* [95].

methane production of the WAS alone showed no improvement.

**3.2. Cyanobacteria**

74 Microalgal Biotechnology

*3.2.1. Arthrospira platensis*

*3.1.7. Selenastrum capricornutum (Chlorophyta) and Isochrysis galbana (Haptophyta)*

Cheng et al. [73] carried out batch experiments to investigate the performance of *O. tenuis* to remove nitrogen, phosphorus, and COD and from the secondary effluents of municipal domestic wastewater. The potential of biogas production was also investigated by applying the co-digestion of *O. tenuis* with pig manure. *O. tenuis* had a good biomass productivity, which ranged from 104 to 150 mg/L·d, and was beneficial for the subsequent anaerobic digestion. A maximum methane yield of 191 mL CH<sup>4</sup> /g VS was achieved through co-digestion of this microalga with pig manure at a mixing ratio of 2.0.

#### **3.3. Binary culture system**

#### *3.3.1. Scenedesmus genus and Chlorella genus*

Zhen et al. [98] used a mixed microalgae culture of *Scenedesmus* sp. and *Chlorella* sp., which were co-digested with food waste in a batch system under mesophilic conditions. The results showed that supplementing food waste with microalga significantly improved the performance of microalga digestion. The highest methane yield achieved was 639.8 ± 1.3 mL/g VS, which was reached at a microalga/food waste ratio of 0.2:0.8, obtaining a 4.99-fold increase with respect to microalgae alone (106.9 ± 3.2 mL/g VS).

#### *3.3.2. Microalgae and bacteria*

Solé-Bundó et al. [99] grew microalgae biomass in wastewater, and subsequently, the algaebacteria biomass was co-digested with wheat straw. Batch systems were used for testing different substrate percentages (20–80%, 50–50% and 80–20%, microalgae and wheat straw, respectively, on a volatile solid basis). The highest synergies in degradation rates were observed by adding at least 50% wheat straw. Therefore, the co-digestion of 50% microalgae biomass and 50% wheat straw was further investigated in mesophilic semi-continuous labscale reactors. The results showed that the methane yield was increased by 77% in the codigestion when compared to microalgae biomass mono-digestion.

**Table 1** summarizes the different microalgae and co-substrates tested in anaerobic co-digestion processes including the improvement in the methane yields observed.


**4. Microalgae growth in anaerobic digestates**

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

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 co-

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

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,

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

+

/TKN ratio, which represents

**methane yield (%)**

http://dx.doi.org/10.5772/intechopen.75914

The Influence of Microalgae Addition as Co-Substrate in Anaerobic Digestion Processes

498.5 (compared to microalga)

77 (compared to microalga)

**Reference**

77

[98]

[99]

present better water release than the digestate from single microalga digestion.

**Microalga Co-substrate Conditions Improvement in** 

Food waste (80%) Batch at mesophilic

Wheat straw (50%) Batch at mesophilic

C, carbon; N, nitrogen; WAS, waste-activated sludge; OMSW, olive mill solid waste; \*, not available.

temperature

temperature

**Table 1.** Improvement of methane yields after anaerobic co-digestion processes of microalgae with different substrates.

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

*Scenedesmus* genus *+ Chlorella*

genus (20%)

*Chlorella* sp. + some *Monoraphidium* sp. (50%)

digestion resulted in a more stable digestate.

co-substrate with a higher carbon content.

microalgae (80 g/kg TS and 56 g/kg TS), although the N-NH<sup>4</sup>

and no difference was observed among the different digestates [100].


**Table 1.** Improvement of methane yields after anaerobic co-digestion processes of microalgae with different substrates.
