Anaerobic Co-Digestion of Microalgae and Industrial Wastes: A Critical and Bibliometric Review

Abstract

Microalgae are photosynthetic organisms able to grow faster than land plants and produce biomass with relatively high energy potential. Accumulated high-value compounds like lipids, minerals, or proteins have focused the attention of scientists due to the potential production of biofuels and other value-added products. However, several drawbacks regarding both the biochemical structure of these organisms and technological difficulties have prevented the industry for implementing a comprehensive low-cost process regarding energy and environmental contamination. Among these technologies, anaerobic digestion (AD) has greatly increased research attention because of its simplicity and the ability to produce easily recycle by-products. Moreover, anaerobic co-digestion (AcoD) has shown promising results as a method to bypass the AD problems of microalgae as a sole substrate. This review is focused on the recent trends and comparison of the AcoD process to maximize energy recovery from microalgae biomass and agro-industrial wastes. The yield of methane gas among the studied bibliography is compared and a critical review of published data and methods used is included.

1. Introduction

Microalgae are a wide family of photosynthetic organisms able to increase their biomass by using CO₂ and sunlight as energy sources by a rate 100 times faster than plants [1]. Moreover, the water and nutrients consumption is lesser than the needed for the same amount of biomass of terrestrial crops and it does not compete with other biomass from land areas [1, 2].

For the above-mentioned reasons, microalgae have been studied for decades for their potential conversion to energy. However, it was not until recently that microalgae have been considered feasible biomass to be used as a feedstock to produce biofuels (e.g. biohydrogen, biodiesel, biomethane, bio-oil, bioethanol, etc.) [1, 2, 3, 4]. This is mainly due to several drawbacks (e.g. the need of solvents to produce biodiesel contributing to greenhouse gas emission, the use of expensive enzymes for bioethanol production, etc.) that have been overpassed thanks to technology and research efforts over the years focusing on the understanding and optimizing those factors that affect the different systems along with a better understanding of the biomass itself (e.g. the effect of the cell wall, the algae growth requirements, etc.) [1, 2, 3, 4, 5].

Among these technologies, anaerobic digestion (AD) has shown promising results. AD is a biological process where the organic compounds from certain biomass are degraded in the absence of oxygen (O₂) by a microbial consortium. The main effluents of this process are biogas (i.e. a gas composed primarily of methane and CO₂) and a nutrient-rich digestate [3]. While the produced biogas is considered a renewable energy source to produce electricity and heat in cogeneration plants, the nutrient-rich digestate could be used as a fertilizer [3, 4].

Microalgae are not only a viable AD feedstock, but can also serve as a means of biogas upgrading and its cultivation in the digestate can reduce the excess of nutrients and mitigate its potential toxicity [5]. However, the mono-digestion of microalgae has shown some concerns regarding its viability at industrial scales. Briefly, these concerns are related to the presence of long-chain organic compounds, mainly in the cell wall, the low carbon to nitrogen (C/N) ratio, and the high retention time needed in the reactors, which led to low methane yields, undigested organic matter in the digestate and more importantly to the inhibition of the AD process [2, 3].

In order to overpass these problems, anaerobic co-digestion (AcoD) of microalgae along with a wide range of co-substrates has been the focus of several research groups recently [1, 2, 3, 4, 5, 6, 7]. AcoD has several benefits due to its capacity to enhance the C/N ratio, the buffer capacity, the nutrient balance, and to dilute inhibitory compounds [4]. These improvements produce higher methane yields which in most cases are higher than the theoretical values obtained from the sole digestion of each co-substrate showing a synergetic effect [1, 2, 3, 4, 5].

Nevertheless, AcoD presents some drawbacks such as a higher organic load in the digestate, the usual need of pretreatments, or the difficulties to maintain a stable feedstock along the seasons [3]. This chapter aims to summarize the state of the art of microalgae used as co-substrate in AcoD processes and to highlight knowledge gaps and potential future developments. Moreover, a comprehensive analysis of the parameters affecting AcoD of microalgae in order to enhance methane yield is included in detail. Lastly, the energetic viability of several scenarios is discussed and future trends proposed.

2. Review methodology

For this bibliographic review, 92 articles have been selected from the 137 articles were carried out on the Scopus with the keywords “microalgae” and “anaerobic co-digestion” during the periods of 2011–2021 [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100]. The bibliometric analysis was realized using the VOSviewer software using these 92-original articles.

Thanks to this software, it has been verified that there are few groups dedicated to the study of AcoD with microalgae. These investigations focus mainly on countries such as Spain (30 articles), the United States (17 articles), China (8 articles), Mexico (8 articles), and Brazil (5 articles). In addition, only eight authors have five or more articles to 321 authors (Figure 1a). In Figure 1b, as can see the connection between different authors and groups. The color and size indicate the group and the citations of these eight authors.

Figure 1.

Bibliometric analysis with VOSviewer: (a) authors with five or more articles, (b) connexion between authors, (c) substrates and (d) microalgae.

3. Microalgae and growth mediums

Since the first study on biogas production with microalgae by Golueke [101], a wide variety of microalgae genera have been studied. Due to the composition of the microalgae, it has been seen that each strain has a very specific biogas production, and has very diverse productions. One of the factors that most affect the AD of microalgae is the structure of its cell wall, which is why the selection of the type of microalgae is important. One of the main characteristics for microalgae to have a good methane production potential is to have a thin or null cell wall, large cytoplasmic components, a high growth rate, and a high tolerance to stress [102]. Other important aspects are whether it has a low content of hollocellulose in the cell walls, metabolic, and growth conditions are favorable, the morphological traits of the microalgae strains [27]. In addition, the selected strains offer a feasible genetic manipulation to control metabolic activities and improve tolerance to nutrient and ecological stress [102].

Nineteen genera of microorganisms have been investigated in the 92 articles found on the AcoD of microalgae. Of these 92 articles, 51 of these works have been studied with the genus Chlorella and 24 of these works with the genus Scenedesmus. The rest of the work carried out was with Chlorophyta as Nannochloropsis (5 studies), Micractinium (3 studies), Dunaliella (2 studies), Dictyosphaerium, Closteriopsis, Desmodesmus, Chlamydomonas, Stigeoclonium, Botryococcus and Tetraselmis genus. Other genus as Tribonema (Ochrophyta), Phaeodactylum (Bacillariophyta) and Tisochrysis (Haptophyta) have been also studied as co-substrate. In addition, the following cyanobacteria have been studied: Arthrospira (4 studies), Spirulina (2 studies), Merismopedia, Oscillatoria.

Another factor that should be considered is the culture medium for microalgae growth. Depending on the medium, it could favor the production of biogas in a later step due to the nutrient requirement of the microorganisms in the AD process [96]. According to the reviewed bibliography, the microalgae used for AcoD are obtained through other research groups or are cultivated using three different types of medium for growing it.

1. Synthetic medium: BG11, Bold’s basal (BBM), Conway enriched medium, Jaworki’s medium, modified Zarrouk medium, BlueBIOTech Ltf. (Table 1).

2. Digestate or effluent anaerobic: Anaerobic sludge, continuous stirred tank reactor (CSTR) digestate, up-flow anaerobic sludge blanket (UASB) digestate, primary effluent and sludge, chicken manure digestate, swine digestate, Anaerobic membrane bioreactor (AnMBR) digestate.

3. Wastewater: Tannery effluents, piggery WW, industry, domestic, municipal, fresh waters, lake waters, natural seawater enriched, soft drink. WW, Winery WW.

As shown in Table 1, microalgae are microorganisms that need certain nutrients to perform vital functions. Therefore, synthetic culture media contain macronutrients such as calcium, sodium, potassium, magnesium, and chloride. In addition to adding micronutrients such as iron, cobalt, molybdenum, manganese, copper, zinc, and vitamins. Finally, ethylenediaminetetraacetic acid (EDTA) is also added to form a complex ring (a chelate) with the trace elements, which, when used in low concentrations, stimulates the growth of microalgae by making this element available in low quantities. These nutrients are found in nature and are bioavailable in the other two natural culture media such as anaerobic digestate and wastewater, but may be found in lower concentrations than necessary [10].

Nutrient	BG11	BBM	Conway	Jaworki’s	Zarrouk
Nitrate	NaNO3	NaNO3	KNO3	Ca(NO3)2·4H2O NaNO3	NaNO3
Potassium	K2HPO4	K2HPO4 KH2PO4	Na3PO4	KH2PO4 Na2HPO4·12H2O	K2HPO4 K2SO4
Sodium	Na2CO2			NaHCO3	NaHCO3 and Na2CO3
Magnesium	MgSO4·7H2O	MgSO4·7H2O		MgSO4·7H2O	MgSO4·7H2O
Chloride	CaCl2·2H2O	NaCl CaCl2·2H2O			NaCl CaCl2
Boron	H3BO3	H3BO3	H3BO3	H3BO3
Zinc	ZnSO4·7H2O	ZnSO4·7H2O	ZnCl2
Manganese	MnCl2·4H2O	MnCl2·4H2O	MnCl2·4H2O	MnCl2·4H2O
Molybdenum	Na2MoO4·2H2O	MoO3	(NH4)6Mo7O24·4H2O	(NH4)6Mo7O24·4H2O
Copper	CuSO4·6H2O	CuSO4·6H2O	CuSO4·6H2O
Cobalt	Co(NO3)2·6H2O	Co(NO3)2·6H2O	CoCl2·6H2O
Iron	Ferric ammonium citrate	FeSO4·7H2O	FeCl3·6H2O	NaFeEDTA	FeSO4·7H2O
EDTA	Na2EDTA	Na2EDTA (KOH)	Na2H2EDTA·2H2O	Na2EDTA	EDTA
Vitamin	Citric acid		Thiamin HCl
Cyanocobalamin	Thiamin HCl
Cyanocobalamin Biotin

Table 1.

Synthetic culture medium for the growth of microalgae.

In addition, using wastewater for the growth of microalgae could prevent eutrophication of the water due to the consumption of excess nutrient of these type of waters. In this sense, microalgae have been succesfully used for removing nitrogen and phosphorous from various wastes as shown in Table 2. Significant amounts of removal of nutrients and biomass production achieved in these studies demonstrate the feasibility of coupled wastewater treatment and microalgae cultivation processes.

Algae	N-NH4		P-PO4		Ref.
	[Conc] mg/L	%Remove	[Conc] mg/L	%Remove
Scenedesmus sp. + Chlorella sp.	7.9	97	2.5	93	[10]
Mix culture	35.5	61	nd*	nd*	[80]
Arthrospira platensis	375	88.4	158	97.01	[42]
Chlorella vulgaris	285	99.6	117	91.2	[13]
Oscillatoria tenius	10.2	96.1	0.8	82.9	[18]
Chlorella 1067	202.68	94.33	7.18	29.67	[38]
Chlorella sp.	1144	58	188	98	[84]
Micractinium nov	2.2	94	4	95	[82]
Chlorella sp.	1.5	96	3.5	95
Micractinium nov	27	92	0.7	51	[88]
Chlorella sp.
Spirulina platensis	120	68	55	30	[92]
Chlorella sp.	225	85	120	20
Cholerella sp.	45	88	9	58	[56]

Table 2.

Removal of N-NH₄ and P-PO₄ in microalgae culture.

*

nd: not determined.

4. Co-substrates

AcoD is the use of a mixture of biomasses to obtain a relatively higher methane yield [10]. This modified technique is considered economically more viable and easier to control mixed biomass compared to traditional mono-digestion systems. Depending on the species, microalgae contain significantly high or low amounts of protein, carbohydrates, and lipids. To balance the nutritional requirements of microorganisms in anaerobic reactors [13]. In addition, it would be possible to improve the stabilization of the process with well-balanced mixtures. With this, it could be possible to increase the organic load capacity, reduce the concentration of possible inhibitors, and increase the buffering capacity of the digestates. Apart from the nutrient balances and net synergistic effects that would occur when co-digesting microalgae with an efficient substrate. In the revised bibliography, synthetic co-substrates, agri-food residues and slurry, and liquid residues can be found.

1. Synthetic substrate: Cellulose and glycerol, and synthetic food wastes.

2. Solid waste: Cattle manure (chicken, cow, pig), cheese whey powder, chicken litter, green willow, chromium tanned leather shavings (SHA), coffee wastes, corn silage, fat, oil and grease (FOG), food waste, millet grass (Pennisetum glaucum), model kitchen waste, olive mill solid waste (OMSW), oil palm empty fruit bunches, Opuntia maxima, papaya waste, potato wastes, rice straw, Sida hermaphrodita (L.), silage and seaweed, sugarcane leaves, teak leaves (Tectona grandis), used cooking oil.

3. Wastewater: Bacteria biomass from anaerobic sludge, biosolids of water resource recovery facilities, catering waste leachate and raw sludge, cow rumen liquid, deproteinated cheese whey, dewatered ww, food waste leachate, mill residue, municipal, paper sludge, palm oil mill effluent, septic tank sludge (STS), sewage sludge, swine wastewaters, waste activated sludge (WAS), and thickened WAS.

The production and processing of food and feed results in the generation of a large amount of waste. AD stands out as a suitable technology to reduce the environmental impact of agro-industrial waste and increase the energy self-sufficiency of these industries [29]. However, agro-waste is characterized by the lack of nutrients in its composition necessary for AD. In addition, having a high C/N ratio can affect the performance of AD. Another waste used as a co-substrate is animal manure (i.e. pig, cattle, and poultry). Contrary to agri-food residues, it has a relatively low C/N ratio, which increases the risk of ammonia inhibition [102]. Finally, one of the most studied co-substrates is sewage sludge, which was studied for the first time in 1983 [103], when it was co-digested with Spirulina maxima. Sludge of various varieties can be used as co-substrate, although like the rest, the interactions between microalgae-substrate must be studied to obtain a good balance between the different parameters that can affect AD [12].

5. Factors influencing anaerobic co-digestion

AD process is affected by several factors which led to a higher or lower biogas yield; these factors can be split into two main sources: operational conditions and substrates composition. Although both sources are widely related, they can be studied and controlled separately. The main operational conditions are temperature, pH, configuration of bioreactors, acclimation of inoculum, hydraulic retention time (HRT), organic load rate (OLR), and inoculum to substrate ratio (ISR) [1, 104]. The main factors related to substrates composition are the C/N ratio and macro and micronutrients [1, 5, 6, 104].

In this sense, AcoD is proposed as a feasible alternative in order to balance these factors and allow a better performance and a higher biogas production.

5.1 Effect of initial conditions

Most studies agree that the best operational temperatures for AD are under mesophilic (20–45°C) or thermophilic (>45°C) conditions [104]. Temperature affects, either directly or indirectly, the solubility of substrate compounds and the specific growth rate of the microorganisms involved, provoking a change in the HRT, the pH, and the methane yield [104]. Among the literature, the most common range is the mesophilic conditions due to its lower energy cost requirements and the similar methane yield when compared with higher temperature conditions [105]. However, temperature variations during AD performance have shown significant reductions in methane yield and the kinetics of the process [104, 106].

The effect of pH is mainly related to the optimum pH of the microorganism performance during AD. Based on that, a pH between 4.6 and 6.0 favored the hydrolysis, acidogenesis, and acetogenesis stages, while a pH between 6.0 and 8.0 favored the methanogenesis phase [1]. Literature shows that the most suitable pH range is between 6.5 and 7.5, where the methane production is most benefited [1, 104]. Furthermore, it has been reported that the initial pH of the substrate had a significant impact on methane yield, being the optimum value in the range of 7.0–7.5 for the co-digestion of swine manure and maize stalk [107]. However, pH is highly affected during the AD process, lowering its value if the buffer system is not strong enough due to the accumulation of volatile fatty acids (VFA) (e.g. acetic, propionic, butyric, and valeric acids) [104] or increasing it if ammonium nitrogen is accumulated (around 5.0 g NH₃-N L⁻¹) [33].

Temperature and pH have been linked to free ammonium nitrogen and ammonium ions equilibrium, showing that when one parameter is fixed there is a linear increase in methane yield when the other two independent variables increased up to a certain limit, after which the methane production decrease [104, 108]. A recent study showed optimal conditions for the mono-digestion of chicken manure of 34.0°C, 5.0 g NH₃-N L⁻¹, and pH 7.5 [108]. Moreover, in an earlier study, an increase of pH from 7 to 8 and above, enhanced the biogas production with similar methane proportion when temperature conditions increased from 37 to 55°C during the AD of buffalo manure [109].

As shown above, temperature and pH have a significant impact on AD performance and biogas production. Thus, co-digestion is presented as a suitable technique able to enhance the buffer system, the substrate pH, and free ammonium nitrogen values, allowing a higher methane yield and a more stable process [67, 77, 78, 99, 100]. Meneses-Reyes et al. showed that increasing the C/N ratio of the substrate by reducing the microalgae ratio provoked an increase in pH, however, the pH of digestate is similar among the different co-digestion ratios studied (7.33–7.51) [41]. AcoD of Chlorella sp. and food waste in batch mode (35°C) showed that methane yield was related to the initial pH of the substrates (7.3–8.7), being the optimum value 8.0 and reporting that the methane yield decreased almost linearly when pH differs from the optimum value, although these results were not conclusive as other variables were also different (e.g. VS_feeding from 8.0 to 9.2 g) [35]. However, a mixture of microalgae biomass with thermally treated wheat straw presented a pH of 12 with no significant effect on AcoD performance when compared with the AcoD of microalgae biomass and untreated wheat straw (pH: 6.82) [71]. Another study assessed the effect of temperature within the mesophilic range (25°C, 30°C, 35°C, 40°C) in biogas production, reporting a significant increase of biogas production (45%) when the temperature was increased up to 35°C, but a reduction of production or no significant improvement (depending on the C/N ratio of substrates) when the temperature was set up at 40°C [24]. This result is in accordance with other studies reporting a decrease of methane yield for the AcoD of microalgae with undigested sewage sludge in batch mode when increasing the temperature from mesophilic (37°C) to thermophilic (55°C) conditions, even though the pH was not affected (6.91–7.03) and the processes were stable [45]. AcoD of corn silage and Nannochloropsis salina in a semi-continuous mode (38°C; C/N: 21.2) do not present any stability deviation by the increasing OLR (2–4.7 g VS L⁻¹ d⁻¹), while the AcoD of two microalgae (Scenedesmus sp. and Opuntia maxima) in semi-continuous mode (37°C; C/N: 15.6) showed that pH was affected by OLR (2–6.67 g VS L⁻¹ d⁻¹) and ISR (6:8% VS basis), is the most stable conditions OLR: 2 g VS L⁻¹ d⁻¹ and ISR: 8% VS basis [60, 110]. Furthermore, a study assessing the effect of alkali, acid, and thermal pretreatment of Oscillatoria tenuis, before the AcoD with pig manure, reported that pH control affected the biogas production rather than physical or chemical pretreatments [18]. Similar results were reported during the AcoD of alkali pretreated microalgae consortium with swine wastewater, where the negative effect of ammonia inhibition at high pH (11) was stronger than the positive effect of the destruction of microalgae’s cell walls [47].

pH is also being widely used as a control parameter able to indicate the stability of the reactors since is strongly linked to VFA accumulation [40, 43]. AcoD of Arthrospira platensis with several carbon-rich co-substrates proved to be stable at low OLR (1 g VS L⁻¹ d⁻¹) in a semi-continuous mode but unstable at higher OLR (2–5 g VS L⁻¹ d⁻¹) due to VFA accumulation and pH dropped, this study is in accordance with the AcoD of Chlorella sp. and glycerol that presented a stable pH range (6.6–7.32) when the HRT was above 5 days, at which point volatile fatty acids (VFA) accumulation inhibit the biogas production [24, 33]. However, AcoD of naturally grown microalgae consortium with WAS in a semi-continuous mode (37°C) showed that when the HRT was increased from 1 to 3 to 4–6, pH was reduced (from 7.51 to 7.04), although VFA accumulation was not observed and the system remained stable with no difference in methane production except at HRT of 6 were slightly dropped [46].

Another important factor is alkalinity, which helps to prevent large changes in pH due to the accumulation of volatile fatty acids, or the generation of ammonia due to protein hydrolysis. Alkalinity provides the necessary buffering capacity to counteract possible changes in pH, produced by the balance between carbonate and bicarbonate. The ideal alkalinity values for AD would be between 2000 and 4000 mg CaCO₃ L⁻¹ [99, 100]. A study assessed the relation between pH and alkalinity, where the initial pH was fixed at 7.0 while the initial alkalinity changed (70–3200 mg CaCO₃ L⁻¹), however, pH remained around neutral values (6.9–7.2) during the AcoD process suggesting that initial alkalinity has no impact avoiding an ammonium concentration from nitrogen-rich substrates as microalgae [30].

The C/N ratio is another factor that influences the AD process [29]. A good substrate C/N ratio can range between 20 and 30, with an optimal value of 25 [102]. With a C/N ratio below 20, there is an imbalance between C and N in the reactor, which ultimately releases a large amount of NH₃, which usually happens with the degradation of microalgae [102]. The high concentration of NH₃ in the digester affects the growth and metabolism of microorganisms and produces an accumulation of volatile fatty acids, which results in a decrease in biogas yield. This factor can also be supplied by choosing a good co-substrate and optimizing this C/N ratio [94].

The ISR is another key parameter that influences AD and methane production [13]. To find the maximum methane potential, a proper balance between the substrate and the microorganisms is necessary so that limitations and inhibitions do not occur due to the loading of the substrate. For biochemical methane potential (BMP) assays, a ISR ≥ 2 is suggested as the default value [111].

5.2 Effect of pretreatments

As a common way to improve the methane yield of AcoD of microalgae, several studies have reported the effect of pretreatments on microalgae biomass. Table 3 shows some of these pretreatments and the effect on AcoD calculated as the increase of biomethane production in percentage over the non-pretreated AcoD.

Pretreatment	Conditions	Microalgae	Co-substrate	Improvement	Reference
Enzymatic	0.1% v/v; 0.5 h	Scenedesmus sp. + Chlorella sp.	WAS	-2%	[97]
Ultrasonication	n/d	Nannochloropsis oculata	Cow manure	16.7%	[57]
Hot water	121°C; 15 psi	Nannochloropsis oculata	Cow manure	36.7%	[57]
Ultrasonication:hot water	121°C; 15 psi	Nannochloropsis oculata	Cow manure	16.7%	[57]
Thermal	60°C, 24 h	Scenedesmus sp. + Chlorella sp.	WAS	−33.8%	[86]
Thermal	55°C, 2d	Chlorella vulgaris	WW	8.3%	[19]
Thermal	120°C, 20 min	Oscillatoria tenius	Pig manure	43%	[18]
Alkaline	NaOH 3% w/v	Scenedesmus sp. + Chlorella sp.	Swine WW	−10.4%	[47]
Thermal	120°C, 1 h	Chlorella sp.	Coffee wastes	13.9%	[49]
Thermo-alkaline	10%CaO, 75°C, 24 h	Chlorella sp.	Wheat straw	9.0%	[71]
Thermo-alkaline	10%CaO, 75°C, 24 h	Chlorella sp.	Wheat Straw	15%	[71].

Table 3.

Effect of pretreatment on AcoD.

It has been reported that although some pretreatments can improve the methane yield of microalgae as a sole substrate, these have a negative effect during the AcoD, mainly due to the high organic matter consumption or the inefficacy of pretreatments breaking down the cell wall [47, 86, 97].

From Table 3 it can be seen that low thermal pretreatments (60–55°C; 1−2d) have none or very little effect on AcoD [19, 86], however, when the temperature increased to 120°C, the biomethane production improve greatly (up to 43%) [18, 49]. Other successfully tested pretreatments are ultrasonication, hot water, and a combination of thermal and alkaline pretreatments [57, 71]. Nevertheless, based on the higher improvement on biomethane production, thermal pretreatment at 120°C is the most effective process, where time and pressure would be the variables to analyze [49, 57].

6. Biomethane potential (BMP) performance

Table 4 summarizes all the BMP assay results up to date complying with the following prerequisites:

• The co-substrates ratio is well described as VS or C/N.

• ISR has been considered.

• Methane yield have been reported under standard conditions.

Co-substrates	Co-substrate ratio (VS)	ISR (VS)	Temperature (°C)	Methane yield ($N{L}_{C{H}_4}k{g}_{VS}{{}^{-}}^1$)	Synergetic effect (%)	Ref.
Dictyosphaerium sp.:synthetic food wastes	1:3	2	35	514	27.6	[29]
Tribonema sp.:pig manure	2:8	2	35	580.4	16.9	[34]
Chlorella sp.:sludge:FOG	4:4:2	2	35	334	3.7	[72]
Scenedesmus quadricauda:OMSW	1:3, C/N: 25.3	2	35	461	36.4	[26]
Chlamydomonas reinhardtii 6145:OMSW	1:1, C/N: 18.3	2	35	542	37.2	[27]
Chlamydomonas reinhardtii cw15:OMSW	1:1, C/N: 18.3	2	35	451	16.5	[27]
Nannochloropsis limnetica:piggery slurry	4:6	5	53	355	12	[77]
Microalgae consortium:WW	1:3	2	35	339	5.9	[74]
Scenedesmus sp.:deproteinated cheese whey	17:83	3.3	35	302	−7.6	[16]
Scenedesmus sp.:cellulose	16:84	3.3	35	272	−2.0	[16]
Microalgae consortium:WW	37:63	2	35	237.1	2.1	[46]
Chlorella vulgaris:cattle manure	4:1	4	55	431	8–15	[40]
Chlorella sp.:swine manure	6:94, C/N: 33.9	2	35	348	11.2	[84]
Scenedesmus sp. + Chlorella sp.:food waste	1:4, C/N: 26.4	2	Mesophilic	639.8	32.8	[99]
Nannochloropsis gaditana:cellulose	1:3, C/N: 20.3	2	37	268	1.5	[11]
Scenedesmus sp and C. vulgaris:sewage sludge	12:88, C/N: 9.4	2	37	388	0	[45]
Dunaliella salina:Olive mill solid waste	1:3, C/N: 26.7	2	37	330	28.7	[28]
Nannochloropsis salina:corn silage	1:6, C/N: 21.2	2.7	40	660	15	[60]

Table 4.

Methane yield of co-digested microalgae with different substrates at different ratios in BMP assays.

The above prerequisites have been selected as they are crucial for comparison purposes. The authors of this review acknowledge the lack of homogenization regarding BMP performance, which difficult the task to evaluate scientific data and provide reliable conclusions. The authors of this review also acknowledge the lack of a widely approved and used standard methodology for BMP tests, although, some are published and proved to be reliable enough [111, 112]. The authors of this review would also like to highlight that only 20 out of 120 reports complies with the above prerequisites, and if other crucial factors were included within the prerequisites (e.g. use of positive control or details on inoculum acclimation) no reports could have been included in this review. Additionally, when more than one co-substrate ratio was measured in the same study, only the one producing the highest methane yield was included in Table 4.

As can be observed in Table 4, methane yield ranged from 237.1 to $639.8N{L}_{C{H}_4}k{g}_{VS}{{}^{-}}^1$. AcoD of microalgae with industrial wastes showed positive synergetic effects in most cases, being the improvement against the theoretical values up to 37%. However, some studies had reported negative effects. This could be due to the limits of batch methodologies and could lead to higher methane productions during continuous tests as pointed out by several authors [16, 33] or could be related to the low C/N ratio [45].

The optimum C/N ratio produced higher synergetic effects than those studies where the C/N ratio was above 30 or below 20. The most common and successful temperature is within the mesophilic range with an ISR of 2–3, although, some studies have reported improvements when applying thermophilic temperature at a higher ISR (4–5) [40, 77].

Regarding the co-substrates ratio, when microalgae were used as a nitrogen source in order to balance the C/N ratio, it was added commonly as a fourth part of the whole influent, or even less. When microalgae were co-digested with other low C/N substrates, it was added at higher concentrations. Nevertheless, the optimum ratio between microalgae and other co-substrates is unique and needs to be assessed through experimentation.

7. Scaling up the AcoD process

Table 5 summarizes all the results obtained by semi-continuous or continuous assays at lab or pilot scale results up to date complying with the following prerequisites:

• The co-substrates ratio is well described as VS or C/N.

• Temperature, OLR, and HRT/ISR has been considered and reported.

• Methane yield have been reported under standard conditions (when values were reported under normal conditions a factor of 0.8871 was applied to obtain the methane yield under standard conditions).

Co-substrate	Co-substrate ratio (VS)	System	Acclimated inoculum	Conditions	Methane yield	Improvement over control	Ref.
Microalgae biomass:WW	26.4:1 (VSS)	UASB (pilot scale)	Yes	25 C; OLR: 1.0 g COD L^−1 d^−1; HRT: 8.1 h	235 NL kgVS^−1	−20% (wastewater)	[32]
Chlorella sp.:WW	38:62, C/N: 7.08	AnMBR (lab scale)	Yes	25 C; OLR: 0.5 g COD L^−1 d^−1; HRT: 30 days; HRT: 70 days	391 NL kgVS^−1	nd*	[62]
Scenedesmus sp. Chlorella sp.:WW	38:62, C/N: 7.08	AnMBR (pilot scale)	Yes	35°C; OLR: 0.5 g COD L^−1 d^−1; HRT: 30 days; HRT: 70 days	370 NL kgVS^−1	nd*	[62]
Chlorella sp.:WW	38:62, C/N: 7.08	CSTR (lab scale)	Yes	55°C; OLR: 0.5 g COD L^−1 d^−1; HRT: 30 days	242 NL kg VS^−1	nd*	[61]
Microalgae biomass:sewage	(Flow rate) algae: 0.5 L h^−1, sewage: 49 L h^−1	UASB (pilot scale)	No	23°C; OLR: 0.7 g VS L^−1 d^−1; HRT: 7 h	211 NL kgVS^−1	34.8% (Sewage only)	[79]
Chlorella sp.:WW	38:62, C/N: 7.08	Two stage: AnMBR, AnR (lab scale)	No	AnMBR; 35°C; OLR: 0.52 g COD L^−1 d^−1; HRT: 30 d,; AnR; 35°C; OLR: 0.15 g COD L^−1 d^−1; HRT: 100 days	291 NL kgCOD^−1	nd*	[65]
Scenedesmus sp.:primary sludge	38:62	AnMBR (lab scale)	Yes	35 C; OLR: 0.5 g COD L^−1 d^−1; HRT: 30 d	241 NL kgCOD^−1	40.1% (algae only)	[93]
Chlorella sp.:primary sludge	38:62	AnMBR (lab scale)	Yes	35 C; OLR: 0.5 g COD L^−1 d^−1; HRT: 30 d	228 NL kgCOD^−1	6.5% (algae only)	[93]
Chlorella vulgaris:chicken litter:glycerol	30:67:3, C/N: 8.15	Semi-continuous (lab scale)	Yes	37 C; OLR: 0.716 g VS L^−1 d^−1; HRT: 30 d	240 NL kgVS^−1	39% (chicken litter only)	[43]
Nannochloropsis limnetica:piggery slurry	2:3	CSTR (lab scale)	nd*	53 C; OLR: 1.4 g VS L^−1 d^−1; HRT: 15 d	216 NL kgVS^−1	22.7% (pig slurry only); −31% (algae only)	[77]
Microalgae biomass:WW	1:3	Semi-continuous (lab scale)	No	37 C; OLR: 1.17 g VS L^−1 d^−1; HRT: 30 days	460 NL kgVS^−1	187.5% (pretreated algae only)	[74]
Stigeoclonium Scenedesmus:papaya waste	1:1 (w/w)	Semi-continuous (lab scale)	Yes	35 C; OLR: 1.1 g VS L^−1 d^−1; HRT: 31 days	230 NL kgCOD^−1	59.7% (algae only); 12.2% (theoretical)	[17]
Microalgae biomass:primary sludge:WAS	2:3, C/N: 8.49	Semi-continuous (lab scale)	nd*	37 C; OLR: 2.4 g COD L^−1 d^−1; HRT: 15 days	168 NL kgVS^−1	−15.8% (WWTP only)	[46]
Chlorella sp.:wheat straw	1:1, C/N: 13.1	Semi-continuous (lab scale)	nd*	37 C; OLR: 1 g VS L^−1 d^−1; HRT: 20 days	240 NL kgVS^−1	15% (no pretreated); 75% (algae only)	[71]
Chlorella sp.:swine manure	6:94	Semi-continuous (lab scale)	nd*	35 C; OLR: 1.16–1.68 g VS L^−1 d^−1; HRT: 21 days	348 NL kgVS^−1	9.8% (swine only)	[84]
Chlorella vulgaris (MACC-755):used cooking oil	1:1, C/N: 477	Semi-continuous (lab scale)	No	38 C; OLR: 4.01 g VS L^−1 d^−1; HRT: 90 days	880 NL kgVS^−1 2.86 L L^−1 d^−1	6.0% (algae only)	[54]
Chlorella vulgaris (MACC-755):maize silage	1:1, C/N: 16	Semi-continuous (lab scale)	No	38 C; OLR: 4.01 g VS L^−1 d^−1; HRT: 90 days	1.99 L L^−1 d^−1	nd*	[54]
Chlorella vulgaris (MACC-755):mill residue	1:1, C/N: 12	Semi-continuous (lab scale)	No	38 C; OLR: 4.01 g VS L^−1 d^−1; HRT: 90 days	1.96 L L^−1 d^−1	nd*	[54]

Table 5.

Methane yield of co-digested microalgae with different substrates at different ratios in semi-continuous or continuous assays.

*

nd: not determined.

Semi-continuous or continuous processes could overpass some drawbacks from batch assays as reported by several authors [71, 77, 84]. This could be due to the acclimation of the inoculum during the experiments. To this sense, these processes had shown successful results in the co-digestion of substrates with low C/N ratios, low pH, and higher ammonium content, allowing a higher concentration of microalgae in the influent. However, some studies had shown negative results when compared with the AD of sole substrates [32, 46, 77]. This low methane production had been related to the difficulty of microalgae cell-wall digestion, accumulation over time of ammonium, or high OLR [32, 46, 77].

Regarding operational parameters, the most common used temperature was within the mesophilic range due to its low energy cost and good performance. OLR ranged from 0.5 to 4 g COD L⁻¹ d⁻¹, although, most studies showed a higher methane production when the OLR is below 1.5 g COD L⁻¹ d⁻¹. HRT was around 30 days, with some studies reporting 90 days when the OLR is around 4 g COD L⁻¹ d⁻¹. OLR and HRT had been discussed as key factors for a complete energy-effective harnessing of microalgae AcoD, to this sense, high OLR and low HRT would be the most effective way for achieving this. However, pH decreases due to VFA accumulation, and ammonium release are the main factors affecting the OLR.

8. Conclusions

The status and current trends of AcoD of microalgae and industrial wastes were reviewed in this chapter. AcoD performance improvements still need further research on varied co-substrates and optimal mix ratios. Operational parameters and their control are key to achieving optimal biogas. Pretreatments of microalgae biomass are a promising way to enhance biogas production. The majority of research investigations are done by a few research groups and centered on biomethane potential tests lacking a common methodology, thus, further research and the application of common criteria need to be implemented. Moreover, pilot-scale assays have shown promising results, however, very few research groups have the ability to implement these studies.

Acknowledgments

The authors wish to express their gratitude to the regional government of Andalucía, Junta de Andalucía, Consejería de Transformación Económica, Industria, Conocimiento y Universidades (Project FEDER UPO-1380782) and to the Spanish national R&D plan (PID2020-114975RB-100/AEI/10.13039/5011000/11033) for providing financial support.

Conflict of interest

The authors declare no conflict of interest.