Algae-Bacteria Consortia as a Strategy to Enhance H2 Production

Biological hydrogen production by microalgae is a potential sustainable, renewable and clean source of energy. However, many barriers limiting photohydrogen production in these microorganisms remain unsolved. In order to explore this potential and make biohydrogen industrially affordable, the unicellular microalga Chlamydomonas reinhardtii is used as a model system to solve barriers and identify new approaches that can improve hydrogen production. Recently, Chlamydomonas–bacteria consortia have opened a new window to improve biohydrogen production. In this study, we review the different consortia that have been successfully employed and analyze the factors that could be behind the improved H2 production.


Introduction
Finding renewable, sustainable and clean energy sources has become one of the main priorities of our society. Hydrogen (H 2 ) is a promising clean and carbon-free energy source with a high energy value (142 kJ/g) that can be easily interconverted with electricity and used for domestic and industrial applications. Currently, H 2 production techniques include steam reforming natural gas/oil, coal gasification, biomass gasification/pyrolysis, and electrolysis and thermolysis of water. All these techniques are either polluting and/or demand a large amount of energy [1,2]. Under this scenario, the biological production of H 2 (bioH 2 ) has garnered considerable attention in recent decades, as it could be a cheap and renewable source of fuel. Different microorganisms such as microalgae, cyanobacteria, photosynthetic bacteria and some heterotrophic bacteria can produce H 2 [3,4]. Algae and cyanobacteria are well-known photoautotrophic organisms able to convert CO 2 into organic matter and release O 2 during this process. Under specific conditions, H 2 production is linked to photosynthetic activity. Non-oxygenic photosynthetic bacteria can also use light and organic acids (and other chemical forms) to obtain energy and produce H 2 , without releasing O 2 . Heterotrophic bacteria, on the other hand, can degrade organic matter and release CO 2 , with some of them also producing H 2 . Among them, photobiological H 2 evolution by green algae and cyanobacteria has attracted considerable attention since, potentially, they do not require organic carbon sources to produce H 2 , only water and sunlight [4][5][6]. Moreover, microalgae and cyanobacteria are the most dominant photosynthetic organisms on Earth, which increases their biotechnological interest. However, photosynthetic H 2 production is still inefficient for industrial implementation due to its low yield and rate of H 2 generation. One of the most important bottlenecks of biological H 2 production is its sensitivity to oxygen (O 2 ). In all the H 2 -producing microorganisms, O 2 is a strong repressor of H 2 production.

H 2 Production in Non-Oxygenic Photosynthetic Bacteria
Some non-oxygenic photosynthetic bacteria can also produce H 2 . In this group of microorganisms, the Purple Non-Sulfur Photosynthetic (PNSP) bacteria are among the best known H 2 producers. As with cyanobacteria, H 2 production by PNSP bacteria is mostly linked to nitrogenase activity. ATP generated during photosynthesis is used by the nitrogenases to produce NH 3 and H 2 . In this case, photosynthesis is not linked to water splitting and thereby O 2 is not produced. Instead, the most common source of electron donors are organic acids, and the process is known as photo-fermentation. For H 2 production, formate, acetate, lactate and butyrate can act as electron donors, with butyrate being the best inducer of H 2 production [8,46,47]. Like cyanobacteria, two of the main factors limiting H 2 production in PNSP bacteria are the simultaneous occurrence of H 2 uptake and the need to establish nitrogen-deficient conditions.

H 2 Production in Heterotrophic Bacteria
Many heterotrophic bacteria can produce H 2 though fermentative pathways (also known as dark H 2 production). Bacterial fermentation of sugars can produce a large variety of fermentative end products, including H 2 . There are two distinctive groups of bacteria that have been extensively studied regarding fermentative H 2 production. One group is composed of strict anaerobes (represented by, e.g., Clostridium spp.), where H 2 production is linked to the oxidation of pyruvate into acetyl CoA by Pyruvate Ferredoxin Oxidoreductase (PFOR). This pathway is known as the PFOR pathway. The second group are facultative anaerobes (represented by, e.g., E. coli), which, under anaerobic conditions, perform so-called mixed acid fermentation, where pyruvate can be used by Pyruvate Formate Lyase (PFL) to produces formate and acetyl CoA. Formate is then converted to CO 2 and H 2 by the Formate Hydrogen Lyase (FHL), and the process is known as the PFL H 2 -production pathway. H 2 production through dark fermentation has several limiting factors, including 1) the existence of other competitive fermentation pathways and, 2) the excessive accumulation of end products (mainly ethanol, formate, acetate, lactate, succinate, glycerol and butyrate) that block H 2 production [48][49][50][51][52][53].
Although numerous efforts have been made to improve H 2 production in algal and bacterial systems, the integration of these two systems to improve bioH 2 production has received less attention [8,21,47]. This review outlines the past and recent achievements obtained when the green algae Chlamydomonas is co-cultivated with different bacterial strains to improve H 2 production.

Current Achievements Obtained with Chlamydomonas-Bacteria Consortia
Several studies have proven the possibility to improve H 2 production when using co-cultures of alga and bacteria [21,54,55], with some of them focusing on the use of the alga Chlamydomonas. Table 1 provides a comparative analysis of all the previously published data about H 2 production in Chlamydomonas-bacteria consortia with their respective algal monocultures in terms of yield, rate and sustainability. Studies are ranked according to the total H 2 production yield. Notably, most of the publications show enhancements in H 2 production parameters (yield, rate and duration) in the co-cultures relative to the monocultures, with many consortia promoting a threefold yield enhancement. Different Pseudomonas sp. and Bradyrhizobium japonicum are bacterial partners that lead to the highest H 2 production yields in cultures incubated in Tris-Acetate-Phosphate (TAP) medium, devoid of S (TAP-S), and they often lead to great enhancements in H 2 production (up to 22.7-fold and 32.3-fold, respectively) ( Table 1). Note that these two bacterial partners are not known to be H 2 producers by themselves. In general, the best condition for H 2 production can be obtained in TAP-S (Table 1), confirming that, as in the case of Chlamydomonas monocultures, S deprivation is a physiological condition that greatly promotes H 2 production in this alga. The light intensity does not seem to be a crucial parameter for H 2 production from consortia incubated in TAP-S (Table 1). H 2 photoproduction in Chlamydomonas Cells 2020, 9, 1353 4 of 22 monocultures in TAP medium is scarce, unless low light intensities (below 22 PPFD) are used [14]. However, different consortia can attain noticeable H 2 production in TAP medium at higher light intensities (Table 1), which open the possibility to further explore H 2 production under non-stressful conditions to avoid S removal and two-phase bioreactors. Finally, the use of H 2 -producing bacterial strains such as wild-type strains of E. coli in media supplemented with glucose brings up the possibility to combine H 2 production from both alga and bacterium [56]. This consortium can produce up to 32.7 mL/L, which is higher than the production reported for other consortia in TAP-S medium (Table 1). Similarly, other bacteria like Pseudomonas putida and Rhizobium etli can also facilitate H 2 production in Chlamydomonas when incubated with sugars as the only carbon sources (Table 1).  1 Chlamydomonas reinhardtii unless otherwise stated; 2 photosynthetic photon flux density (PPFD) (µmol photons · m 2 −1 · s −1 ); 3 Avogadro's law for ideal gas is considered to estimate H 2 productivity in the unit of (mL/L culture) 1 mole H 2 gas (at pressure = 101.325 kPa and temperature = 273.15 K), equal to 22.41 liters of H 2 ; 4 the average of the lowest and the highest chlorophyll concentration was considered to estimate the H 2 productivity from "per mg chlorophyll" to "per liter culture"; 5 enhancements in co-cultures compared with monocultures are presented as fold changes in parentheses; 6 sugar is added when acetic acid is depleted in the culture media; 7 folds cannot be calculated because either H 2 production in alga monocultures are zero or are not reported; 8 data for chlorophyll concentration was not reported; 9 reported fold change; Modified Bristol Medium (MBM); information not provided in the original report (NP); glucose (glu); mannitol (man); light-dark cycles (L-D); "≈": data estimated from the original study (rounded values). To contextualize the achievements obtained using Chlamydomonas-bacteria consortia, Table 2 lists some of the most successful strategies described in Chlamydomonas for H 2 production, including monocultures and co-cultures, and ranks them by the total H 2 yield obtained. Monocultures using genetically modified strains and S deprivation can lead to the highest H 2 yields. However, the use of Chlamydomonas wild-type strains co-cultured with different bacterial partners under S deprivation are also ranked within the top list. For example, different co-cultures incubated in TAP-S employing Pseudomonas sp. or Bradirizhobium japonicum have achieved ≈165-170 mL H 2 /L culture [57][58][59], and there is a published patent for H 2 production using Chlamydomonas and Pseudomonas fluorescens co-cultures claiming to produce 196 mL/L [68]. These values obtained using co-cultures are a bit far from the maximal Chlamydomonas H 2 production reported (850 mL/L) using a proton gradient mutant (pgrl5) affecting the cyclic electron transfer [69]. However, co-culturing techniques could have a great potential to further improve H 2 production if genetically modified Chlamydomonas (or bacterial) strains are employed in co-cultures. Moreover, it should be noted that most studies exploring H 2 production in Chlamydomonas co-cultures are very recent and there are much more possibilities to explore in this field.

Characteristics of the Algae-Bacteria Association for H 2 Production
In recent years, an increased interest in the study of algal-bacterial interactions has emerged not only due to their ecological significance, but also for their biotechnological potential. It is known that algae and bacteria affect one another's physiology and metabolism. In natural ecosystems, algal-bacterial interactions cover a whole range of relationships: mutualism, commensalism and parasitism, depending on specific species and living requirements [77]. These interactions are omnipresent in all ecosystems. Moreover, microorganisms have complex and very versatile metabolisms, allowing them to grow or to simply survive in non-optimal environments. In this sense, Chlamydomonas, for example, apart from its photoautotrophic metabolism, has a fermentative metabolism that allows this alga to consume internal reserves such as starch under anaerobic conditions, releasing H 2 and other end products to the medium. Moreover, Chlamydomonas can also grow heterotrophically and is able to consume acetic acid as a carbon source. Noticeably, acetic acid is the only organic carbon form that Chlamydomonas can uptake and, under hypoxic conditions, it has also been suggested that the assimilation of this compound is connected to H 2 production in this alga [14][15][16].
At a physiological level, the production of H 2 by microorganisms is considered as an escape valve for the electrons generated in excess during either photosynthetic or fermentative processes. The activation of hydrogenases (or nitrogenases) occurs under very specific environmental conditions, and for most microorganisms, H 2 production can be considered as a transitory event. When cultivating axenic cultures of H 2 -producing microorganisms in the laboratory, different growth conditions are used to maximize H 2 production. However, the complex interplay between the different microorganisms has not been not studied. Understanding this interplay can provide valuable information to overcome some of the bottlenecks associated with biological H 2 production.
A straightforward advantage of co-culturing heterotrophic bacteria with algae is that they can efficiently remove O 2 from the media, which is the most critical bottleneck associated with H 2 photoproduction. At the same time, the CO 2 released during bacterial fermentation can support algae and cyanobacteria growth, while the photosynthetic O 2 production can support the growth of facultative anaerobic bacteria. In addition, algae and photosynthetic bacteria can theoretically combine their sunlight wavelength absorption ranges to increase the overall light-to-energy conversion efficiency for H 2 production or for biomass generation. Finally, several photosynthetic and fermentative metabolites can be exchanged between microorganisms, establishing specific nutrient fluxes that can benefit H 2 production and/or growth. Among these nutrient fluxes, carbon fluxes are quantitatively the most prominent, although nitrogen, phosphorous and S sources, and growth factors like Vitamin B12, have also been reported as favoring algae-bacteria interactions [78][79][80][81][82].
In the following sections, the potential mechanisms influencing H 2 production in algae-bacteria cultures are discussed. They are categorized according to the impact on (1) biomass, accumulation of internal reserves, and metabolite exchange supporting H 2 production, (2) net O 2 evolution, and (3) the possibility to extend the solar spectrum absorption range.

Starch Accumulation could be Promoted in Co-Cultures
Starch reserves in Chlamydomonas can be connected to photobiological H 2 production through the PSII-independent pathway ( Figure 1). This pathway relies on the non-photochemical reduction in the PlastoQuinone (PQ) pool using the electrons derived from NAD(P)H [9][10][11]83]. The glycolytic degradation of starch is proposed to be the main source of electrons for this H 2 -producing pathway during S deprivation conditions [12]. Moreover, starch degradation can also feed the fermentative or dark H 2 production in Chlamydomonas via the PFR pathway ( Figure 1) [8,17,18]. Different nutrient stresses (mainly N and S) can promote starch accumulation in Chlamydomonas cultures under both light and dark conditions [84,85], which, in turn, can favor H 2 production.
japonicum [58], Azotobacter chroococcum [67], Pseudomonas sp. [59] and Thuomonas intermedia [65]. However, the precise reasons explaining why the starch accumulation occurs in these co-cultures have not been elucidated. In any case, co-culturing Chlamydomonas with certain bacterial strains could be used as an approach to promote starch accumulation, which potentially can enhance algal H2 production through the PSII-independent pathway or through metabolite exchange (see below sections) (Figure 1). Figure 1. Potential starch-derived relationships between Chlamydomonas and other microorganisms during H2 production. Starch accumulated in Chlamydomonas cells can be used to feed the PIIindependent (1) and fermentative (2) pathways. The accumulation of starch in Chlamydomonas can be favored when co-cultured with some bacterial strains (3). Different end products derived from Chlamydomonas starch mobilization can be excreted and used by Purple Non-Sulfur Photosynthetic (PNSP) bacteria for H2 production (4). Starch-enriched Chlamydomonas biomass can be used directly by some heterotrophic bacteria to produce H2 (5a) or in collaboration with PNSP bacteria (5b). Pyruvate Ferredoxin Reductase (PFR); PlastoQuinone (PQ); hydrogenase A (HydA).

Mobilization of the Algal Starch Reserves can Provide Organic Acids for H2 Producing Bacteria
Chlamydomonas has a very versatile fermentative metabolism and is able to quickly degrade starch reserves under anaerobic conditions to different fermentative end products including H2 [86][87][88] (Figure 1). Some end products are secreted to the medium by wild-type Chlamydomonas cultures, including acetate, ethanol and formate. Glycerol, succinate and lactate are minor end products secreted by most wild-type Chlamydomonas cells; however, the noticeable excretion of these fermentative products can be found in some Chlamydomonas mutants [86] or in some strains considered to be wild-type [89]. All these secreted end products can be theoretically used by bacteria Recently, it has been observed that co-culturing Chlamydomonas with different bacterial strains can lead to high starch accumulation in this alga. These bacterial strains include Bradyrhizobium japonicum [58], Azotobacter chroococcum [67], Pseudomonas sp. [59] and Thuomonas intermedia [65]. However, the precise reasons explaining why the starch accumulation occurs in these co-cultures have not been elucidated. In any case, co-culturing Chlamydomonas with certain bacterial strains could be used as an approach to promote starch accumulation, which potentially can enhance algal H 2 production through the PSII-independent pathway or through metabolite exchange (see below sections) (Figure 1).

Mobilization of the Algal Starch Reserves Can Provide Organic Acids for H 2 Producing Bacteria
Chlamydomonas has a very versatile fermentative metabolism and is able to quickly degrade starch reserves under anaerobic conditions to different fermentative end products including H 2 [86][87][88] (Figure 1). Some end products are secreted to the medium by wild-type Chlamydomonas cultures, including acetate, ethanol and formate. Glycerol, succinate and lactate are minor end products secreted by most wild-type Chlamydomonas cells; however, the noticeable excretion of these fermentative products can be found in some Chlamydomonas mutants [86] or in some strains considered to be wild-type [89]. All these secreted end products can be theoretically used by bacteria as electron donors for H 2 production (Figure 2), and some of them have been probed at an empirical level using Chlamydomonas-PNSP bacteria cultures [62,90]. Miyamoto et al. [62] reported that, when co-culturing Chlamydomonas and Rhodospirillum rubrum, they both produced H 2 in dark conditions. In the case of Chlamydomonas, H 2 originated from the fermentative degradation of the starch reserves, while, in the case of R. rubrum, H 2 originated from the Formate Hydrogen Lyase pathway using the formate excreted by the alga as a substrate. Similarly, Miura et al. [90] reported that after incubating Chlamydomonas in the dark, the resulting medium broth was used by a marine photosynthetic bacterium, Rhodopseudomonas sp., to photoproduce H 2 . This Chlamydomonas medium broth was enriched with acetic acid and ethanol.

Acetic Acid Exchange Can Promote H 2 Production in both Algae and Bacteria
As mentioned before, the donation of fermentative metabolites from Chlamydomonas to different bacteria can promote bacterial H 2 production. However, the opposite flux (from bacteria to alga) can also benefit both algal and bacterial H 2 production, especially when acetic acid is produced and secreted by the bacteria [56]. In Figure 2, some of the metabolites that can be potentially exchanged between algae and other microorganisms during both growth and H 2 production conditions are depicted.
Many bacteria can produce H 2 though fermentative pathways (dark H 2 production). In organisms using the PFOR H 2 -production pathway (e.g., Clostridium spp.), the highest yield is obtained when acetate is the main fermentation end product. Similarly, in organisms using the PFL H 2 -production pathway (e.g., E. coli), the highest yield is obtained when acetic acid and ethanol are the end products ( Figure 2) [48,49]. The maximum theoretical yield of dark H 2 production is assumed to be 2 to 4 mol of H 2 per mol of glucose, depending on the kind of microorganisms (2 moles for facultative aerobes and 4 moles for strict anaerobes). To obtain this theoretical maximum yield, glucose must be fully converted to acetate as the terminal end product. In summary, the process in strict anaerobes, such as Clostridium sp., consists of the conversion of pyruvate to acetyl CoA and CO 2 through PFOR, and electrons are donated to the hydrogenases via reduced FDX. This results in a maximum yield of 2 mol of H 2 per mol of glucose. Two additional moles of H 2 can be produced from the NADH produced during glycolysis via NADH:ferredoxin oxidoreductase (NFOR) which can donate electron to the FDX hydrogenase system, making an overall theoretical maximum yield of 4 mol of H 2 per mol of glucose for this kind of bacteria. In facultative anaerobes, because a maximum of two molecules of formate are produced from two pyruvate molecules, the theoretical maximum yield for the PFL pathway is 2 mol of H 2 per mol of glucose [48][49][50][51]91]. However, different constraints make the actual yields of dark fermentation much reduced. Two of the main drawbacks of dark H 2 production are a) the existence of other fermentative competing pathways that lower the yield and b) the excessive accumulation of fermentative end products, especially acetic acid, which impairs microbial growth and H 2 production [48][49][50][51]91]. Numerous studies have focused on the manipulation of Clostridium spp. and E. coli to enhance the H 2 production by redirecting the fermentative pathways and reducing the accumulation of some undesired end products such as lactate, succinate or butyrate. However, the accumulation of acetic acid cannot be avoided since, in both pathways, this compound is directly linked to the production of H 2 , and its production is crucial to maintain an optimal energy/redox balance for the cells [48][49][50][51]. Cells 2020, 9, x FOR PEER REVIEW 15 of 26 Figure 2. Potential metabolites exchanged among different H2-producing microorganisms during growth conditions (A) and H2-producing conditions (B). The secretion and uptake of metabolites are indicated with plain and dotted arrows, respectively. Depending on the specific culture conditions the same metabolites can be secreted or accumulated. Organic Acids (OAs) mainly include ethanol, glycerol, formate, acetic acid, lactate, succinate and butyrate. When predominant, the specific OA is indicated next to the arrow. In order to solve the problematic acetic acid accumulation, integrative strategies combining dark bacteria and non-sulfur photosynthetic bacteria have been assessed. In these bacteria consortia, the organic acids generated by the dark bacteria can feed the photosynthetic bacteria for H 2 production, resulting in increased H 2 production yields ( Figure 2) [47]. Theoretically, maximum yields in these integrative cultures can be obtained if acetic acid is the only secreted end product. Two molecules of acetate can be generated from glucose, in both facultative and strict anaerobes, which can then be converted into H 2 by the PNSP bacteria, producing, theoretically, a maximum of 8 extra mol of H 2, and, making the overall theoretical yield of the integrative systems 10 to 12 mol of H 2 per mol of glucose. Again, these theoretical values are not reached since different limitations exist. Among others, the use of photosynthetic bacteria in these integrative systems often requires two-stage bioreactors due to the growth incompatibility and the removal of nitrogen, which strongly inhibits the H 2 -evolving nitrogenases [47].
The literature concerning the use of integrative systems has considered, almost exclusively, photosynthetic bacteria as the only partners able to use and remove the acetic acid resulting from dark fermentation. However, some microalgae can be used instead of (or with) photosynthetic bacteria ( Figure 2). When co-culturing Chlamydomonas with different non-H 2 producing bacteria in acetate-free media supplemented with sugars (glucose or mannitol), algal H 2 production can be observed if acetic acid is excreted by the bacteria. The amount of acetic acid excreted by the bacteria directly correlates with the capacity of Chlamydomonas to produce H 2 [56]. Moreover, as demonstrated by Fakhimi et al. [56] using E. coli and Chlamydomonas co-cultures incubated with glucose as the sole carbon source, it is possible to produce H 2 in a synergetic way (60% more H 2 than the sum of the respective control monocultures), with acetic acid probably being the metabolite linking dark H 2 production with H 2 photoproduction (Figure 2). This study entails a proof-of-concept linking dark bacteria and algae H 2 production. Nonetheless, the H 2 production yield obtained in E. coli-Chlamydomonas co-cultures was very low and optimizations are required.
As mentioned before, acetic acid is the only compound that Chlamydomonas can uptake as the sole carbon source for heterotrophic growth. Note that in, Chlamydomonas monocultures, no other source of organic carbon (e.g., glucose) can be used for growth or to trigger H 2 production ( Figure 2). Apart from growth promotion, acetic acid plays a significant role in H 2 production in this alga. The presence of acetate in the medium promotes O 2 consumption, represses CO 2 fixation, and decreases the photosynthetic rates [92][93][94][95]; all of these factors favor H 2 production. In addition, the presence of acetic acid in the culture media has been reported as a key parameter for photo-H 2 production in Chlamydomonas monocultures [14] and co-cultures [56], whose role is partially independent of its capacity to promote hypoxia [14]. It has been suggested that, under light, nutrient-repleted conditions and hypoxia, the assimilation (or photoassimilation) of acetic acid, and not starch mobilization, can provide, directly or indirectly, electrons for the PSII-independent H 2 production pathway [13,14]. Physiologically, the photoassimilation of acetate under hypoxia could be equivalent to the H 2 photo-fermentation described in photosynthetic bacteria.
Overall, the use of microalgae such as Chlamydomonas leads to photo-H 2 production, while helping to bypass the drawbacks of the acetic acid accumulation and pH acidification that prevent bacterial H 2 production. The use of algae instead of photosynthetic bacteria or cyanobacteria has the advantage of avoiding the concomitant occurrence of H 2 uptake and the nitrogen removal from the medium, which is required to induce nitrogenases. Moreover, compared with PNSP bacteria, algae have more compatible growth conditions with some dark bacteria. Moreover, algae, but not photosynthetic bacteria, can provide extra acetate-independent H 2 production via direct H 2 production (PSII-dependent pathway) or via the mobilization of the starch reserves (PSII-independent pathway). The two latter pathways can potentially surpass the theoretical maximum H 2 yield of 10-12 mol H 2 per mol of glucose in the solely bacterial integrative systems. However, more research is still needed to explore the potential of algae-bacteria co-cocultures for H 2 production, and to better understand how the acetate metabolism is linked to H 2 production in Chlamydomonas anaerobic cultures.

Co-Culturing Chlamydomonas with Bacteria Can Alleviate the Negative Effect of S Deprivation while Promoting H 2 Production
S deprivation is a strategy widely used to enhance photobiological-H 2 production in Chlamydomonas [35,36], which can lead to the highest H 2 yields (Table 1). However, this strategy has several drawbacks, including growth inhibition and the loss of the cell viability (caused by the S prolonged deficiency), which reduce the potential for H 2 generation. Previous studies have partially overcome the harmful effects of S deprivation using continuous or semi-continuous regimes of cultivation [96][97][98]. Recently, different studies using batch co-cultures in TAP-S [58,59,65] have obtained similar results to these previous studies, although avoiding the use of continuous or semi-continuous strategies, which can greatly simplify the overall process. For example, co-culturing Chlamydomonas with Pseudomonas sp. [59] or with Bradyrhizobium japonicum [58] in TAP-S can slow the reduction in chlorophyll, enhance starch accumulation, and maintain protein content, while favoring algal H 2 production relative to algal monocultures. However, the precise reasons why these bacteria prolonged the viability of Chlamydomonas cells in TAP-S is uncertain. Interestingly, when Chlamydomonas is incubated with the sulfur-oxidizing bacterium Thuomonas intermedia [65], a considerable increase in H 2 production and algal growth are observed; these effects are even more pronounced when the cultures are treated with the oxygen scavenger Na 2 S 2 O 3 ( Table 1). Authors propose that T. intermedia is able to oxidize S 2 O 3 2− to SO 4 2− , providing a S source for the alga to satisfy the minimum requirement for algal growth and, at the same time, maintain the S-deprived environment required for H 2 photoproduction [65]. Overall, co-cultures in TAP-S require less energy inputs than continuous or semi-continuous alga monocultures and, more importantly, can support algae growth and H 2 production simultaneously.

Starch-Enriched Alga Biomass Can Be Used as Substrate for H 2 Producing Bacteria
Besides the direct supply of excreted fermentative metabolites to H 2 -producing bacteria by living algal cultures, algal biomass can also support H 2 production by strict or facultative anaerobic bacteria. Different bacteria consortia have been probed to produce H 2 from Chlamydomonas biomass. These consortia are often composed of a fermentative bacterium and a photosynthetic bacterium. The fermentative bacteria can degrade the Chlamydomonas biomass and excrete organic acids such as ethanol, formate, acetate, propionate and butyrate, which can be used by the photosynthetic bacteria to photoproduce H 2 via photo-fermentation. For instance, Lactobacillus amylovorus is able to hydrolize starch from algae biomass to lactic acid, which can feed the photo-H 2 production in Rhodobacter sphaeroides, Rhodobacter capsulata, Rhodospirillum rubrum and Rhodobium marinum [99,100]. Similarly, Vibrio fluvialis converted starch accumulated in Chlamydomonas to acetic acid and ethanol, which drove H 2 production in Rhodobium marinum under high salt condition [101]. Likewise, Rhodobacter sphaeroides produced H 2 from formate, acetate and butyrate secreted by Clostridium butyricum after anaerobic fermentation of Chlamydomonas biomass [102]. In this example, direct H 2 production from Clostridium butyricum fed with Chlamydomonas biomass was also attained, which can illustrate the potential of producing H 2 from algal biomass using bacteria consortia and two-step processes (Figure 1).

Net O 2 Evolution
In Chlamydomonas, the photoproduction of H 2 is unavoidably linked to the photosynthetic electron chain and thereby to O 2 generation. As mentioned before, O 2 is a strong inhibitor of both the expression and activity of the Chlamydomonas HYDAs [103]. The measurements of O 2 in co-cultures can be indistinctly done in the liquid phase as dissolved O 2 (DO 2 ), or in the headspace. The DO 2 measurements are more accurate to predict HYDAs activity in Chlamydomonas co-cultures, as demonstrated by Ban et al. [59]. Nevertheless, a good correlation between these two O 2 indices and their relationship with H 2 production has been observed in Chlamydomonas co-cultures [60].
In algae monocultures, the net O 2 evolution is a result of the O 2 inputs and outputs. The O 2 inputs include initial O 2 in the headspace, the DO 2 in the culture media, and photosynthetic O 2 generation.
The light intensity directly influences the activity of the photosynthesis processes and thereby O 2 generation. The O 2 outputs are due to the respiratory activity. Chlamydomonas respiratory activity greatly increases when growing heterotrophically (or mixotrophically) in acetate-containing media due to the capability of this alga to use acetate as carbon source. This is the reason why most publications use either TAP or TAP-S to study H 2 production in this alga.
A very simple relationship between O 2 evolution and H 2 production in H 2 -producing acetate-containing cultures is shown in Figure 3. In algal monocultures incubated in TAP medium, the O 2 level quickly drops during the first 24 h. Under moderate light intensities (<50 PPFD), the photosynthetic O 2 evolution is lower than the O 2 consumption and the cultures remain under hypoxia for a few days, while the H 2 production starts within the first 24 h. The hypoxic condition is maintained as far as acetic acid remains in the media. Once the acetic acid is fully consumed, the O 2 levels rise and H 2 production stops. Light intensity directly impacts the acetic acid uptake: the higher the light intensity, the faster the acetic acid uptake and the shorter the H 2 production phase. At higher light intensities (>50 PPFD), there is a net positive O 2 evolution and cultures do not reach hypoxia. [14] ( Figure 3A). In co-cultures, the O 2 outputs can be significantly increased if aerobic or facultative anaerobic bacteria are incubated in media containing organic carbon sources, which can greatly benefit H 2 production. Again, most of the studies about H 2 production using Chlamydomonas co-cultures are done in TAP or TAP-S media. In TAP co-cultures, the respiration rate can increase from 18% to 64% relative to Chlamydomonas monocultures, depending on the algal strain and the bacterial partners [57,61,66]. Unlike algal monocultures, no net O 2 evolution is obtained in TAP co-cultures under moderate to high light intensity (50-100 PPFD), which allows H 2 production at these light intensities [57,60,61,63]. A direct correlation between the presence of acetic acid in the media and the capacity to produce H 2 have been observed in different Chlamydomonas-bacteria cultures [56,60,63,64]. Recently, Fakhimi et al. [63] have shown that the positive effect of the bacterial partners on H 2 production can be linked to a decrease in acetate assimilation by the alga. Slower acetic acid uptake allows for a longer presence of this compound in the TAP culture medium, which, in turn, results in longer hypoxia and H 2 production phases. This effect also allows for the use of higher light intensities compatible with H 2 production. Distinct bacteria partners can impact the acetic acid uptake rates of Chlamydomonas differently; out of the different bacteria tested, Pseudomonas sp. showed the highest capacity to decrease the acetic acid uptake. All these data reveal that the use of co-cultures in TAP medium can help to reach hypoxia at higher light intensities than in monocultures, and they can increase the H 2 yield by the means of more sustained H 2 production.
On the other hand, S deficiency causes a decline in PSII activity and thereby in photosynthetic O 2 evolution [104]. In Chlamydomonas monocultures incubated in TAP-S, at light intensities above 50 PPFD, there are 1-3 days where the cultures remain aerobic (termed as lag or oxic phase). Afterwards, an anerobic phase starts and H 2 is produced; the H 2 production yield in TAP-S is often higher than in TAP. In TAP-S cultures, the acetic acid is never fully consumed, and its level is neither linked to the aerobic or anaerobic phases nor to the H 2 production phase [36,105] (Figure 3B). Dark incubation prior illumination or purging with noble gases are often used as strategies to quickly deplete O 2 levels in the TAP-S cultures and shorten the lag phase. In co-cultures incubated in TAP-S, the respiration rate is enhanced by three to eight times during the first day compared with algal monocultures, depending on the light intensity [57,59,61,66,67]. Lakatos et al. [60] observed that after just 4h of illumination, the O 2 level in the co-cultures (4-5%) were lower than in monocultures (15-16%). These observations demonstrated that, in the case of TAP-S cultures, the co-incubation with bacteria can reduce the lag phase and avoid the dark incubation or purging required to reach hypoxia and initiate H 2 production. results in longer hypoxia and H2 production phases. This effect also allows for the use of higher light intensities compatible with H2 production. Distinct bacteria partners can impact the acetic acid uptake rates of Chlamydomonas differently; out of the different bacteria tested, Pseudomonas sp. showed the highest capacity to decrease the acetic acid uptake. All these data reveal that the use of co-cultures in TAP medium can help to reach hypoxia at higher light intensities than in monocultures, and they can increase the H2 yield by the means of more sustained H2 production. In TAP cultures (A), H2 production occurs only in the presence of acetic acid, which is necessary to establish hypoxic conditions. In TAP-S cultures (B), the H2 production phase and the hypoxic phase are independent of the acetic acid concentration. Under the same light conditions, TAP cultures show faster acetic acid uptake and shorter lag phase than in TAP-S. H2 production yield and duration in TAP-S cultures is often higher than in TAP cultures. Tlag, lag phase before H2 production; tp, H2 production phase; thypoxia, hypoxia/anaerobic phase; (O2)in, initial O2 levels; (O2)hyp, minimal O2 levels compatible with H2 production.
On the other hand, S deficiency causes a decline in PSII activity and thereby in photosynthetic O2 evolution [104]. In Chlamydomonas monocultures incubated in TAP-S, at light intensities above 50 PPFD, there are 1-3 days where the cultures remain aerobic (termed as lag or oxic phase). Afterwards, an anerobic phase starts and H2 is produced; the H2 production yield in TAP-S is often higher than in TAP. In TAP-S cultures, the acetic acid is never fully consumed, and its level is neither linked to the aerobic or anaerobic phases nor to the H2 production phase [36,105] (Figure 3B). Dark incubation prior illumination or purging with noble gases are often used as strategies to quickly deplete O2 levels in the TAP-S cultures and shorten the lag phase. In co-cultures incubated in TAP-S, the respiration rate is enhanced by three to eight times during the first day compared with algal monocultures, depending on the light intensity [57,59,61,66,67]. Lakatos et al. [60] observed that after just 4h of illumination, the O2 level in the co-cultures (4-5%) were lower than in monocultures (15-16%). These observations demonstrated that, in the case of TAP-S cultures, the co-incubation with bacteria can reduce the lag phase and avoid the dark incubation or purging required to reach hypoxia and initiate H2 production.
Overall, elevating the O2 consumption rate by bacteria can improve H2 production by a) allowing the implementation of hypoxic conditions compatible with H2 production, b) decreasing the time required to establish hypoxia, c) extending the duration of the hypoxia phase, which directly influences the production phase, and d) tolerating higher light intensities without impairing the hypoxic conditions [59,60,63].
Finally, among the important aspects to be considered when setting up algae-bacteria cocultures are the initial cell number ratios, which are one of the main concerns of many studies In TAP cultures (A), H 2 production occurs only in the presence of acetic acid, which is necessary to establish hypoxic conditions. In TAP-S cultures (B), the H 2 production phase and the hypoxic phase are independent of the acetic acid concentration. Under the same light conditions, TAP cultures show faster acetic acid uptake and shorter lag phase than in TAP-S. H 2 production yield and duration in TAP-S cultures is often higher than in TAP cultures. T lag , lag phase before H 2 production; t p , H 2 production phase; t hypoxia , hypoxia/anaerobic phase; (O 2 ) in , initial O 2 levels; (O 2 ) hyp , minimal O 2 levels compatible with H 2 production.
Overall, elevating the O 2 consumption rate by bacteria can improve H 2 production by a) allowing the implementation of hypoxic conditions compatible with H 2 production, b) decreasing the time required to establish hypoxia, c) extending the duration of the hypoxia phase, which directly influences the production phase, and d) tolerating higher light intensities without impairing the hypoxic conditions [59,60,63].
Finally, among the important aspects to be considered when setting up algae-bacteria co-cultures are the initial cell number ratios, which are one of the main concerns of many studies [57,59,61,65]. Different ratios can impact the O 2 inputs and outputs and thereby the net O 2 concentration in the cultures. Moreover, due to the light shading effect of the bacteria, the initial algae-bacteria ratios and light intensities should be considered and optimized at the same time [67]. According to Ban et al. [59], there is an optimum initial cell number of algae which results in the highest H 2 production.

Extension of the Solar Spectrum Absorption Range
An important aspect of the association between microalgae and photosynthetic bacteria is the possibility to increase the range of the solar spectrum for conversion to H 2 . Microalgae and cyanobacteria can capture the visible portion of sunlight (400-700 nm) and generate H 2 , while PNSP bacteria can also capture near-infrared emissions (700-1010 nm) to produce H 2 . Therefore, an integrated system can lead to a better solar irradiation utilization. However, few studies have been carried in this sense using Chlamydomonas. Following this idea, Melis and Melnicki [106] studied a consortium of Chlamydomonas with Rhodospirillum rubrum to improve biomass generation. However, the light irradiance performance of this co-culture was weakly analyzed and H 2 production was not reported for this co-culture. It would be interesting to perform a more thorough investigation of the light irradiance efficiency in similar co-cultures and their suitability for H 2 production.

Final Remarks
H 2 production by microalgae is being studied due for its potential to provide a clean and renewable biofuel. However, this technology is still far from industrial application due to its low rates and yields, which make it economically unviable. In the context of improving bio-H 2 production, strategies based on algae-bacteria consortia are still poorly explored; however, they show great potential and could be some of the best strategies to improve H 2 production. Indeed, despite the limited number of publications, the combination of Chlamydomonas with different non-H 2 producing bacteria is already among the most successful strategies to attain H 2 production in this alga (Table 2). However, the future of algae-bacteria consortia remains in their capacity to integrate co-cultures with other successful strategies such as physiological treatments (e.g., S or Mg deprivation), O 2 scavengers, cell immobilization or light modulation. Importantly, co-cultures using genetically modified strains of both algae and bacteria could also offer great potential to further improve H 2 production.
Improved H 2 production in Chlamydomonas co-cultures can be explained by multiple factors, including an increase in the starch content, a decline in net O 2 evolution, a decrease in the algal acetic acid uptake, metabolite exchanges, and the utilization of higher light intensities compatible with H 2 production. However, there are still many questions that remain uncertain regarding how non-H 2 producing bacteria promote algal H 2 production.
In any case, the use of integrative systems combining different H 2 -producing microorganisms (alga, cyanobacteria, PNS bacteria and heterotrophic bacteria) could be the real challenge in the bio-H 2 field. Combining fermentative, photofermentative and photosynthetic pathways for H 2 production could be the most feasible approach to overcome the low bio-H 2 production yields and make them compatible with industrial applications. In the case of microalgae, this is a very promising approach that needs to be further explored and extensively improved. A few studies have already confirmed the possibility to achieve collaborative [62,90] and even synergetic H 2 production [56] when using Chlamydomonas together with different kinds of H 2 -producing microorganisms. This prospect can provide a new perspective on how to produce H 2 from cheap raw materials or waste, taking advantage of microbial metabolic collaborations, while, at the same time, bypassing some H 2 production barriers identified in both algae and bacteria (e.g., O 2 withdrawal, acetic acid accumulation, pH control, or organic carbon and other nutrient supplies).
However, H 2 -producing microorganisms have complex and very versatile metabolisms. Unravelling the metabolic and physiological relationships that they develop in natural ecosystems is the key to creating properly designed strategies to improve H 2 production when co-culturing. Finding the appropriate algal and bacterial partners, suitable raw materials, and culture conditions could be the next challenge to address efficient and sustainable H 2 production.