The biomass optimization in the first step showed that mainly the pH and the light intensity interfered in the biomass yield, corroborating with previous research that claims that physical and chemical factors affect the growth of microalgae [35–38]. Thus, a higher amount of biomass was obtained in the first step at a lower pH (6.2) and light intensity of 200 µmol m− 2 s− 1. It is believed that this biomass optimization provided the inoculation of a greater amount of biomass in the photobioreactors and favored the hydrogen production variable test as studies have reported that an increased amount of biomass can improve the hydrogen production yield [6–8, 21].
The results described above demonstrate a significant influence of sulfur concentration and light intensity in hydrogen production by C. reinhardtii (CC425). The best results were obtained with sulfur-deprived, and higher light intensity supported better hydrogen production, 3.57 mmol L− 1, and 2.62 mmol L− 1 at the lowest light intensity (conditions 2 and 1, respectively) for 180 hours, agree with previous literature that used similar methodology, and were even higher compared to some of the reported results [39, 40].
In order to improve photobiological hydrogen production, there must be a balance between respiration and photosynthesis, and this can be achieved by varying the concentration of nutrients in the culture medium, such as that of sulfur, nitrogen and phosphorus [19]. Sulfur deprivation decreases photosynthetic activity and consequently decreases oxygen production, which is consumed by respiration. There is a compensatory point between photosynthesis and respiration that results in an anaerobic environment [6, 8, 10, 41]. Reports show that low sulfur concentrations (around 12.5 to 100 µmol L− 1) can improve the compensatory point between photosynthesis and respiration and obtain better results in hydrogen production [13, 14].
However, other researchers obtained negative results when low sulfur concentration was added compared to complete sulfur deprivation [42] and addition above 50 µmol L− 1 can delay the onset of hydrogen production and decrease the final yield of hydrogen produced [13]. The same occurred in this research, and when comparing the conditions just by observing the variation in the sulfur concentration (comparison of conditions 1 with 3 and 2 with 4) it was noticed that the hydrogen productivity was on average 6 times higher when there was total sulfur deprivation.
These results could be due to increased photosynthetic rate upon sulfur addition, and consequently an increase in oxygen production, thus resulting in the inhibition of hydrogen production. Another factor associated with sulfur concentration is light intensity, which must be varied in parallel with the sulfur concentration, so that the best compensatory point between photosynthesis and respiration to make the anaerobic environment can be achieved [7, 8, 41]. Wild and mutant strains may also respond differently to sulfur concentrations and light intensities, and variations in this compensatory point may also occur. It has been stated that approximately 27 µmol m− 2 s− 1 is the best light intensity to achieve this compensation point with the CC425 strain [39], which is lower than the intensities used in this study and which possibly prevented us from obtaining positive results upon sulfur addition.
In this research, it was evident that the light intensity was another factor that interfered in hydrogen production, mainly in sulfur-deprived conditions in which the production was, respectively, 73.4% higher under 200 µmol m− 2 s− 1 compared to 60 µmol m− 2 s− 1. Although higher light intensities can inhibit the hydrogenase due to higher photosynthetic rate [43, 44], in sulfur-deprived cultures there is low evolution of oxygen through photosynthesis, then this inhibition by light is not expected to occur [20]. Several researchers analyzed the influence of light intensity in hydrogen production in sulfur deprived cultures, with different strains of Chlamydomonas, and some obtained better results at lower intensities (around 12 to 40 µmol m− 2 s− 1) [20, 45] and others at higher intensities (around 100 to 300 µmol m− 2 s− 1) [6, 46, 47], although researchers affirm that the optimal light intensity is 50 to 200 µmol m− 2 s− 1 for hydrogen production [35].
Different light intensities, light:dark cycles, light absence, and sulfur deprivation are also factors that aid in the investigation of metabolic pathways involved in hydrogen and other fermentation by-products. In this study, acetic acid was used as a source of organic carbon and it was consumed in all conditions, although the consumption was higher in the photobioreactors containing sulfur (average consumption of 57.2% under sulfur-free conditions and 97.2% with sulfur addition). As discussed above, under these conditions hydrogen production was lower, probably due to the increase in the photosynthetic rate, as previously discussed. This consumption is one of the factors responsible for maintaining the anaerobic environment and providing reductant for hydrogen production via indirect photoproduction (photofermentation) [10]. Furthermore, acetic acid can also be used by microalgae as a substrate for respiration and starch accumulation, and after 24 hours of anoxia, acetate production by the fermentative acidogenic pathways is observed, and there is a superposition between its consumption and production [8]. Therefore, the results suggest that in the presence of additional sulfur, the higher acetic acid consumption rate is probably due to its lower production by the fermentation pathway, justifying and corroborating the lower hydrogen production that occurs under these conditions.
As the system becomes anaerobic, the ability of microalgae to modify their metabolism to anaerobic fermentation can lead to the generation of organic acids, ethanol, carbon dioxide and hydrogen through multiple fermentation pathways through anaerobic decomposition of pyruvate [6, 8, 23, 48]. However, considering that an important part of the pyruvate anaerobic metabolism occurs in the chloroplast, where the hydrogenase is located, there is competition for a reductant between the hydrogen production and other fermentation pathways [7, 9].
The production of ethanol measured under all experimental conditions in this work confirms the occurrence of the alternative fermentative metabolic pathways. Ethanol production was higher under higher light intensity and with sulfur (average of 203.2 mg L− 1). The results of this research were better, compared to those of other researchers that utilized similar methodology and the same strain, who reported 37 mg L− 1 [8] and 57 mg L− 1 of ethanol [49], but under lower light intensity (100 µmol m− 2 s− 1) and a more basic pH (around 7.2 to 7.7). It is known that low pH favor ethanol production, which explains the better ethanol production results in this study. However, it has also been shown that high ethanol production decreases hydrogen photoproduction due to the competition for the reductant that occurs between these two pathways [18, 50].
Besides hydrogen and ethanol production by the fermentative pathways, the more acidic pH and nutritional sulfur limitation modify the protein metabolic pathway causing an increase in the degradation of proteins and carbohydrates [18, 51]. It has been reported that the decrease in carbohydrates and protein content occurs concomitantly with hydrogen photoproduction in sulfur-deprived cultures [17, 18, 52], although the carbohydrate accumulation can occur during the hydrogen production due to the presence of acetic acid and mixotrophic metabolism of Chlamydomonas [48, 53, 54]. This information corroborates the results obtained in this research, in which there was a decrease in protein concentrations in all photobioreactors, especially under deprived sulfur, which show higher hydrogen production (consumption of protein 111 times higher in deprived sulfur conditions than conditions with sulfur). Carbohydrate consumption, also observed in all conditions, probably occurred for ATP generation needed to conduct metabolic processes that require energy during anaerobiosis [9, 17, 52].
In addition to the efficient hydrogen and ethanol production by the CC425 strain in this research, the results also demonstrated that it is possible to associate the cultivation of microalgae, such as C. reinhardtii, with wastewater treatment, as has been recently studied by some researchers, mainly using Chlorophyceae [24, 25, 55]. However, there is a lack of studies on this species in the literature.
The results demonstrated mainly the removal of acetic acid, as previously discussed, and the occurrence of phosphate uptake, with an average removal of 72.8%, which may have occurred by its adsorption to the cell surface and assimilation into biomass [56, 57]. Phosphate enters the cell by active transport through the cell membrane and can be used in the formation of ATP molecules [25]. Nitrogen uptake was not observed at significant levels, possibly due to the increased accumulation of nitrogen compounds in the liquid medium caused by the decrease in biomass protein under all tested conditions. Furthermore, it was observed that the nitrogen removal occurred under the conditions, in which there was less decrease in biomass protein, corroborating the results. Indeed, organic matter uptake was greater under conditions of lower hydrogen production. Under higher hydrogen production conditions, an increase in acetic acid production, as previously discussed, may have occurred due to the acidogenic pathway and may have influenced this result, as previously discussed.
In general, the greatest removal of nutrients occurred under conditions with sulfur presence, in addition to less hydrogen production. These data can be an indication that under these conditions there was a higher photosynthetic rate, and consequently it did not provide a totally anaerobic environment [7, 13]. Thus, it may have contributed to the greater removal of nutrients, since photosynthetic microorganisms can be efficient for this purpose in aerobic conditions, although a high concentration of dissolved oxygen may limit the removal [25, 55, 56]. Therefore, a microaerobic environment may have reduced the hydrogen production, due to the inhibition of hydrogenase, consequently causing metabolic deviations to fermentative pathways and higher production of ethanol in these conditions, in addition to having provided greater nutrient removal.
It has been shown that microalgae biomass bioflocculation, or immobilized-cells cultivation systems, can improve the nutrient removal efficiency [24, 58, 59], and our assays were performed with suspended-cell cultivation systems, which may have decreased the nutrient uptake efficiency. Therefore, research with CC425 with flocculated biomass is the aim of future research in the wastewater treatment to improve the nutrient removal efficiency by this strain, in addition to using these microalgae in association with other microorganisms, which has given positive results for the wastewater treatment [25, 60]. Algal-bacterial consortia can be combined with biohydrogen generation and wastewater treatment and aerobic bacteria can contribute to this process by consuming the oxygen produced by the algae in photosynthesis, providing an anaerobic environment [60]. Furthermore, if there is an anaerobic digestion pathway, fermentative bacteria break down organic matter in wastewater into soluble acids, alcohols, hydrogen gases and carbon dioxide. Acetate can be generated from these products by acidogenic bacteria through the process of acetogenesis during the dark fermentation step [26, 61]. In the second step, microalgae having mixotrophic or heterotrophic metabolism, such as Chlamydomonas, are able to grow in closed systems and use this acetate produced as an organic carbon source to produce hydrogen by photoheterotrophic pathway (indirect biophotolysis) [9, 16, 61, 62].
Therefore, the results of this research confirm that hydrogen production occurs through direct water biophotolysis and from carbohydrates breakdown, from the starch reserve or from acetic acid. The occurrence of each pathway may vary according to the different conditions studied. The relative contribution of each of the electron sources may depend on factors such as the strain, extent of damage of PSII, culture conditions and metabolic restrictions [63], such as light intensity age and sulfur concentration, all of which affected our results. In addition, the results of this research demonstrate applicability of C. reinhardtii cultivation (CC425) in anaerobic photobioreactors and provide an important discussion of factors that influence hydrogen production, as well as the contribution of using this strain for other purposes of environmental and economic interest.