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Biochemical methane potential of microalgae biomass using different microbial inocula
© The Author(s) 2018
- Received: 17 April 2018
- Accepted: 25 June 2018
- Published: 29 June 2018
Microalgae biomass is regarded as a potential feedstock for bioenergy purposes through anaerobic digestion (AD). Even though AD is a well-proven technology, the use of new feedstocks requires in-depth studies. A lot of research has been conducted assessing methane yield without paying attention to the anaerobic microbiome and their activities. For such a goal, the present investigation was designed to link methane yield to those two later sludge characteristics. In this sense, different anaerobic sources were tested, namely adapted to microalgae biomass and adapted to sewage sludge.
Despite the registered differences for the anaerobic microbiome analysis and specific methane activities towards model substrates, sludge adapted to digest sewage sludge did not affect the methane yield of Chlorella sorokiniana and Scenedesmus sp. Opposite to that, sludge samples adapted to digest microalgae exhibited a concomitant increase in methane yield together with increasing digestion temperatures. More specifically, the values attained were 63.4 ± 1.5, 79.2 ± 3.1 and 108.2 ± 1.9 mL CH4 g COD in−1 for psychrophilic, mesophilic and thermophilic digestions, respectively. While psycro- and mesophilic digestion supported similar yields (most probably linked to their anaerobic microbiome resemblance), the values attained for thermophilic digestion evidenced the usefulness of having a highly specific microbiome. The relative abundance of Firmicutes, particularly Clostridia, and Proteobacteria together with an important abundance of hydrogenotrophic methanogens was highlighted in this inoculum.
Overall, this study showed that working with tailored anaerobic microbiome could help avoiding pretreatments devoted to methane yield enhancement.
- Anaerobic digestion
- Anaerobic microbiome
- Specific methanogenic activity
Renewable energy will play a key role in limiting CO2 emissions while decreasing as well fossil fuel dependency. Renewable resources such as biomass and organic wastes are envisaged as promising feedstock for energy purposes. Anaerobic digestion is a proven technology for obtaining biogas via conversion of organic matter. In this regard, anaerobic digestion (AD) has been implemented in a large number of facilities and in fact, this number increases over the years . Facilities already running use mainly traditional feedstocks such as manure or energy crops (corn). However, the need of expanding AD to a range of new substrates has raised attention in key points that should be taken into account when new feedstocks are going to be used. Microalgae biomass has been the focus of interest in AD during the last decade. Microalgae biomass is a substrate of particular interest since it contributes to wastewater bioremediation and CO2 bio-mitigation. Additionally, microalgae biomass can be grown in non-arable land and are more productive than terrestrial plant due to their higher photosynthetic efficiency . All these features render microalgae biomass as a promising feedstock for energy purposes.
Out of the different energy forms that can be produced using microalgae biomass as feedstock, biogas production via anaerobic digestion is probably the most economically feasible since it does not require highly concentrated biomass  and anaerobes can use the three biomass macromolecules (proteins, carbohydrates and lipids) for methane production purposes . As a limiting step, anaerobic hydrolysis appears to be one of the most challenging steps to reach a positive economic balance and to completely exploit the potential of microalgae for biogas. Once evidenced the resilience of easily growing microalgae strains towards AD, a great number of investigations have been devoted to pretreat this biomass prior to anaerobic digestion. A summary of the research efforts that have been undertaken to date can be found elsewhere [5, 6]. Several authors have found that the energy consumption associated to the pretreatment of microalgae biomass is equal to or higher than the energy gained [7, 8].
One alternative to the use of pretreatments is the selection of appropriate anaerobic inocula. In fact, it has been shown that the use of one or another inoculum influences the biochemical methane potential of different substrates [9–11]. A consortium of interdependent microorganisms composes the anaerobic inoculum. The prevalence of determined anaerobes might affect the hydrolytic and methanogenic activity of the sludge and thus, different microbial population might lead to different anaerobic digestion efficiencies. Moreover, not only the inherent microbial activity of the sludge might be of importance but this activity can also be tuned by microbial adaptation. Anaerobic microbial community has the ability to adapt to changing conditions. Microbial adaptation to the new situation entails most of the time a lower microorganism diversity but more specified . In this manner, some of the adaptation mechanisms include genetic changes, selective population enrichment  and enzymatic activity modification .
Although a number of studies have addressed the limited anaerobic biodegradability of this biomass and potential pretreatment methods to enhance it, controversial results might be found in the literature dealing with microalgae biomass digestion. One of the reasons of different results has been attributed to the different anaerobic sludge inocula used in the digestions. In this sense, to the best of our knowledge, no investigation has been made on the effect of different anaerobic inocula in the methane yield achievable when using microalgae as substrates. As a matter of fact, when comparing different inocula, studies mostly focus on methane production or substrate degradation while inocula themselves are not paid enough attention. For this reason, the aim of this investigation was to evaluate the effect of different inoculum sources on microalgae biomass anaerobic digestion. Six different inocula were tested in terms of anaerobic microbiome, degradation activity towards model substrates and methane production yield using microalgae as feedstocks. Those inocula included four operating at mesophilic range (35 °C), one psychrophilic (ambient temperature, 15–25 °C) and one thermophilic (55 °C). Moreover, the psychro-, thermo- and one of the mesophilic sludge were adapted to digest microalgae biomass while the rest of them were adapted to sewage sludge degradation.
Microalgae biomass used as substrate
The substrate consisted in two types of microalgae biomass, namely Scenedesmus gender and the specie Chlorella sorokiniana. Scenedesmus inoculum was grown in synthetic media in 1 L flask under continuous artificial light supply (TL-D 36W, PHILIPS, Holland). Cultures were kept at room temperature (22–24 °C) with magnetic stirring. Culture media consisted in (all compounds in mg L−1): 160 NH4Cl, 25 CaCl2·2H2O, 75 MgSO4·7H2O, 75 K2HPO4, 175 KH2PO4, 25 NaCl, 50 EDTA disodium, 31 KOH, 4.98 FeSO4·7H2O, 11.42 H3BO3, 17.64 ZnSO4·7H2O, 2.88 MnCl2·4H2O, 1.42 MoO3, 3.14 CuSO4·5H2O, and 0.98 CoNO3·6H2O.
Scenedesmus sp. was grown in a 200 L raceway reactor. The raceway was operated without temperature control (20–24 °C) and the illumination consisted in 12 fluorescent lamps. Culture was conducted in semicontinuous condition; harvested and fed once per week. Fresh urban wastewater from Rey Juan Carlos University was used as culture media for Scenedesmus sp. The specific composition of the influent consisted in (all elements in mg L−1): chemical oxygen demand (COD); total fraction 498 ± 190 (out of which 58% belongs to soluble fraction), N–NH4+ of 34.1 ± 16.3, N–NO2− of 0.9 ± 0.8, N–NO3− of 1.2 ± 1.1 and P–PO43− of 4.9 ± 3.6 and total suspended solids (TSS) and volatile suspended solids (VSS) of 0.17 ± 0.06 and 0.15 ± 0.04 g L−1, respectively. Scenedesmus sp. biomass was harvested with an industrial centrifuge (OTC3-02-137, 10,000 rpm, WESTFALIA).
On the other hand, C. sorokiniana was grown in synthetic media (previously described) in 1 L flasks for biochemical methane potential assay. Previously to the AD, this biomass was concentrated with the centrifuge MegaFUGE 16R (Thermo Scientific, EEUU) at 5000 rpm during 15 min. Second, C. sorokiniana biomass was frozen to compare the biogas production using a more easily degradable biomass than Scenedesmus sp. .
Inocula: anaerobic sludge
Characteristics and chemical properties of sludge samples (S1–S6) employed as inoculum for anaerobic digestion
Substrates: model compounds and microalgae biomass
Bovine serum albumin (BSA) as protein substrate and cellulose as carbohydrate substrate were chosen as model compounds for assessing the specific methanogenic activity (SMA, g COD consumed/g VSS day) of the different anaerobic inocula. These substrates (proteins and carbohydrates) were selected based on their prevalence in microalgae biomass grown in wastewater . These SMAs were used as an indicator of proteolytic and cellulolytic activity of anaerobic sludge samples. Second, all inocula were employed to digest Scenedesmus sp. (fresh biomass and poorly biodegradable) and C. sorokiniana (previously frozen and thus, easily biodegradable biomass). The fresh Scenedesmus sp. biomass was stored at 5 °C during less than 5 days to preserve its physiological characteristics.
Biochemical methane potential (BMP)
The anaerobic digestion was carried out in enclosed reactors in batch mode during approximately 20–30 reaction days. BMPs were considered finished when cumulative methane production was stable. Digestion bottles had a total volume of 0.12 L and were incubated at different temperatures depending on the inoculum. Temperatures were set at room temperature (22–24 °C) for the psychrophilic assay (S5), at 35 °C for the mesophilic range (S1–S4) and at 50 °C for the thermophilic digestion (S6). Agitation was provided with an orbital shaker at 150 rpm. To ensure anaerobic conditions, bottles were purged with helium, closed with butyl rubber and aluminum caps. Calculations were set to achieve a final working volume of 0.07 L, allowing a head space of 42% for biogas production. The inocula and anaerobic sludge samples were mixed with the different model substrates and algae biomass in a ratio of CODsubstrate/VSinoculum of 0.5 (g g−1). Biodegradability assays were carried out in triplicates. The methane volume generated was calculated with the pressures determined in the head space bottle. The net methane production at standard temperature and pressure (STP) was calculated by subtracting the blank methane production to the amount of methane measured in each sample. To determine the endogenous methane production, bottles containing only anaerobic sludge samples were run as a blank.
Total solids (TS), volatile solids (VS), total suspended solids (TSS), volatile suspended solids (VSS) and the total Kjeldahl nitrogen (TKN) were measured according to standard methods . Total proteins were calculated by multiplying TKN values by 5.95. Carbohydrates content were measured employing phenol sulphuric . Colorimetric commercial methods were used for quantification of COD and ammonium (Merck, ISO 15705 y ISO 7150-1, respectively). N–NO2−, N–NO3−, and P–PO43− were determined by ion chromatography (IC 9030, METHROM, Switzerland). Soluble fractions were obtained by centrifuging at 5000 rpm for 15 min with the centrifuge MegaFUGE 16R (Thermo Scientific, USA) and later filtering by nylon filter of 0.45 µm porous diameter. Biogas composition was determined by gas chromatography (Agilent 7820A) equipped with a 30 m column (HP-PLOTQ, 0.53 mm, 40 µm) connected to a thermal conductivity detector at 250 °C and helium flow of 4.5 mL min−1 as gas carrier.
Inocula microbial community analysis: pyrosequencing
Total DNA extraction of anaerobic sludge samples was performed using FastDNA Spin kit for soil (MPBiomedicals, LLC). Before DNA amplification reaction, samples were purified using commercial kit QiAmp DNA Microkit (QIAGEN, USA). Library preparation was made using set primer based on the capturing of 16s rRNA region as described in Klindworth et al. . Sequencing samples were loaded in the MiSeq Platform from Illumina using a 300PE combination. Sequences were compared against the built rRNA database using a BLAST local alignment approach to associate each cluster to one taxonomical group from the database. Pyrosequencing was performed by Lifesequencing S.L. (University of Valencia, Spain).
Data were presented as means values ± standard deviation of the mean and statistical significances were assessed by analysis of variance (ANOVA). Values were considered statistically significant when p value was lower than 0.05.
Sludge samples and microalgae chemical characteristics
All anaerobic sludge samples exhibited a standard VS/TS ratio ranging 0.66 ± 0.05, VSS/TSS = 0.80 ± 0.04 and VSS/VS = 0.57 ± 0.07. These values were in good agreement with other sludge samples used for BMP tests [20, 21]. In this manner, despite the different feedstock used during their previous activity, namely sewage sludge (mixture of primary and secondary sludge) (S1, S2 and S3) or microalgae biomass (S4, S5 and S6), all of them were within the usual values.
Macromolecular distribution of the microalgae biomass employed as feedstock for anaerobic digestion
TS g L−1
26.7 ± 0.7
37.5 ± 0.2
VS g L−1
23.0 ± 1.2
34.1 ± 0.0
33.8 ± 2.4
10.4 ± 0.7
41.0 ± 0.7
56.3 ± 0.7
11.5 ± 3.4
24.1 ± 0.7
13.8 ± 2.5
9.2 ± 0.3
Sludge samples: anaerobic microbiome
Pyrosequencing was performed to characterize the anaerobic microbiome of all sludge samples. With regard to Shannon’s index of diversity at genera level, all the sludge samples were in the range of 1.36–1.75, exception made for S1 and S6, which exhibited 3.07 and 0.32, respectively. This indicated that the diversity of S1 was considerably higher than the rest of sludge samples while S6 displayed a highly specific consortium in which the biodiversity was really reduced.
The second main phyla retrieved from the sludge samples was Firmicutes. Non-adapted sludge (S1–S3) showed a relative abundance of approximately 20% of Firmicutes (Fig. 1a). This bacterial community is also normally present in anaerobic sludge digesting sewage sludge. Interestingly, adapted sludge samples (S4–S6) decreased their abundance of Firmicutes in psychrophilic and mesophilic sludge to around 10% while that of the thermophilic displayed 28% relative abundance. The sequences were mainly affiliated to the order Clostridiales. More specifically, the relative abundance of Clostridia in S2–S5 were in the narrow range of 62–66% of the Firmicutes, while S1 and S6 showed markedly higher percentages (86 and 96%, Fig. 1b). It is important to note that Clostridia are responsible of conducting macromolecules hydrolysis  and syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis . Clostridiales is an order of obligate anaerobic bacteria with chemoorganotrophic fermentative metabolisms. Members of this group of microorganisms are frequent in soils, sediments, rumen and intestinal tracts of animals and insects. In addition, Clostridiaceae family members have in common their saccharolytic fermentative metabolism in environmental substrate decomposition .
Bacteroidetes were present at really low relative abundance (2–6% for all sludge samples). Bacteroidetes are anaerobic microorganisms which are included in animal intestinal microflora  due to its importance in cellulose and protein degradation . When digesting raw and thermally pretreated Chlorella, Bacteroidetes abundancy increased to 20% with regard to 12% registered in the inoculum used as seed of the reactor (adapted to digest sewage sludge, ). Likewise, Bacteroidetes also found the dominant phylum in the digestion of Scenedesmus obliquus at mesophilic range . Nevertheless, in the present study, their abundance remained low regardless of the inoculum source.
Chloroflexi phylum are aerobic bacteria commonly found in activated sludge systems , and therefore, this phylum usually ends up in the anaerobic digesters of wastewater treatment plants. Due to their filamentous morphology, their presence is normally associated to bulking phenomena. In this manner, no trend could be withdrawn in sludge samples collected from wastewater treatment and their relative abundance varied from 6% for S3 to 27% for S1. A striking feature was the fact that the thermophilic adapted sludge showed 1% of this phylum (Fig. 1a). This value was really low when compared to the rest of sludge samples. Nevertheless, this feature was in agreement with Greses et al.  who also reported extremely low relative abundance (1.1%) in a thermophilic CSTR fed with Scenedesmus. Authors attributed this fact to the low-ammonium tolerance of this phyla and the operational temperature. It should be also highlighted that S6 displayed 16% Thermotogae relative abundance (classified as other in Fig. 1a). The presence of this phylum was negligible in the rest of the sludge samples. Thermotogae have been described to release hydrolytic enzymes catalyzing the degradation of polysaccharides into acetate, carbon dioxide and hydrogen  and also to play a key role in interspecies hydrogen transfer . Thermotogae species are obligate anaerobes and hyperthermophiles bacteria found in large range extremophile environments, including mammalian, ruminant and termite digestive tracts. All those ecophysiology characteristics explain this abundance in S6 sludge .
There are no other studies published that focused in the comparison of the microbial population inocula adapted to digest microalgae and sewage sludge at different temperatures ranges. As an example, many other articles have characterized the microbial composition of inocula adapted to digest microalgae and activated sludge only at one temperature range [24, 25, 29, 34]. The novelty of this article is the comparison of different microbiome composition adapted to three different operational temperature ranges and feedstocks (microalgae and raw sewage). Overall, the anaerobic microbiome analysis of all sludge samples used in the present study evidenced different bacterial and archaeal population not only in terms of relative abundances but also in the phyla and genera identified. In this manner, it could be concluded that the different feedstocks fed in the digesters (sewage sludge and microalgae biomass) as well as the operational temperature had an impact on the inoculum sources.
Specific methanogenic activity of sludge samples toward model substrates: BSA and cellulose
Since hydrolysis is the first step on anaerobic digestion, it can be assumed that these data reflected a higher proteolytic activity of S1 with regard to the other tested sludge samples. Proteolytic bacteria occur in high numbers in anaerobic digesters receiving raw sewage sludge . This would support the fact that S1-SMA was the highest since the other sludge samples were adapted to digest mainly microalgae biomass. More interestingly, S1 was the sludge with lower Proteobacteria relative abundance (35% out of the total bacterial number vs. 46–51% for the adapted sludge, Fig. 1a). In this manner, it can be highlighted that despite the lower relative abundance, the enzymes secreted by the proteolytic bacteria of S1 were more suitable and/or active for BSA hydrolysis. The SMA using BSA as a substrate was diminished along digestion time. After approximately 7 days of digestion, the SMA attained for BSA was minimal (Fig. 3a) while the one corresponding to cellulose as model substrate slowly increased (Fig. 3b). Thus, it could be concluded that all sludge samples had a higher proteolytic activity than cellulolytic toward the model substrates. One or other prevalent hydrolytic activity strongly depend on the selected inoculum. In principle, protein hydrolysis is slower than the hydrolysis of carbohydrates . Nevertheless, hydrolysis constants and, therefore, methane production is highly dependent on several factors such as microbial community, selected model substrate and digestion temperature. In this manner, a wide range of hydrolytic constant might be found in the literature .
As observed for S1 and the adapted sludge samples (S4–S6), the SMA obtained when using cellulose model substrate was also clearly different (Fig. 3b). The only common behavior for all the sludge samples was the lack of methane production during the 2–3 first days of digestion. Similarly to what it was observed in the previous case, S1 exhibited three-fold higher SMA than the adapted sludge samples after 5 days of digestion (0.07 g COD consumed/g VS day for S1 vs ± 0.022 g COD consumed/g VS day for S4–S6). Once again, the higher biodiversity determined in S1 mediated higher SMA for cellulose than the rest of the sludge samples. While in the case of the proteolytic activity, SMA steadily decreased along digestion, in the case of cellulolytic activity, the sludge samples responded differently. Almost barely any activity was observed after 7 days of digestion for S1, while the adapted sludge samples maintained an SMA of 0.021–0.024 along 19 days of digestion regardless of the digestion temperature. This fact resulted in a similar methane yield but lower methane productivity of the adapted sludge samples.
Microalgae digestion using different anaerobic sludge inocula
This research demonstrated that despite the differences related to their anaerobic microbiome and SMA towards model substrates, the anaerobic digestion of microalgae biomass was not influenced by different inocula sources adapted to digest sewage sludge. Opposite to that, the sludge samples adapted to digest microalgae biomass exhibited better performances. Mesophilic sludge adapted to the digestion of microalgae consortium mainly composed by Scenedesmus showed greater methane yields than adapted to digest sewage sludge. The same could not be concluded with other microalgae biomass (Chlorella). Thus, it could be concluded that the anaerobic microbiome was tailored to degrade mainly Scenedesmus. With regard to the adapted inocula, psychrophilic digestion displayed lower methane productivity while methane yield was comparable to mesophilic digestion with adapted sludge. Most remarkably was the methane yield achieved by the thermophilic adapted sludge. Even though, a high microbial diversity might play a positive role in maintaining the stability of the system, the anaerobic microbiome of thermophilic digester presented a low diversity but highly efficient for the anaerobic digestion of Scenedesmus sp. The relative abundance of Firmicutes, particularly Clostridia, and Proteobacteria together with an important abundance of hydrogenotrophic methanogens was highlighted in this inoculum. Linking process engineering to microbial community in AD reactors could bring new insights to pay the way out to a better digester performance and avoid pretreatments by working with a highly specific anaerobic microbiome.
CGF designed, analyzed the data and wrote the manuscript. SBV performed the experiments. IG, MB, MF and AZ contributed to the writing and critical reviewing of the manuscript. All authors discussed the results and commented on the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
The authors agree to publish in the journal.
The authors wish to thank the Spanish Ministry of Economy and Competitiveness for the financial support provided through the grants ENE2013-45416-R and RYC-2014-16823. We would also like to acknowledge the Community of Madrid for the support offered in the framework of the project INSPIRA-1 (S2013/ABI-2783) and in addition to the WWTP of Valladolid (Spain) for kindly supplying the anaerobic sludge sample.
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