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Microalgae are a promising feedstock for biofuel and bioenergy production due to their high photosynthetic efficiencies, high growth rates and no need for external organic carbon supply. However, microalgal biomass cultivation for energy production purposes is still rare in commercial scale. Further research and development is needed to make microalgal derived energy sustainable and economically competitive. This work investigated cultivation of fresh water microalga Chlorella vulgaris and marine microalga Dunaliella tertiolecta and their utilization in production of hydrogen, methane, electricity, butanol and bio-oil after bulk harvesting the biomass. Growth of the two microalgae was studied in five different photobioreactor (PBR) configurations especially concentrating on the quantification and characterization of heterotrophic bacteria in non-axenic microalgal cultivations and microalgal utilization of different nitrogen sources. Anaerobic cultures used for the energy conversion processes were enriched from a mesophilic municipal sewage digester separately for production of H₂, CH₄ and electricity from the two microalgal species. After culture enrichment, energy conversion yields of microalgal biomass to the different energy carriers were compared. Anaerobic microbial consortia utilizing microalgal biomass were characterized based on the 16S rRNA gene sequence analysis. H₂ and CH₄ production potentials were tested in anaerobic serum bottles and electricity and butanol production in fed-batch operated two-chamber microbial fuel cells (MFCs).
All the PBR configurations tested were amenable to C. vulgaris and D. tertiolecta biomass production. Highest biomass concentrations and productivities by C. vulgaris and D. tertiolecta were 3.8 and 3.2 g L⁻¹, and 0.60 and 0.83 g L⁻¹ d⁻¹,, respectively. They were obtained in bubble column PBRs at 12% CO₂, 10 mM NO₃⁻ and 350 μmol photons m⁻² s⁻¹. Static mixers used in the flat plate PBRs did not generally enhance the growth of either C. vulgaris or D. tertiolecta at the low light intensities (50 μmol photons m⁻² s⁻¹) used. However, the low light intensity resulted in high growth rates at the early stages of growth. The highest specific growth rates were obtained in the flat plate PBRs and were 2.0 and 1.4 d⁻¹ for C. vulgaris and D. tertiolecta, respectively.
Bacterial growth occurred simultaneously with microalgal growth and correlated generally well with algal exuded dissolved organic carbon (DOC) concentrations. The bacterial communities were relatively stable and reproducible in both C. vulgaris and D. tertiolecta cultivations. However, algal associated bacterial communities were vastly different in C. vulgaris and D. tertiolecta cultures due to different growth medium salinity (<0.05% and 3%, respectively). C. vulgaris cultures were dominated with Alphaproteobacteria, especially Sphingomonas spp., which are typical in freshwater environments, whereas D. tertiolecta cultures were accompanied with halotolerant or halophilic bacteria belonging to classes Gammaproteobacteria, Flavobacteria and Alphaproteobacteria.
The choice of nitrogen source (ammonium, nitrate and urea) did not affect the biomass productivity or growth rate of D. tertiolecta at 1.3 mM N concentration. Nitrate and urea supported growth of C. vulgaris to a similar extent. The use of ammonium resulted in acidification and collapse of the culture due to algal and bacterial uptake of NH₄⁺, bacterial nitrification and a lower buffering capacity of fresh water medium used for C. vulgaris compared to the marine medium used for D. tertiolecta. Growth responses of C. vulgaris and D. tertiolecta to N limitation were also different. Total lipid content and heating value of C. vulgaris increased under N limitation, whereas no significant changes occurred in the lipid content of D. tertiolecta when conditions changed from sufficient N to N limitation. The energy embedded in the N source was low compared to power consumption of gas sparged photobioreactors but was still a significant penalty in the overall energy balance. The maximum net energy ratios were 1.85 and 2.16 for C. vulgaris and D. tertiolecta biomass production, respectively, when the energy consumption of gas sparging and N source was taken into account. However, there is a need to further optimize cultivation strategies to reduce the mixing as well as CO₂ and N consumption.
Low level of H₂ was produced from both microalgal feedstocks by the anaerobic sewage sludge enrichment cultures. H₂ was, however, subsequently consumed even in presence of 2-bromoethanesulfonic acid, a specific inhibitor of methanogenesis. Some H₂ accumulated from both C. vulgaris and D. tertiolecta biomass in cultures with no added anaerobic inoculum. This was attributed to anaerobic activities of bacteria associated with the harvested algal biomass slurries. H₂ production by these satellite heterotrophs was similar with both feedstocks; 0.52 mmol H₂ g-VS⁻¹ from D. tertiolecta and 0.45 mmol H₂ g-VS⁻¹ from C. vulgaris. Methanogenesis and butanol production by the anaerobic enrichments were higher from C. vulgaris (11.9 mmol CH₄ g-VS⁻¹and 0.56 mmol butanol g-VS⁻¹) than from D. tertiolecta (1.0 mmol CH₄ g-VS⁻¹ and 0.11 mmol butanol g-VS⁻¹). Electricity generation in MFCs was also higher from C. vulgaris (15 vs. 5.3 mW m⁻²), whilst power generation was more sustained from D. tertiolecta (13 vs. 9.8 J g-VS⁻¹). In addition, the highest lipid content was higher in C. vulgaris (28%) than in D. tertiolecta (19%) biomass. Methanogens were likely inhibited in D. tertiolecta cultures by high salinity of D. tertiolecta slurry. Salinity also resulted in formation of Ca- and Mg-precipitates on the cathodes of the D. tertiolecta-fed MFCs, revealing a mechanism which may impair electricity generation from salt water algae during long-term MFC operation.
Microbial community analysis of the anaerobic serum bottle and MFC cultures indicated that the anaerobic decomposition of microalgal biomass supported a high diversity of bacteria. Microbial communities enriched separately for the different energy conversion processes and for the two microalgal biomass feedstocks were different. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiling demonstrated the presence of H₂ producing bacteria (e.g. Clostridium spp., Hafnia alvei) among the satellite bacteria of both microalgal biomass samples and both H₂ producing (e.g. Petrimonas spp., Syntrophobacter spp) and H₂ consuming bacteria (e.g. Bilophila wadsworthia, Wolinella succinogenes) in the anaerobic serum bottle enrichments. MFC cultures contained bacteria previously reported in bioeletrochemical systems or with a demonstrated ability to transfer electrons to anode electrode. These included W. succinogenes, Bacteroides and Synergistes spp. in C. vulgaris enriched MFC cultures and Geovibrio thiophilus, W. succinogenes, Bacteroides, Synergistes, and Desulfomicrobium spp. in D. tertiolecta enriched MFC cultures.
The highest calorific yield of methanogenic conversion of microalgal biomass to energy was 10.3 kJ g-VS⁻¹. The highest H₂, simultaneous electricity and butanol as well as lipid production yields were 0.14, 1.4 and 4.8 kJ g-VS⁻¹ from the similarly grown microalgal biomass. Calorific yield of lipids increased up to 10.4 kJ g-VS⁻¹ by growing the microalgal biomass under conditions favoring lipid production. The effect of higher lipid content of biomass on H₂, CH₄, electricity or butanol production was not studied. Maximum areal energy productions obtained as H₂ and CH₄ were significantly higher from microalgal biomass (3.15 and 246 GJ ha⁻¹ y⁻¹) than from the reference lignocellulosic plant material, non-pretreated reed canary grass (0.38 and 51.0 GJ ha⁻¹ y⁻¹).
In summary, this study demonstrated that both C. vulgaris and D. tertiolecta can be used for production of H₂, CH₄, electricity, butanol and lipids. Based on this study C. vulgaris is more suitable for bioenergy production than D. tertiolecta. Depending on cellular lipid content, lipid utilization for bio-oil production and anaerobic digestion were the most potent means of converting C. vulgaris biomass to energy. The study also revealed diverse microbial communities in non-axenic microalgal photobioreactor cultures and in anaerobic consortia converting microalgal biomass to energy carriers. |