Evaluation of microalgae production coupled with wastewater treatment

<p>In the present study, the feasibility of microalgae production coupled with wastewater treatment was assessed. Continuous cultivation of <i>Chlorella sorokiniana</i> with wastewater was tested in lab-scale flat-panel photobioreactors. Nitrogen and phosphorus removals were found to be inversely proportional to the four dilution rates, while chemical oxygen demand removal was found to be 50% at all the tested conditions. The biomass obtained at the highest dilution rate was characterized for its content of lipids, proteins and pigments. The average yields of fatty acid methyl esters (FAMEs), protein, lutein, chlorophylls and β-carotene was 62.4, 388.2, 1.03, 11.82 and 0.44 mg per gram dry biomass, respectively. Economic analysis revealed that potentially more than 70% of revenue was from the production of pigments, that is, chlorophyllin (59.6%), lutein (8.9%) and β-carotene (5.0%) while reduction in discharging costs of the treated wastewaters could account for 19.6% of the revenue. Due to the low market price of biodiesel, the revenue from the above was found to be the least profitable (1.4%). Even when combining all these different revenues, this cultivation strategy was found with the current prices to be uneconomical. Power consumption for artificial light was responsible for the 94.5% of the production costs.</p

A novel bioinformatic strategy to characterise microbial communities in biogas reactors

none5Sequences encoding ribosomal Bacterial and Archeal genes are very similar among species of the same genus, in fact, in some cases, similarity is 99% or higher among the 1500 bp that compose the 16S rDNA. For this reason today it is still a challenge to gain the species level characterisation using 16S hypervariable regions, especially when working with the not high quality very short reads characteristics of next generation sequencers (Mande S.S. et al., 2012). Previous works analysed the microbial community composition in biogas reactors via 16S rDNA sequencing (Luo, G. et al., 2013; Werner, J.J. et al., 2011). For this reason we developed a bioinformatics strategy in order to create a tool to review the generated dataset and to obtain a more strict control on the bacterial composition at the species level, with estimation of its reliability. The program perform local similarity search and evaluate the results with high stringency (95 up to 100%) and returns all the possible candidate species with unique or multiple matches for each genus. In the process of species identification, different categories of reliability can be generated: certain can lead to univocal species identification even in the same genus, while others give multiple matches with the same probability. The software was used to analyse samples taken during the digestion process in three independent biogas reactors continuously fed with raw cattle manure. Among the most represented (>1%) considering the relative abundance of the community Clostridium resulted to be the most complex genus to elucidate. Some species in this genus, Clostridium ultunense and Clostridium irregular, have been assigned with high probability (100% and 99.7% of unique matches) while other 11 have only few unique matches (0.1 to 10%). Bacteroides, Acetobacterium and Pseudomonas genera had difficulties in the assignment, gathering medium-low probability as well (1 to 50%). On the contrary several other genera were assigned with high probability and no multiple matches. Some of them including only one species uniquely identified, some other including more than one (i.e. Dialister succinatiphilus with 37% and Dialister propionicifaciens with 63%, Tissierella praeacuta with 26% and Tissierella creatinophila with 74%, Proteiniphilum acetatigenes with 100%, Halothermothrix orenii with 100%, Thermoflavimicrobium dichotomicum with 100%). Furthermore comparative analyses with MG-RAST (Meyer F. et al., 2008) results have been performed to test our strategy. We also found that our method can be used to understand which hypervariable region of 16S rDNA is more efficient in the identification at the species level in different genera. Our conclusion is that the identification at the species level remains a challenge of major interest but it can be done reliably for specific genera. In fact we uniquely identified the species of up to 67% of the most abundant genera and we obtained a less reliable identification for the remaining 33%. References 1. Mande, S.S., Mohammed, M.H. and Ghosh, T.S. (2012) Classification of metagenomic sequences: methods and challenges. Briefings in bioinformatics. 13(6):669-81. 2. Luo, G., Wang, W. and Angelidaki, I. (2013). Anaerobic digestion for simultaneous sewage sludge treatment and CO biomethanation: process performance and microbial ecology. Environmental Science & Technology. 47(18):10685-93 3. Werner, J.J., Knights, D., Garcia, M.L., Scalfone, N.B., Smith, S., Yarasheski, K., Cummingse, T.A., Beerse, A.R., Knightf, R. and Angenent, L.T. (2011) Bacterial community structures are P37 unique and resilient in full-scale bioenergy systems. Proceedings of the National Academy of Sciences. 108(10), 4158-4163. 4. Meyer, F., Paarmann, D., D'Souza, M., Olson, R., Glass, E.M., Kubal, M., Paczian, T., Rodriguez, A., Stevens, R., Wilke, A., Wilkening, J. and Edwards, R.A. (2008) The metagenomics RAST server - a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics. 19 (9), 386.Conference topic: Advanced methods for microbiological analysis (POSTER PRESENTATION)noneL. Treu; S. Campanaro; D. De Francisci; P.G. Kougias; I. AngelidakiTreu, Laura; Campanaro, Stefano; D., De Francisci; P. G., Kougias; I., Angelidak

Comparative analysis of the microbial diversity in liquid and foaming layer in biogas reactors

Foaming incidents have been recorded in many biogas plants causing severe operational, economical and environmental problems (Kougias et al., 2014). However, the foaming phenomenon in biogas reactors fed with agro-industrial wastes has not been extensively investigated, especially with respect to the microbial composition of the digesters (Moeller et al., 2012). In the cited literature, it has been reported that specific microorganisms, which are mainly filamentous (e.g. Gordonia species, Microthrix parvicella), are attached to biogas bubbles and transferred to the air/liquid interface of sludge reactors or wastewater treatment works (Ganidi et al., 2009). Once these microorganisms accumulate on the liquid surface, they initiate biosurfactants production due to their metabolic activity, leading to the decrease of the surface tension and thus generate foaming. The aim of the present study was to investigate the microbial diversity in the liquid versus the foaming layer in manure-based biogas reactors suffering by foaming incidents in order to elucidate potential role and contribution of the microorganisms in foam promotion. The experimental work was carried out in three thermophilic continuous stirred tank reactors (CSTR) fed with manure and supplemental amounts of lipids, proteins and carbohydrates. Once foaming was formed in the reactors, samples from the liquid and foaming layer were obtained and screened using 16S rDNA sequencing. The results of these analyses revealed that there are indeed some species that significantly vary their relative abundance in the foaming layer compared to the liquid one (e.g. Methanoculleus sp., Dialister sp.). However, based on the cited literature and to the best of our knowledge there was not a direct correlation of these species with foaming. Further investigation is needed in order to define the properties of these species on foam generation. Finally, it was observed that particles of barley plant (that was contained in the raw manure as ingredient of animal nutrition) were accumulated in the foaming layer. It has been previously documented that barley contributes in stabilization of beer foam due to the activity of one of its proteins (Brey et al., 2003). For that reason, it could be hypothesised that the existence of barley particles in our reactors could contribute in foaming although their presence was also prior to the foaming incidents; their effect on foam formation or stabilization might be enhanced in correlation with other parameters (i.e. presence of specific microorganisms). References 1. Brey, S. E., de Costa, S., Rogers, P. J., Bryce, J. H., Morris, P. C., Mitchell, W. J.,Stewart, G. G., 2003. The Effect of Proteinase A on Foam-Active Polypeptides During High and Low Gravity Fermentation. Journal of the Institute of Brewing. 109, 194-202. 2. Ganidi, N., Tyrrel, S.,Cartmell, E., 2009. Anaerobic digestion foaming causes – A review. Bioresource Technology. 100, 5546-5554. 3. Kougias, P. G., Boe, K., Tsapekos, P.,Angelidaki, I., 2014. Foam suppression in overloaded manure-based biogas reactors using antifoaming agents. Bioresource Technology. 153, 198-205. 4. Moeller, L., Goersch, K., Neuhaus, J., Zehnsdorf, A.,Mueller, R., 2012. Comparative review of foam formation in biogas plants and ruminant bloat. Energ Sustain Soc. 2, 1-9

Comparative microbial analysis before and after foaming incidents in biogas reactors

Foaming is one of the major problems that occasionally occurring in biogas plants affecting negatively the overall anaerobic digestion (AD) process. According to a recent survey, 15 out of 16 full-scale biogas plants, which were surveyed in Denmark, faced foaming incidents in the main reactor and/or in the pre-storage feeding tank, resulting in 30-50% biogas production loss (Kougias et al., 2014). In activated sludge systems and in wastewater treatment plants the major causes of foaming are organic overload, the presence of surface active agents, operational parameters (e.g. digester’s shape, mixing system etc) and filamentous microorganisms (e.g. Gordonia species, Microthrix parvicella) (Ganidi et al., 2009). However, the contribution of specific microorganisms on foam generation in biogas reactors fed with agro-industrial wastes has not been previously investigated. The aim of the present study was to elucidate the microbiology of biogas reactors fed with different substrates prior and after foaming incidents. The experiment was carried out in three continuous stirred tank reactors (CSTR) denoted as R1, R2 and R3. The total and the working volume of each reactor was 2 and 1.5 L, respectively. Each reactor was continuously stirred using a magnetic stirrer. The operating temperature was maintained at 54 ± 1 °C using thermal jackets. Each reactor was fed with a different mixed substrate, which was found to have an influence on foam formation in our previous study (Kougias et al., 2013). The hydraulic retention time (HRT) of all reactors was kept constant at 15 days. The whole experiment was divided into two periods. During the first period, the reactors were fed only with cattle manure. Once steady state conditions were reached, liquid sample from all reactors was obtained for DNA extraction and metagenomic analysis. After sampling, the feedstock composition of each reactor was changed by the addition of gelatine or Na-Oleate or glucose (second experimental period). As a consequence, foam formation was observed in all reactors approximately after one HRT period. Once the daily volume of the formed foam was steady, samples were taken again for DNA extraction and metagenomic analysis. Results from the present study revealed significant variations in the microbiology of the manure-based biogas reactors after foam initiation. A number of genera could be linked to foaming as they produce biosurfactants (Lactobacillus, Bacillus, Pseudomonas, Thermotoga), others contain mycolic acid in their cell wall (Thermoactinomyces, Pseudonocardia) or decrease the surface tension of the media (Micrococcus, Streptococcus). Frankia, Dialister and Paenibacillus are known to be correlated to this phenomenon but their mechanism is still unclear. Finally, microorganisms that have a widely known association with foaming were identified when the identification threshold for the microorganisms was decreased to similar levels reported in the cited literature; however, the latter, due to its high importance, needs to be further investigated. References 1. Ganidi, N., Tyrrel, S.,Cartmell, E., 2009. Anaerobic digestion foaming causes – A review. Bioresource Technology. 100, 5546-5554. 2. Kougias, P., Boe, K., O-Thong, S., Kristensen, L.,Angelidaki, I., 2014. Anaerobic digestion foaming in full-scale biogas plants: a survey on causes and solutions. Water Science and Technology. 69, 889-895. 3. Kougias, P. G., Boe, K.,Angelidaki, I., 2013. Effect of organic loading rate and feedstock composition on foaming in manure-based biogas reactors. Bioresource Technology. 144, 1-7