Impact of organic acids supplementation to HSSL as substrate for selection of
6.2. Material and Methods
6.3.4. Microbial Community Characterization
The goal of the present study was also to establish the link between microbial population relative abundance and the performance of PHA-producing capacity of the selected MMC. The differences should be a function of the operational conditions applied and the type of substrate used. Several works that address the microbial characterization of their enrichments revealed diverse communities, due to both parameters (Queirós et al., 2015). Such characterization can contribute towards the development of process operational strategies designed to optimize the structure of the microbial community (Carvalho et al., 2014).
FISH analysis was performed to original sludge and samples collected from the SBR on days 17, 32, 45, 51, 60 and 66 of operation. Initially, specific probes for the main phyla within Bacteria domain were applied. The microbial community was mostly composed of
Bacteria since no Archaea were detected. Several FISH probes were used taking into consideration bacteria previously reported as PHA-accumulating organisms.
To identify the microbial community and to follow the MMC composition throughout the reactor operation, specific probes for the main groups within Bacteria domain were applied: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Chloroflexi, Flavobacteria, Bacteroidetes, Sphingobacteria, Actinobacteria, Firmicutes, Planctomycetales. Fig. 6.5 shows the evolution of the composition during the reactor operation. Since the beginning of the process, Betaproteobacteria were the dominant group and their relative abundance only suffered mild fluctuations during the whole reactor operational time. Since Betaproteobacteria were reported as PHA-producers, this result showed that the operational conditions applied to the SBR, favored the maintenance of this bacterial group within the MMC.
Alphaproteobacteria were the second most dominant group in the bacterial community throughout the SBR operational time, contrary to what was described so far with HSSL (Queirós et al., 2014). This proves that a small change, the introduction of different SCOA profile, led to a different community enrichment with good PHA accumulation capacity.
Using solely HSSL as substrate at high OLR and shorter cycle lengths, 17 gCOD L−1 d−1 and 8 h, respectively, a different community was selected (chapter 5). Dominated by Alphaproteobacteria, the PHA production was low despite the presence of a considerable number of PHA-storing microorganisms. A second preferred reserve polymer was accumulated, a glucose polymer (chapter 5).
As previously mentioned, the operational conditions were changed from 24 h to 12 h cycle on day 44. The MMC took 10 days to stabilize and reach a pseudo-stationary phase. By analyzing the bacterial composition during 12 h cycles, it is possible to verify that the bacterial community did not suffer significant changes during this period, at the class level. Betaproteobacteria remained the dominant group, and their relative abundance was constant during this period (42.99 ± 0.38% on day 60 and 40.76 ± 2.15%
on day 66). Results obtained in this study are in line with previous studies that identified organisms belonging to Alphaproteobacteria, Betaproteobacteria or
Gammaproteobacteria classes as PHA-accumulating organisms selected under ADF (Albuquerque et al., 2013; Ferreira et al., 2015; Jiang et al., 2012; Moita et al., 2014).
Fig. 6.5. Bacterial community evolution throughout the SBR operation.
Regarding the Alphaproteobacteria class, specific probes for Amaricoccus, Sphingomonas, Defluvicoccus and Defluvicoccus related Tetrad-Forming Organism (TFO-DF), all PHA-accumulating organisms, were applied. Positive results were only obtained for TFO-DF (0.78 ± 0.38%), in far less extent than in others enrichments with HSSL (Queirós et al., 2014).
For the Betaproteobacteria group, specific probes for Thauera and Azoarcus genera were applied due to their previous identification as PHA-accumulating organisms (Queirós et al., 2015). Positive results were obtained for both genera, but only small amounts of Thauera (0.72 ± 0.25%) and Azoarcus (0.62 ± 0.02%) were found. After applying the available specific probes for genera belonging to Betaproteobacteria, the main part of this population remained unidentified. Nevertheless, almost whole biomass could store PHA as shown by Nile Blue staining (data not shown).
To identify the main bacteria responsible for PHA accumulation in the selected MMC, a 16S rRNA gene clone library was constructed. The sample used to extract the DNA was collected from the SBR on the 66th day of operation. 60 clones were chosen for
15,45 13,28 15,42 12,14 12,73 12,68
Alphaproteobacteria Betaproteobacteria Deltaproteobacteria Gammaproteobacteria Actinobacteria Chloroflexi Bacteroidetes
partial sequencing. The obtained 16S rDNA partial sequences were then analyzed by BLAST and grouped by genera. After analyzing the results of the partial sequencing, 15 clones were chosen for complete sequencing and refinement of their taxonomic affiliation. The major part of the clones presents in the clone library belonged to Betaproteobacteria class. These results were expected since, during the pseudo-stationary phase, Betaproteobacteria was the dominant group of the bacterial community.
The representatives of the fifteen most abundant clones are shown in Table 6.2 and their phylogenetic relations are presented in Fig. 6.7. Almost all of them are known PHA producers previously found in MMC selection processes for PHA production (Queirós et al., 2015).
During the analysis of 16S rRNA gene sequences, six of the 15 sequences were related to Acidovorax. Moreover, Acidovorax was previously shown to be capable of PHA storage (Schulze et al., 1999). Positive results for Acidovorax were obtained for all the screened samples and its content in the microbial community increased from 2.62 ± 0.93% (in day 17) to 28.98 ± 2.51% (in day 66). During the pseudo-stationary phase, the Acidovorax genus became the major constituent of Betaproteobacteria, composing 71%
of this class population on day 66 (Fig. 6.7).
Fig. 6.6. Phylogenetic tree based on full-length nucleotide sequences of 16S rRNA gene of clones CR1-15 (in the tree preceded by a filled red dot). Molecular Phylogenetic analysis by Maximum Likelihood method. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis
involved 42 nucleotide sequences. Evolutionary analyses were conducted in MEGA6.
Table 6.2. Results of the complete sequencing.
Sequence name Highest Similarity % Identity Accession number
CR1 Acidovorax caeni 94% KT262954
CR2 Acidovorax wautersii 98% KT262955
CR3 Fluviicola taffensis 92% KT262951
CR4 Leadbetterella byssophila 95% KT262956
CR5 Comamonas testosteroni 90% KT262952
CR6 Paracoccus siganidrum 98% KT262957
CR7 Agrobacterium tumefaciens 99% KT262958
CR8 Acidovorax radicis 98% KT262959
CR9 Simplicispira metamorpha 98% KT262960
CR10 Comamonas testosteroni 99% KT262961
CR11 Alcaligenes aquatilis 94% KT262962
CR12 Shinella zoogloeoides 99% KT262953
CR13 Pseudoxanthomonas kaohsiungensis 99% KT262963
CR14 Acidovorax delafieldii 98% KT262964
CR15 Simplicispira metamorpha 97% KT262965
Fig. 6.7. a) Acidovorax content evolution throughout the reactor operation. Percentages are about the relative abundance of Betaproteobacteria. b) FISH picture of Acidovorax on day 66. Green cells are
hybridized with EUBmix and the yellow cells also hybridized with ACI145 probe.
Clones related to Comamonas spp. and Novosphingobium spp. were also detected. Ferreira et al. (2016) isolated and characterized organisms able to store PHA, from an MMC selected under the same conditions as the ones of this study, same OLR and 12 h cycle length, but using non SCOA-supplemented HSSL. Without this supplementation, the culture was dominated by microorganisms belonging to
Alphaproteobacteria. This observation suggested that the simple addition of known SCOA precursors of PHA lead to marked differences in the microbial community composition.
Several microorganisms present in the clone library were already described as PHA-accumulating organisms in pure culture: Comamonas acidovorans using 1,4- butanediol and glucose (Lee et al., 2004), Paracoccus denitrificans consuming ethanol and n-pentanol (Chanprateep et al., 2001), Cupriavidus necator using soya wastes from a soya milk dairy (Doi et al., 1990) and Alcaligenes latus using malt wastes from a beer brewery plant as substrate (Yu et al., 1999).
There were also sequences related to Fluviicola taffensis, Leadbetterella byssophila, Paracoccus siganidrum, Agrobacterium tumefaciens, Alcaligenes aquatilis, Shinella zoogloeoides and Pseudoxanthomonas kaohsiungensis. Other clones had phylogenetic relationships with Lampropedia hyalina, Azoarcus sp., Thauera sp., Bacillus megaterium, Plasticicumulans acidivorans, Thiocystis violacea and Zoogloea sp..
6.4. Conclusion
The supplementation of HSSL with different SCOA reduced greatly the adaptation period of the culture and allowed to produce a copolymer of P(3HB-co-3HV) without GB accumulation.
A pseudo-stationary state was reached after 34 days of SBR operation, which indicated that the MMC could successfully adapt to the different conditions imposed. A stable PHA accumulating MMC was obtained with a low organic loading rate and with no ammonia limiting conditions, thus providing the culture with the selective pressure for PHA accumulation. OLR and cycle length were also shown to have affected the kinetics of substrate consumption and the type of polymer stored. Substrate uptake rates and polymer production rates were found to increase with increasing substrate concentrations in the selection reactor, without the consumption of any HSSL’s sugars.
For the accumulation step, the presence of nitrogen showed to negatively affect the PHA production in the accumulation step. Under ammonia limitation the culture reached 34.6% P(3HB-co-3HV) cdw.
FISH analysis revealed a dominant Betaproteobacteria class, around 41% since the beginning of the reactor operation. Acidovorax, a PHA producer, was the dominant genus, overcoming the remaining population when the OLR and cycle length were changed.
Furthermore, clone library results presented several bacteria that were already described as PHA-storing microorganisms.
Introducing a pre-fermentation step of HSSL would be the next step to be tested in the selection of PHA-producing MMC. By testing different operational conditions during the acidogenic fermentation, different mixtures of SCOA would be obtained and the impact of feeding these mixtures on the selection step should be evaluated.
6.5. References
Albuquerque, M.G.E., Carvalho, G., Kragelund, C., Silva, A.F., Barreto Crespo, M.T., Reis, M.A.M., Nielsen, P.H., 2013. Link between microbial composition and carbon substrate-uptake preferences in a PHA-storing community. ISME J. 7, 1–12.
doi:10.1038/ismej.2012.74
Albuquerque, M.G.E., Eiroa, M., Torres, C., Nunes, B.R., Reis, M.A.M., 2007. Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses. J. Biotechnol. 130, 411–21.
doi:10.1016/j.jbiotec.2007.05.011
Albuquerque, M.G.E., Martino, V., Pollet, E., Avérous, L., Reis, M.A.M., 2011. Mixed culture polyhydroxyalkanoate (PHA) production from volatile fatty acid (VFA)-rich streams: Effect of substrate composition and feeding regime on PHA productivity, composition and properties. J. Biotechnol. 151, 66–76.
doi:10.1016/j.jbiotec.2010.10.070
Albuquerque, M.G.E., Torres, C.A. V., Reis, M.A.M., 2010. Polyhydroxyalkanoate (PHA) production by a mixed microbial culture using sugar molasses: effect of the influent substrate concentration on culture selection. Water Res. 44, 3419–33.
doi:10.1016/j.watres.2010.03.021
Alexandri, M., Papapostolou, H., Vlysidis, A., Gardeli, C., Papanikolaou, S., Koutinas, A.A., Nutrition, H., 2016. Extraction of phenolic compounds and succinic acid production
from spent sulphite liquor. J. Chem. Technol. Biotechnol.
Amann, R.I., Ludwig, W., Schleifer, K.H., 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation . Phylogenetic Identification and In Situ Detection of Individual Microbial Cells without Cultivation.
Microbiol. Rev. 59, 143–169.
Bengtsson, S., Werker, A., Christensson, M., Welander, T., 2008. Production of polyhydroxyalkanoates by activated sludge treating a paper mill wastewater.
Bioresour. Technol. 99, 509–16. doi:10.1016/j.biortech.2007.01.020
Carta, F., Beun, J.J., Van Loosdrecht, M.C.M., Heijnen, J.J., 2001. Simultaneous storage and degradation of PHB and glycogen in activated sludge cultures. Water Res. 35, 2693–
2701. doi:10.1016/S0043-1354(00)00563-7
Carvalho, G., Oehmen, A., Albuquerque, M.G.E., Reis, M.A.M., 2014. The relationship between mixed microbial culture composition and PHA production performance from fermented molasses. N. Biotechnol. 31, 257–263.
doi:10.1016/j.nbt.2013.08.010
Cavaillé, L., Grousseau, E., Pocquet, M., Lepeuple, A.-S., Uribelarrea, J.-L., Hernandez-Raquet, G., Paul, E., 2013. Polyhydroxybutyrate production by direct use of waste activated sludge in phosphorus-limited fed-batch culture. Bioresour. Technol. 149, 301–9. doi:10.1016/j.biortech.2013.09.044
CEPI, 2016. Preliminary Statistics 2015 Paper and board production [WWW Document].
URL http://www.cepi.org/taxonomy/term/26
Chanprateep, S., Abe, N., Shimizu, H., Yamane, T., Shioya, S., 2001. Multivariable control of alcohol concentrations in the production of polyhydroxyalkanoates (PHAs) by Paracoccus denitrificans. Biotechnol. Bioeng. 74, 116–124. doi:10.1002/bit.1101 Chua, A.S.M., Takabatake, H., Satoh, H., Mino, T., 2003. Production of
polyhydroxyalkanoates (PHA) by activated sludge treating municipal wastewater:
Effect of pH, sludge retention time (SRT), and acetate concentration in influent.
Water Res. 37, 3602–3611. doi:10.1016/S0043-1354(03)00252-5
Clesceri, L.S., Greenberg, A.E., Eaton, A.D., 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association.
Dionisi, D., Carucci, G., Petrangeli Papini, M., Riccardi, C., Majone, M., Carrasco, F., 2005.
Olive oil mill effluents as a feedstock for production of biodegradable polymers.
Water Res. 39, 2076–84. doi:10.1016/j.watres.2005.03.011
Dionisi, D., Majone, M., Papa, V., Beccari, M., 2004. Biodegradable polymers from organic acids by using activated sludge enriched by aerobic periodic feeding. Biotechnol.
Bioeng. 85, 569–79. doi:10.1002/bit.10910
Dionisi, D., Majone, M., Vallini, G., Gregorio, S. Di, Beccari, M., 2007. Effect of the Length of the Cycle on Biodegradable Polymer Production and Microbial Community Selection in a Sequencing Batch Reactor. Biotechnol. Prog. 23, 1064–1073.
doi:10.1021/bp060370c
Doi, Y., Segawa, A., Kunioka, M., 1990. Biosynthesis and characterization of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in Alcaligenes eutrophus. Int. J. Biol.
Macromol. 12, 106–111.
Fava, F., Totaro, G., Diels, L., Reis, M., Duarte, J., Carioca, O.B., Poggi-Varaldo, H.M., Ferreira, B.S., 2015. Biowaste biorefinery in Europe: opportunities and research &
development needs. N. Biotechnol. 32. doi:10.1016/j.nbt.2013.11.003
Ferreira, A.M., Queirós, D., Gagliano, M.C., Serafim, L.S., Rossetti, S., 2015.
Polyhydroxyalkanoates-accumulating bacteria isolated from activated sludge acclimatized to hardwood sulphite spent liquor. Ann. Microbiol. 1–10.
Jiang, Y., Marang, L., Tamis, J., van Loosdrecht, M.C.M., Dijkman, H., Kleerebezem, R., 2012. Waste to resource: Converting paper mill wastewater to bioplastic. Water Res.
46, 5517–30. doi:10.1016/j.watres.2012.07.028
Koutinas, A. a, Vlysidis, A., Pleissner, D., Kopsahelis, N., Lopez Garcia, I., Kookos, I.K., Papanikolaou, S., Kwan, T.H., Lin, C.S.K., 2014. Valorization of industrial waste and by-product streams via fermentation for the production of chemicals and biopolymers. Chem. Soc. Rev. 43, 2587–627. doi:10.1039/c3cs60293a
Laycock, B., Halley, P., Pratt, S., Werker, A., Lant, P., 2013. The chemomechanical properties of microbial polyhydroxyalkanoates. Prog. Polym. Sci. 38, 536–583.
doi:10.1016/j.progpolymsci.2012.06.003
Lee, W.H., Azizan, M.N.M., Sudesh, K., 2004. Effects of culture conditions on the
composition of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) synthesized by Comamonas acidovorans. Polym. Degrad. Stab. 84, 129–134.
doi:10.1016/j.polymdegradstab.2003.10.003
Lemos, P., Serafim, L., Reis, M., 2006. Synthesis of polyhydroxyalkanoates from different short-chain fatty acids by mixed cultures submitted to aerobic dynamic feeding. J.
Biotechnol. 122, 226–238.
Lemos, P.C., Levantesi, C., Serafim, L.S., Rossetti, S., Reis, M.A.M., Tandoi, V., 2008.
Microbial characterisation of polyhydroxyalkanoates storing populations selected under different operating conditions using a cell-sorting RT-PCR approach. Appl.
Microbiol. Biotechnol. 78, 351–60. doi:10.1007/s00253-007-1301-5
Marang, L., Jiang, Y., van Loosdrecht, M.C.M., Kleerebezem, R., 2014. Impact of non-storing biomass on PHA production: An enrichment culture on acetate and methanol. Int. J. Biol. Macromol. 71, 74–80. doi:10.1016/j.ijbiomac.2014.04.051 Moita, R., Freches, A., Lemos, P.C., 2014. Crude glycerol as feedstock for
polyhydroxyalkanoates production by mixed microbial cultures. Water Res. 58, 9–20.
doi:10.1016/j.watres.2014.03.066
Norgren, M., Edlund, H., 2014. Current Opinion in Colloid & Interface Science Lignin : Recent advances and emerging applications. Curr. Opin. Colloid Interface Sci. 19, 409–416. doi:10.1016/j.cocis.2014.08.004
Ostle, A.G., Holt, J.G., 1982. Fluorescent Stain for Poly-3- Hydroxybutyrate. Appl. Environ.
Microbiol. 44, 238–241.
Pereira, S.R., Ivanuša, S., Evtuguin, D. V, Serafim, L.S., Xavier, A.M.R.B., 2012. Biological treatment of eucalypt spent sulphite liquors: a way to boost the production of second generation bioethanol. Bioresour. Technol. 103, 131–5.
doi:10.1016/j.biortech.2011.09.095
Philip, S., Keshavarz, T., Roy, I., 2007. Polyhydroxyalkanoates: biodegradable polymers with a range of applications. J. Chem. Technol. Biotechnol. 82, :233–247.
Queirós, D., Fonseca, A., Lemos, P.C., Serafim, L.S., 2016. Long-term operation of a two-stage polyhydroxyalkanoates production process from hardwood sulphite spent liquor. J. Chem. Technol. Biotechnol. 91, 2480–2487. doi:10.1002/jctb.4841