Paulo C. Lemos

Strategies for PHA production by mixed cultures and renewable waste materials.

Production of polyhydroxyalkanoates (PHA) by mixed cultures has been widely studied in the last decade. Storage of PHA by mixed microbial cultures occurs under transient conditions of carbon or oxygen availability, known respectively as aerobic dynamic feeding and anaerobic/aerobic process. In these processes, PHA-accumulating organisms, which are quite diverse in terms of phenotype, are selected by the dynamic operating conditions imposed to the reactor. The stability of these processes during long-time operation and the similarity of the polymer physical/chemical properties to the one produced by pure cultures were demonstrated. This process could be implemented at industrial scale, providing that some technological aspects are solved. This review summarizes the relevant research carried out with mixed cultures for PHA production, with main focus on the use of wastes or industrial surplus as feedstocks. Basic concepts, regarding the metabolism and microbiology, and technological approaches, with emphasis on the kind of feedstock and reactor operating conditions for culture selection and PHA accumulation, are described. Challenges for the process optimization are also discussed.

Model for carbon metabolism in biological phosphorus removal processes based on in vivo13C-NMR labelling experiments

In vivo13C-NMR, 31P-NMR techniques were applied to study phosphorus and carbon metabolism in activated sludge during both the anaerobic and the aerobic stages. By supplying a 13C label on the methyl group of acetate at the beginning of the anaerobic stage, the fate of the label through the subsequent aerobic/anaerobic stages was traced in vivo. It was possible to follow the flux of label from acetate to hydroxybutyrate/hydroxyvalerate co-polymer in the first anaerobic stage, then to monitor the conversion of these units into glycogen in a subsequent aerobic stage, and afterwards, by submitting the same sludge to a second anaerobic stage, to observe the flux of labelled carbon from glycogen to the hydroxyvalerate and hydroxybutyrate units. The uptake/release of inorganic phosphate and the extracellular pH were monitored by 31P-NMR in the same experiments. The data provide an unequivocal demonstration of the involvement of glycogen in the biological phosphorus removal process. On the basis of these 13C labelling data, a biochemical model for the synthesis of polyhydroxyalkanoates from acetate and glycogen was elaborated in which the tricarboxylic acid cycle is proposed as an additional source of reduction equivalents. According to this study, from 1 C-mol acetate, 1.48 C-mol P(HBHV) are synthesized and 0.70 C-mol glycogen are degraded anaerobically, while 0.16 P-mol phosphate is released. In the aerobic stage, 1 C-mol of P(HBHV) is converted to 0.44 C-mol glycogen.

Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms.

Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus (P) removal. Considerable effort has been devoted to understanding the biochemical nature of enhanced biological phosphorus removal (EBPR) and it has been shown that intracellular polymer storage plays an important role in PAOs metabolism. The storage capacity of PAOs gives them a competitive advantage over other microorganisms present that are not able to accumulate internal reserves. Intracellular polymers stored by PAOs include polyphosphate (poly-P), polyhydroxyalkanoates (PHAs) and glycogen. Staining procedures for qualitative visualization of polymers by optical microscopy and combinations of these procedures with molecular tools for in situ identification are described here. The strengths and weaknesses of widely used polymer quantification methods that require destruction of samples, are also discussed. Finally, the potential of in vivo nuclear magnetic resonance (NMR) spectroscopy for on-line measurement of intracellular reserves is reported.

Molecular weight and thermal properties of polyhydroxyalkanoates produced from fermented sugar molasses by open mixed cultures

Polyhydroxyalkanoates (PHAs) produced from fermented molasses and synthetic feeds containing single volatile fatty acids (VFAs) by an open mixed culture enriched in glycogen accumulating organisms (GAOs) were characterized with regards to molecular weight and thermal properties. The polymer contained five types of monomers, namely 3-hydroxybutyrate, 3-hydroxy-2-methylbutyrate, 3-hydroxyvalerate, 3-hydroxy-2-methylvalerate and 3-hydroxyhexanoate in different ratios depending on the VFA composition of the substrate. Polymers produced from fermented molasses had weight average molecular weights (M(w)) in the range (3.5-4.3)x10(5)g/mol and polydispersity indexes (PDI) of 1.8-2.1 while polymers produced from synthetic VFAs had M(w) of (4.5-9.0)x10(5)g/mol and PDI of 1.7-3.9. Thermal properties such as glass transition temperature (-14 degrees C to 4.8 degrees C), melting temperature (89-174 degrees C) and melting enthalpy (0-82.1J/g) were controlled in broad ranges by the monomer composition. The decomposition temperatures of the polymers produced were between 277.2 degrees C and 294.9 degrees C, and independent of monomer composition and molecular weight.

Production of polyhydroxyalkanoates from fermented sugar cane molasses by a mixed culture enriched in glycogen accumulating organisms.

Batch production of polyhydroxyalkanoates (PHAs) under aerobic conditions by an open mixed culture enriched in glycogen accumulating organisms (GAOs) with fermented sugar cane molasses as substrate was studied. The produced polymers contained five types of monomers, namely 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxy-2-methylbutyrate (3H2MB), 3-hydroxy-2-methylvalerate (3H2MV) and the medium chain length monomer 3-hydroxyhexanoate (3HHx). With fermented molasses as substrate, PHA was produced under concurrent consumption of stored glycogen with yields of 0.47-0.66 C-mol PHA per C-mol of total carbon substrate and with rates up to 0.65 C-mol/C-molX h. In order to investigate the role of glycogen during aerobic PHA accumulation in GAOs, synthetic single volatile fatty acids (VFAs) were used as substrates and it was found that the fate of glycogen was dependent on the type of VFA being consumed. Aerobic PHA accumulation occurred under concurrent glycogen consumption with acetate as substrate and under minor concurrent glycogen production with propionate as substrate. With butyrate and valerate as substrates, PHA accumulation occurred with the glycogen pool unaffected. The composition of the PHA was dependent on the VFA composition of the fermented molasses and was 56-70 mol-% 3HB, 13-43 mol-% 3HV, 1-23 mol-% 3HHx and 0-2 mol-% 3H2MB and 3H2MV. The high polymer yields and production rates suggest that enrichment of GAOs can be a fruitful strategy for mixed culture production of PHA from waste substrates.

Metabolic pathway for propionate utilization by phosphorus-accumulating organisms in activated sludge: 13C labeling and in vivo nuclear magnetic resonance.

ABSTRACT In vivo 13C and 31P nuclear magnetic resonance techniques were used to study propionate metabolism by activated sludge in enhanced biological phosphorus removal systems. The fate of label supplied in [3-13C]propionate was monitored in living cells subjected to anaerobic/aerobic cycles. During the anaerobic phase, propionate was converted to polyhydroxyalkanoates (PHA) with the following monomer composition: hydroxyvalerate, 74.2%; hydroxymethylvalerate, 16.9%; hydroxymethylbutyrate, 8.6%; and hydroxybutyrate, 0.3%. The isotopic enrichment in the different carbon atoms of hydroxyvalerate (HV) produced during the first anaerobic stage was determined: HV5, 59%; HV4, 5.0%; HV3, 1.1%; HV2, 3.5%; and HV1, 2.8%. A large proportion of the supplied label ended up on carbon C-5 of HV, directly derived from the pool of propionyl-coenzyme A (CoA), which is primarily labeled on C-3; useful information on the nature of operating metabolic pathways was provided by the extent of labeling on C-1, C-2, and C-4. The labeling pattern on C-1 and C-2 was explained by the conversion of propionyl-CoA to acetyl-CoA via succinyl-CoA and the left branch of the tricarboxylic acid cycle, which involves scrambling of label between the inner carbons of succinate. This constitutes solid evidence for the operation of succinate dehydrogenase under anaerobic conditions. The labeling in HV4 is explained by backflux from succinate to propionyl-CoA. The involvement of glycogen in the metabolism of propionate was also demonstrated; moreover, it was shown that the acetyl moiety to the synthesis of PHA was derived preferentially from glycogen. According to the proposed metabolic scheme, the decarboxylation of pyruvate is coupled to the production of hydrogen, and the missing reducing equivalents should be derived from a source other than glycogen metabolism.

Biopolymers production from mixed cultures and pyrolysis by-products

Polyhydroxyalkanoates (PHAs) production from low value substrates and/or byproducts represents an economical and environmental promising alternative to established industrial manufacture methods. Bio-oil resulting from the fast-pyrolysis of chicken beds was used as substrate to select a mixed microbial culture (MMC) able to produce PHA under feast/famine conditions. In this study a maximum PHA content of 9.2% (g/g cell dry weight) was achieved in a sequencing batch reactor (SBR) operated for culture selection. The PHA obtained with bio-oil as a carbon source was a copolymer composed by 70% of hydroxybutyrate (HB) and 30% of hydroxyvalerate (HV) monomers. Similar results have been reported by other studies that use real complex substrates for culture selection indicating that bio-oil can be a promising feedstock to produce PHAs using MMC. To the best of our knowledge this is the first study that demonstrated the use of bio-oil resulting from fast pyrolysis as a possibly feedstock to produce short chain length polyhydroxyalkanoates.

Community Structure Evolution and Enrichment of Glycogen-Accumulating Organisms Producing Polyhydroxyalkanoates from Fermented Molasses

ABSTRACT An open mixed culture was enriched with glycogen-accumulating organisms (GAOs) by using a sequencing batch reactor and treating an agroindustrial waste (sugar cane molasses) under cyclic anaerobic-aerobic conditions. Over a 1-year operating period, the culture exhibited a very stable GAO phenotype with an average polyhydroxyalkanoate (PHA) content of 17% total suspended solids. However, the GAO microbial community evolved over the course of operation to a culture exhibiting unusual characteristics in producing PHAs comprised of short-chain-length monomers, namely, 3-hydroxybutyrate, 3-hydroxy-2-methylbutyrate, 3-hydroxyvalerate, and 3-hydroxy-2-methylvalerate, and also, up to 31 mol% of the medium-chain-length (MCL) monomer 3-hydroxyhexanoate (3HHx). Microbial community analysis by fluorescence in situ hybridization revealed a concurrent long-term drift in the GAO community balance, from mainly “Candidatus Competibacter phosphatis” to mainly Defluviicoccus vanus-related organisms. The production of 3HHx was confirmed by 13C nuclear magnetic resonance (NMR) and appeared to be related to the increased presence of D. vanus-related GAOs. These results suggest a broadened spectrum of material, chemical, and mechanical properties that can be achieved for biopolymers produced by open mixed cultures from fermented waste. The increased spectrum of polymer properties brings a wider scope of potential applications.

Influence of produced acetic acid on growth of sulfate reducing bacteria

SummaryA culture of SRB growing in lactate was incubated at different pH values in the range of 5.8 to 7.0. Highest growth rates were observed at pH 6.6. Under gás (H2S) stripping conditions the specific growth rate decreased with the undissociated acetic acid produced. An inhibition of SRB growth of 50% was observed for undissociated acetic acid concentrations of approximately 54 mg/L.

Evidence for the intrinsic toxicity of H2S to sulphate-reducing bacteria

SummaryA sulphate-reducing culture of the genus Desulfovibrio was directly inhibited by the hydrogen sulphide (H2S) produced. Batch experiments carried out at pH 6.2 and 6.6 show that complete inhibition is achieved for almost the same H2S concentration of approximately 550 mg/l.