Anaerobic treatment of Phthalates : microbiological and technological aspects

Phthalic acid isomers (dicarboxy benzenes) play an important role in our human environment as constituents of polyester fibres, films, polyethylene terephthalate (PET) bottles and other plastics. Due to the use and generation of water during phthalic acid production from the corresponding xylenes, a concentrated wastewater is generated. The generated wastewater consists of a mixture of phthalic acid isomers, acetic acid, benzoic acid, and toluic acids. The aim of the work described in this thesis was to elucidate if anaerobic biological treatment may represent an attractive alternative for conventionally applied aerobic treatment methods. With regard to the anaerobic biodegradability of the phthalate isomers it was demonstrated that all three phthalate isomers could be degraded by two types of methanogenic granular sludge and digested sewage sludge. Lag-phases prior to degradation ranged from 17 to 156 days. More reduced aromatic analogues of the phthalates were not degraded or only at extremely low rates.Kinetic properties of the anaerobic degradation of the phthalate isomers were studied using enrichment cultures obtained from the biodegradability experiments or bioreactor biomass. The phthalate isomers grown cultures were capable of degrading only one of the phthalate isomers and degraded benzoate without a lag-period. A three species kinetic model enabled the dynamic description of intermediate acetate and molecular hydrogen accumulation and final formation of methane from the phthalate isomers and benzoate. It was shown that the syntrophic biomass cultivated had a low growth rate on the phthalate isomers (μ max≈ 0.09 day -1 ). The energetic efficiency for growth on the phthalate isomers was found to be significantly smaller when compared to growth on benzoate, suggesting that an energetic inefficiency prevails in the degradation pathway of the phthalate isomers. The cultures were furthermore strongly inhibited and even deactivated by co-incubation with acetate or benzoate, or a short period of a few hours without substrate.Despite these unfavourable microbiological characteristics, it was demonstrated that highly active terephthalate degrading biomass could be cultivated at high concentrations in both UASB (Upflow Anaerobic Sludge Bed) reactors and hybrid reactors, resulting in high terephthalate removal capacities (15-20 g­COD(Chemical Oxygen Demand) · l ­1· day ­1 ). High-rate terephthalate degradation in the UASB-reactors was strictly dependent on inoculation of the reactor with granular biomass.After demonstrating that terephthalate as sole substrate could be degraded at high-rates, we studied the feasibility of a two-stage reactor concept for the treatment of terephthalic acid production wastewater, consisting of a mixture of readily degradable substrates (acetate and benzoate) and slowly degradable substrates (terephthalate and para -toluate). It was demonstrated that through pre-removal of acetate and benzoate in the first stage the lag-phase prior terephthalate degradation in the second stage could be significantly reduced (from 300 to approximately 50 days) and the wastewater could be treated at high volumetric removal rates and short hydraulic retention times (25 g­COD · l ­1· day ­1 and 6 hours respectively). For start-up of a two-stage anaerobic bioreactor system for treatment of terephthalic acid production wastewater, a gradual transition from initial operation in parallel to operation in series is suggested.<br/

Molecular characterization of the CmbR activator-binding site in the metC-cysK promoter region in Lactococcus lactis

The metC-cysK operon involved in sulphur metabolism in Lactococcus lactis is positively regulated by the LysR-type protein CmbR. Transcription from the metC promoter is activated when concentrations of methionine and cysteine in the growth medium are low. The metC promoter region contains two direct and three inverted repeats. Deletion analysis indicated that direct repeat 2 (DR2) is required for activation of the metC promoter by CmbR. Gel mobility shift assays confirmed that CmbR binds to a 407 bp DNA fragment containing the rnetC promoter. This binding was stimulated by O-acetyl-L-serine. Competition experiments with deletion variants of the metC promoter showed that CmbR binding only occurred with fragments containing an intact DR2, confirming that DR2 is the CmbR binding site within the metC promoter

Making sense of quorum sensing in lactobacilli: a special focus on Lactobacillus plantarum WCFS1

In silico identification criteria were defined to predict if genes encoding histidine protein kinases (HPKs) and response regulators (RRs) could be part of peptide-based quorum sensing (QS) two-component regulatory systems (QS-TCSs) in Firmicutes. These criteria were used to screen HPKs and RRs annotated on the completed genome sequences of Lactobacillus species, and several (putative) QS-TCSs were identified in this way. The five peptide-based QS-TCSs that were predicted on the Lactobacillus plantarum WCFS1 genome were further analysed to test their (QS) functionality. Four of these systems contained an upstream gene encoding a putative autoinducing peptide (AIP), of which two were preceded by a double-glycine-type leader peptide. One of these was identical to the plnABCD regulatory system of L. plantarum C11 and was shown to regulate plantaricin production in L. plantarum WCFS1. The third TCS was designated lamBDCA for Lactobacillus agr-like module, where the lamD gene was shown to encode a cyclic thiolactone peptide. The fourth TCS was paralogous to the lam system and contained a putative AIP-encoding gene but lacked the lamB gene. Finally, a genetically separated orphan HPK and RR that showed clear peptide-based QS characteristics could form a fifth peptide-based QS-TCS. The predicted presence of multiple (peptide-based) QS-TCSs in some lactobacilli and in particular in L. plantarum might be a reflection of the ability of these species to persist in a diverse range of ecological niches

Microbial catabolic activities are naturally selected by metabolic energy harvest rate

The fundamental trade-off between yield and rate of energy harvest per unit of substrate has been largely discussed as a main characteristic for microbial established cooperation or competition. In this study, this point is addressed by developing a generalized model that simulates competition between existing and not experimentally reported microbial catabolic activities defined only based on well-known biochemical pathways. No specific microbial physiological adaptations are considered, growth yield is calculated coupled to catabolism energetics and a common maximum biomass-specific catabolism rate (expressed as electron transfer rate) is assumed for all microbial groups. Under this approach, successful microbial metabolisms are predicted in line with experimental observations under the hypothesis of maximum energy harvest rate. Two microbial ecosystems, typically found in wastewater treatment plants, are simulated, namely: (i) the anaerobic fermentation of glucose and (ii) the oxidation and reduction of nitrogen under aerobic autotrophic (nitrification) and anoxic heterotrophic and autotrophic (denitrification) conditions. The experimentally observed cross feeding in glucose fermentation, through multiple intermediate fermentation pathways, towards ultimately methane and carbon dioxide is predicted. Analogously, two-stage nitrification (by ammonium and nitrite oxidizers) is predicted as prevailing over nitrification in one stage. Conversely, denitrification is predicted in one stage (by denitrifiers) as well as anammox (anaerobic ammonium oxidation). The model results suggest that these observations are a direct consequence of the different energy yields per electron transferred at the different steps of the pathways. Overall, our results theoretically support the hypothesis that successful microbial catabolic activities are selected by an overall maximum energy harvest rate

Can probiotics modulate human disease by impacting intestinal barrier function?

The expert group received funding from the ILSI Europe Probiotic Task Force. Industry members of this task force are listed on the ILSI Europe website at www.ilsi.eu. d P. D. C. is the recipient of grants from FNRS and the French Cancer Research Association (ARC). This work was supported by the Fonds de la Recherche Scientifique – FNRS for the FRFS-WELBIO under grant no. WELBIO-CR-2012S-02R. This work is supported in part by the Funds InBev-Baillet Latour (Grant for Medical Research 2015). P. D. C. is a recipient of an ERC Starting Grant 2013 (European Research Council, Starting grant no. 336452-ENIGMO)

Microbial diversity arising from thermodynamic constraints

The microbial world displays an immense taxonomic diversity. This diversity is manifested also in a multitude of metabolic pathways that can utilize different substrates and produce different products. Here, we propose that these observations directly link to thermodynamic constraints that inherently arise from the metabolic basis of microbial growth. We show that thermodynamic constraints can enable coexistence of microbes that utilise the same substrate but produce different end products. We find that this thermodynamics-driven emergence of diversity is most relevant for metabolic conversions with low free energy as seen for example under anaerobic conditions, where population dynamics is governed by thermodynamic effects rather than kinetic factors such as substrate uptake rates. These findings provide a general understanding of the microbial diversity based on the first-principles of thermodynamics. As such they provide a thermodynamics-based framework for explaining the observed microbial diversity in different natural and synthetic environments

Fine Tuning of the Lactate and Diacetyl Production through Promoter Engineering in Lactococcus lactis

Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H2O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H2O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15±0.08 mM to 9.94±0.07 mM, and the corresponding diacetyl production increased from 1.07±0.03 mM to 4.16±0.06 mM with the intracellular NADH/NAD+ ratios varying from 0.711±0.005 to 0.383±0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD+ ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H2O-forming NADH oxidase activity led to 76.95% lower H2O2 concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H2O2 accumulation and prolong cell survival

High local substrate availability stabilizes a cooperative trait

Cooperative behavior is widely spread in microbial populations. An example is the expression of an extracellular protease by the lactic acid bacterium Lactococcus lactis, which degrades milk proteins into free utilizable peptides that are essential to allow growth to high cell densities in milk. Cheating, protease-negative strains can invade the population and drive the protease-positive strain to extinction. By using multiple experimental approaches, as well as modeling population dynamics, we demonstrate that the persistence of the proteolytic trait is determined by the fraction of the generated peptides that can be captured by the cell before diffusing away from it. The mechanism described is likely to be relevant for the evolutionary stability of many extracellular substrate-degrading enzymes