Spatial and Temporal Dynamics in Attached and Suspended Bacterial Communities in Three Drinking Water Distribution Systems with Variable Biological Stability.

Microbial presence and regrowth in drinking water distribution systems (DWDSs) is routinely monitored to assess the biological stability of drinking water without a residual disinfectant, but the conventional microbiological culture methods currently used target only a very small fraction of the complete DWDS microbiome. Here, we sequenced 16S rRNA gene amplicons to elucidate the attached and suspended prokaryotic community dynamics within three nonchlorinated DWDSs with variable regrowth conditions distributing similarly treated surface water from the same source. One rural location, with less regrowth related issues, differed most strikingly from the other two urban locations by the exclusive presence of Pseudonocardia (Actinobacteria) in the biofilm and the absence of Limnobacter (Betaproteobacteriales) in the water and loose deposits during summer. There was a dominant seasonal effect on the drinking water microbiomes at all three locations. For one urban location, it was established that the most significant changes in the microbial community composition on a spatial scale occurred shortly after freshly treated water entered the DWDS. However, summerly regrowth of Limnobacter, one of the dominant genera in the distributed drinking water, already occurred in the clean water reservoir at the treatment plant before further distribution. The highlighted bacterial lineages within these highly diverse DWDS communities might be important new indicators for undesirable regrowth conditions affecting the final drinking water quality.

Enrichment of PHA-producing bacteria under continuous substrate supply.

To enrich polyhydroxyalkanoate (PHA) producing microbial communities, generally, a feast-famine regime is applied. Here we investigated the impact of continuous substrate feeding on the enrichment of PHA-producing bacteria in two sequencing batch reactors (SBRs). In the first reactor, the substrate (acetate) was dosed continuously and Zoogloea sp. was enriched. The culture accumulated PHA upon exposure to excess carbon, but the PHA production rate and storage capacity (53 wt.%) were one-fifth of that observed for enrichment cultures in a standard, pulse-fed SBR dominated by the PHA producer Plasticicumulans acidivorans. In the second reactor, half the acetate was dosed at the beginning of the cycle and the other half continuously. Having a true feast phase, the enrichment of P. acidivorans was not impeded by the continuous supply of acetate and the culture accumulated 85 wt.% PHA. This shows that for the enrichment of bacteria with a superior PHA-producing capacity periodic substrate excess - a true feast phase - is essential, while periodic substrate absence - a true famine phase - is not.

Combining the enrichment and accumulation step in non-axenic PHA production: Cultivation of Plasticicumulans acidivorans at high volume exchange ratios.

The process for non-axenic polyhydroxyalkanoate (PHA) production from organic waste generally comprises three steps: acidogenic fermentation of the waste stream, enrichment of a PHA-producing culture, and production of the PHA. This study assesses the feasibility of combining the enrichment and production step. Harvesting PHA-rich biomass directly from the sequencing batch reactor (SBR) used for enrichment of the microbial culture reduces capital cost, but may increase downstream-processing cost if the PHA content is significantly lowered. Operating an acetate-fed SBR at a volume exchange ratio of 0.75 (18h cycles, 1 d SRT) allowed the production of biomass with 70wt% poly(3-hydroxybutyrate) (PHB) in a single-step process. By increasing the exchange ratio to 0.83 (20h cycles) the PHB content of the harvested biomass increased to 75wt%, but the operational stability decreased. SBR operation at these high exchange ratios makes that bacteria have to increase their growth rate and external substrate is available for relatively long periods. This allows the establishment of larger flanking populations and negatively affected the kinetic properties of Plasticicumulans acidivorans, the predominant organism. Maximizing the volume exchange ratio is, therefore, a suitable strategy to produce large amounts of PHA in the SBR, but does not ensure the enrichment of a culture with superior PHA productivity.

Modeling the competition between PHA-producing and non-PHA-producing bacteria in feast-famine SBR and staged CSTR systems.

Although the enrichment of specialized microbial cultures for the production of polyhydroxyalkanoates (PHA) is generally performed in sequencing batch reactors (SBRs), the required feast-famine conditions can also be established using two or more continuous stirred-tank reactors (CSTRs) in series with partial biomass recirculation. The use of CSTRs offers several advantages, but will result in distributed residence times and a less strict separation between feast and famine conditions. The aim of this study was to investigate the impact of the reactor configuration, and various process and biomass-specific parameters, on the enrichment of PHA-producing bacteria. A set of mathematical models was developed to predict the growth of Plasticicumulans acidivorans-as a model PHA producer-in competition with a non-storing heterotroph. A macroscopic model considering lumped biomass and an agent-based model considering individual cells were created to study the effect of residence time distribution and the resulting distributed bacterial states. The simulations showed that in the 2-stage CSTR system the selective pressure for PHA-producing bacteria is significantly lower than in the SBR, and strongly affected by the chosen feast-famine ratio. This is the result of substrate competition based on both the maximum specific substrate uptake rate and substrate affinity. Although the macroscopic model overestimates the selective pressure in the 2-stage CSTR system, it provides a quick and fairly good impression of the reactor performance and the impact of process and biomass-specific parameters.

Impact of non-storing biomass on PHA production: an enrichment culture on acetate and methanol.

The use of enrichment cultures for polyhydroxyalkanoate (PHA) production from substrate mixtures such as wastewater inevitably results in the establishment of a non-PHA-storing population besides the PHA-producing bacteria. This reduces the maximum PHA content that can be established, and increases downstream-processing costs. The aim of this study was to investigate the impact of non-storing biomass on the PHA production process. A microbial culture was enriched in a sequencing batch reactor fed with acetate and methanol. Methanol served as model substrate for compounds unsuitable for PHA production. The enrichment was dominated by Plasticicumulans acidivorans, a known PHA producer, and Methylobacillus flagellatus, an obligate methylotroph that cannot store PHA. As expected, the presence of the non-storing population lowered the maximum PHA content of the culture, from more than 80 to 66wt.%. To mimic a nitrogen-rich waste stream, additional accumulation experiments were performed with continuous supply of carbon and ammonium. In these experiments P. acidivorans still accumulated large amounts of PHA, but unrestricted growth of the non-storing, methylotrophic population reduced the maximum overall PHA content to 52wt.%. Besides ammonium limitation, other strategies to restrict the fraction of non-storing biomass should be developed. The mixture of acetate and methanol is a useful model substrate for the development of such strategies.

Modeling PHA-producing microbial enrichment cultures--towards a generalized model with predictive power.

Polyhydroxyalkanoate (PHA) production from waste streams using microbial enrichment cultures is a promising option for cost price reduction of this biopolymer. For proper understanding and successful optimization of the process, a consistent mechanistic model for PHA conversion by microbial enrichment cultures is needed. However, there is still a lack of mechanistic expressions describing the dynamics of the feast-famine process. The scope of this article is to provide an overview of the current models, investigate points of improvement, and contribute concepts for creation of a generalized model with more predictive value for the feast-famine process. Based on experimental data available in literature we have proposed model improvements for (i) modeling mixed substrates uptake, (ii) growth in the feast phase, (iii) switching between feast and famine phase, (iv) PHA degradation and (v) modeling the accumulation phase. Finally, we provide an example of a simple uniform model. Herewith we aim to give an impulse to the establishment of a generalized model.

Butyrate as preferred substrate for polyhydroxybutyrate production.

In this study, the suitability of butyrate as substrate for polyhydroxyalkanoate (PHA) production by microbial enrichment cultures was assessed. Two sequencing batch reactors were operated under feast-famine conditions: one fed with butyrate, and another with mixed acetate and butyrate. The obtained results were compared to previous results with acetate as sole substrate. In all three reactors Plasticicumulans acidivorans dominated the enrichment culture. The carbon uptake rate and PHA yield were significantly higher on butyrate than on acetate, resulting in a higher PHA production rate. When both substrates were available the bacteria strongly preferred the uptake of butyrate. Only after butyrate depletion acetate was taken up at a high rate. The molar substrate uptake rate remained the same, suggesting that substrate uptake is the rate-limiting step. The results show that for optimized waste-based PHA production the pre-fermentation process should be directed towards butyrate production.

Waste to resource: Converting paper mill wastewater to bioplastic.

In this study we investigated the feasibility of producing polyhydroxyalkanoate (PHA) by microbial enrichments on paper mill wastewater. The complete process includes (1) paper mill wastewater acidogenic fermentation in a simple batch process, (2) enrichment of a PHA-producing microbial community in a selector operated in sequencing batch mode with feast-famine regime, (3) Cellular PHA content maximization of the enrichment in an accumulator in fed-batch mode. The selective pressure required to establish a PHA-producing microbial enrichment, as derived from our previous research on synthetic medium, was validated using an agro-industrial waste stream in this study. The microbial enrichment obtained could accumulate maximum up to 77% PHA of cell dry weight within 5 h, which is currently the best result obtained on real agro-industrial waste streams, especially in terms of biomass specific efficiency. Biomass in this enrichment included both Plasticicumulans acidivorans, which was the main PHA producer, and a flanking population, which exhibited limited PHA-producing capacity. The fraction of P. acidivorans in the biomass was largely dependent on the fraction of volatile fatty acids in the total soluble COD in the wastewater after acidification. Based on this observation, one simple equation was proposed for predicting the PHA storage capacity of the enrichment. Moreover, some crucial bottlenecks that may impede the successful scaling-up of the process are discussed.

Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae.

Dicarboxylic acids that are produced from renewable resources are becoming attractive building blocks for the polymers industry. In this respect, fumaric acid is very interesting. Its low aqueous solubility facilitates product recovery. To avoid excessive waste salt production during downstream processing, a low pH for fumaric acid fermentation will be beneficial. Studying the influence of pH, working volume and shaking frequency on cell cultivation helped us to identify the best conditions to obtain appropriate pellet morphologies of a wild type strain of Rhizopus oryzae. Using these pellets, the effects of pH and CO(2) addition were studied to determine the best conditions to produce fumaric acid in batch fermentations under nitrogen-limited conditions with glucose as carbon source. Decreasing either the fermentation pH below 5 or increasing the CO(2) content of the inlet air above 10% was unfavourable for the cell-specific productivity, fumaric acid yield, and fumaric acid titer. However, switching off the pH control late in the batch phase did not affect these performance parameters and allowed achieving pH of 3.6. A concentration of 20 gL(-1) of fumaric acid was obtained at pH 3.6 while the average cell mass specific productivity and fumaric acid yield were the same as at pH 5.0. Consequently, relatively modest amounts of inorganic base were required for pH control, while recovery of the acid should be relatively easy at pH 3.6.

Polyhydroxybutyrate production from lactate using a mixed microbial culture.

In this study we investigated the use of lactate and a lactate/acetate mixture for enrichment of poly-3-hydroxybutyrate (PHB) producing mixed cultures. The mixed cultures were enriched in sequencing batch reactors (SBR) that established a feast-famine regime. The SBRs were operated under conditions that were previously shown to enable enrichment of a superior PHB producing strain on acetate (i.e., 12 h cycle length, 1 day SRT and 30°C). Two new mixed cultures were eventually enriched from activated sludge. The mixed culture enriched on lactate was dominated by a novel gammaproteobacterium. This enrichment can accumulate over 90 wt% PHB within 6 h, which is currently the best result reported for a bacterial culture in terms of the final PHB content and the biomass specific PHB production rate. The second mixed culture enriched on a mixture of acetate and lactate can produce up to 84 wt% PHB in just over 8 h. The predominant bacterial species in this culture were Plasticicumulans acidivorans and Thauera selenatis, which have both been reported to accumulate large amounts of PHB. The data suggest that P. acidivorans is a specialist on acetate conversion, whereas Thauera sp. is a specialist on lactate conversion. The main conclusion of this work is that the use of different substrates has a direct impact on microbial composition, but has no significant effect on the functionality of PHB production process.

Effect of temperature and cycle length on microbial competition in PHB-producing sequencing batch reactor.

The impact of temperature and cycle length on microbial competition between polyhydroxybutyrate (PHB)-producing populations enriched in feast-famine sequencing batch reactors (SBRs) was investigated at temperatures of 20 °C and 30 °C, and in a cycle length range of 1-18 h. In this study, the microbial community structure of the PHB-producing enrichments was found to be strongly dependent on temperature, but not on cycle length. Zoogloea and Plasticicumulans acidivorans dominated the SBRs operated at 20 °C and 30 °C, respectively. Both enrichments accumulated PHB more than 75% of cell dry weight. Short-term temperature change experiments revealed that P. acidivorans was more temperature sensitive as compared with Zoogloea. This is particularly true for the PHB degradation, resulting in incomplete PHB degradation in P. acidivorans at 20 °C. Incomplete PHB degradation limited biomass growth and allowed Zoogloea to outcompete P. acidivorans. The PHB content at the end of the feast phase correlated well with the cycle length at a constant solid retention time (SRT). These results suggest that to establish enrichment with the capacity to store a high fraction of PHB, the number of cycles per SRT should be minimized independent of the temperature.