Emergent behaviour in a chlorophenol-mineralising three-tiered microbial `food web'

Anaerobic digestion enables the water industry to treat wastewater as a resource for generating energy and recovering valuable by-products. The complexity of the anaerobic digestion process has motivated the development of complex models. However, this complexity makes it intractable to pin-point stability and emergent behaviour. Here, the widely used Anaerobic Digestion Model No. 1 (ADM1) has been reduced to its very backbone, a syntrophic two-tiered microbial food chain and a slightly more complex three-tiered microbial food web, with their stability analysed as function of the inflowing substrate concentration and dilution rate. Parameterised for phenol and chlorophenol degradation, steady-states were always stable and non-oscillatory. Low input concentrations of chlorophenol were sufficient to maintain chlorophenol- and phenol-degrading populations but resulted in poor conversion and a hydrogen flux that was too low to sustain hydrogenotrophic methanogens. The addition of hydrogen and phenol boosted the populations of all three organisms, resulting in the counterintuitive phenomena that (i) the phenol degraders were stimulated by adding hydrogen, even though hydrogen inhibits phenol degradation, and (ii) the dechlorinators indirectly benefitted from measures that stimulated their hydrogenotrophic competitors; both phenomena hint at emergent behaviour.Comment: 19 pages, 8 figure

Method and timing of grassland renovation affects herbage yield, nitrate leaching, and nitrous oxide emission in intensively managed grasslands

Managed grasslands are occasionally ploughed up and reseeded in order to maintain or increase the sward productivity. It has been reported that this renovation of grassland is associated with a flush of soil organic nitrogen (N) mineralization and with a temporary increase in soil mineral N contents. Here, we report on the effects of method and time of grassland renovation on herbage yield, nitrate (NO3 -) leaching and nitrous oxide (N2O) emission. Field experiments were carried out at three sites (two sandy soils and a clay soil) in the Netherlands for three years. Renovation of grassland increased the percentage of Perennial ryegrass from 48–70% up to more than 90%. However, averaged over three years, dry matter yields were higher for the reference (not reseeded) swards (on average 13.6 Mg ha-1 for the highest N application rate) than for the renovated grasslands (12.2–13.1 Mg ha-1 dry matter). Grassland renovation in April did not increase N leaching in comparison to the reference. However, renovation in September increased the risk of leaching, because mineral N contents in the 0–90 cm were in November on average 46–77 kg N ha-1 higher than in the reference. Contents of dissolved organic N (DON) in the soil were not affected by renovation. Renovation increased N2O emissions by a factor of 1.8–3.0 relative to the reference grassland. Emissions of N2O were on average higher after renovation in April (8.2 kg N2O-N ha-1) than in September (5.8 kg N2O-N ha-1). Renovation without ploughing (i.e. only chemically destruction of the sward) resulted in a lower percentage of perennial ryegrass (60–84%) than with ploughing (>90%). Moreover, N2O emissions were higher after renovation without ploughing than with ploughing. Clearly, farmers need better recommendations and tools for determining when grassland renovation has beneficial agronomic effects. Losses of N via leaching and N2O emission after renovation can probably not be avoided, but renovation in spring in stead of autumn in combination with ploughing and proper timing of fertilizer application can minimize N losses

Temperature, inocula and substrate: contrasting electroactive consortia, diversity and performance in microbial fuel cells

The factors that affect microbial community assembly and its effects on the performance of bioelectrochemical systems are poorly understood. Sixteen microbial fuel cell (MFC) reactors were set up to test the importance of inoculum, temperature and substrate: Arctic soil versus wastewater as inoculum; warm (26.5°C) versus cold (7.5°C) temperature; and acetate versus wastewater as substrate. Substrate was the dominant factor in determining performance and diversity: unexpectedly the simple electrogenic substrate delivered a higher diversity than a complex wastewater. Furthermore, in acetate fed reactors, diversity did not correlate with performance, yet in wastewater fed ones it did, with greater diversity sustaining higher power densities and coulombic efficiencies. Temperature had only a minor effect on power density, (Q10: 2 and 1.2 for acetate and wastewater respectively): this is surprising given the well-known temperature sensitivity of anaerobic bioreactors. Reactors were able to operate at low temperature with real wastewater without the need for specialised inocula; it is speculated that MFC biofilms may have a self-heating effect. Importantly, the warm acetate fed reactors in this study did not act as direct model for cold wastewater fed systems. Application of this technology will encompass use of real wastewater at ambient temperatures

COD/sulfate ratio does not affect the methane yield and microbial diversity in anaerobic digesters.

Anaerobic digestion of organic matter is the major route of biomethane production. However, in the presence of sulfate, sulfate-reducing bacteria (SRB) typically outcompete methanogens, which may reduce or even preclude methane production from sulfate-containing wastewaters. Although sulfate-reduction and methanogenesis can occur simultaneously, our limited understanding of the microbiology of anaerobic digesters treating sulfate-containing wastewaters constrains improvements in the production of methane from these systems. This study tested the effects of carbon sources and chemical oxygen demand-to-sulfate ratio (COD/SO42-) on the diversity and interactions of SRB and methanogens in an anaerobic digester treating a high-sulfate waste stream. Overall, the data showed that sulfate removal and methane generation occurred in varying efficiencies and the carbon source had limited effect on the methane yield. Importantly, the results demonstrated that methanogenic and SRB diversities were only affected by the carbon source and not by the COD/SO42- ratio

Microbiological aspects of granular methanogenic sludge

The settling characteristics of anaerobic sludge are enhanced by the formation of microbial conglomerates. Various types of conglomerates having different structures, were distinguished in the present study, viz. granules, pellets and flocs (chapter 1). Granular methanogenic sludge, often developing in upflow anaerobic sludge blanket reactors, is optimal for an economic application of the system. One of the hypotheses to explain the formation of these granules is that bacteria would excrete extracellular polymers, responsible for the formation of stable, well settling aggregates. Scanning electron micrographs showed that indeed extracellular material can be detected in granular methanogenic sludge grown on waste water of a liquid sugar plant (chapter 3). Various methods were used to quantify the fraction of this material and to elucidate its chemical composition: 1 - 2% of the dry weight of the granules appeared to consist of extracellular sugars. The relative proportions of these sugars varied somewhat with the extraction methods used and the batch of granules analysed.Granular methanogenic sludge grown on waste water of a sugar refinery consisted of a diverse microflora composed of various types of bacteria (chapter 3). As these granules had been formed on a complex waste water the growth of granular methanogenic sludge was studied under better defined conditions with respect to substrate supply. In chapter 2 it was shown that growth of methanogenic sludge granules is possible in bench scale UASB reactors with a volume of one liter. The first series of experiments was carried out with waste water of the sugar factory on which granular sludge had been found to develop in a full scale UASB reactor. Development and growth of granular methanogenic sludge was possible when a UASB reactor with a volume of one liter had been seeded with disintegrated granular sludge. However, when the reactor had been seeded with digested sewage sludge more fluffy pellets developed. Subsequent experiments showed that various compounds could be omitted from artificially prepared waste waters that originally reflected the composition of the waste water of the sugar refinery. Cultivation of methanogenic sludge granules proved possible on ethanol or propionate as most simple substrates. Feeding granular methanogenic sludge for prolonged periods with acetate as sole energy source resulted in weaker, more fluffy pellets.Incubation of granular methanogenic sludge in batch culture under growth- supporting conditions generally resulted in the overgrowth of the original seed granules by dispersed growing bacteria and development of fluffy pellets. Cultivation of granular methanogenic sludge in batch culture was only possible in experiments where propionate was stoichiometrically converted to methane and acetate (and probably carbon dioxide).By studying the potential methanogenic activities of granular methanogenic sludges (chapter 4) it was shown that the biomass consisted for a significant part of acetoclastic methanogens. These potential specific activities showed that obligately acetoclastic methanogens were quantitatively more important than hydrogenotrophic methanogens. These conclusions were drawn from a comparison between the potential methanogenic activities measured in the present experiments, and literature data on the potential activities of various groups of anaerobic bacteria. This approach provided a reliable tool for the estimation of the microbial composition of methanogenic consortia and was faster and more accurate than MPN counts. The resulting estimates of the acetoclastic biomass with respect to the occurrence of obligately and facultatively acetoclastic methanogens were corroborated by microscopic observations and indicated that Methanothrixsoehngenii -like organisms are the most numerous acetoclastic methanogens in granular sludge.An indirect method for the determination of the potential methanogenic activity of anaerobic biomass as suggested by others, viz. the estimation of the amount of F 420 present in methanogenic consortia was tested with granular sludge in the present study. It was shown (chapter 5) that a good correlation existed between the F 420 content of different granular consortia and their potential methanogenic activity on formate. No correlation, however, was observed between the F 420 content and the potential acetoclastic methanogenic activity of such consortia. This indicates that F 420 content is not a reliable parameter for assessing the overall potential methanogenic activity of anaerobic biomass.In densely packed biolayers mass transfer limitation will influence the conversion capacity of the biomass at low substrate concentrations. The results reported in chapter 6 show that the effect of mass transfer limitation in granular methanogenic sludge is dependent on the composition of the biolayer, viz. its potential specific activity, the thickness, and on the type of substrate. To detect significant effects methanogenic granules had to be cultivated on specific substrates which resulted in the development of uniform biolayers. Even with this material mass transfer resistance was of no significance unless biolayer thickness reached values above 1 mm. These results indicate that mass transfer resistance will generally not be effective in industrial reactors. They furthermore show that not only the outer parts, but also the inner parts of the granules consisted of actively catabolizing bacteria

Increasing sulfate levels show a differential impact on synthetic communities comprising different methanogens and a sulfate reducer

Methane producing microbial communities are of ecological and biotechnological interest. Syntrophic interactions among sulphate reducers and aceto/hydrogenotrophic and obligate hydrogenotrophic methanogens form a key component of these communities, yet, the impact of these different syntrophic routes on methane production and their stability against sulphate availability are not well understood. Here, we construct model synthetic communities using a sulphate reducer and two types of methanogens representing different methanogenesis routes. We find that tri-cultures with both routes increase methane production by almost two-fold compared to co-cultures, and are stable in the absence of sulphate. With increasing sulphate, system stability and productivity decreases, and does so faster in communities with aceto/hydrogenotrophic methanogens despite the continued presence of acetate. We show that this is due to a shift in these methanogens’ metabolism towards co-utilisation of hydrogen with acetate. These findings indicate the important role of hydrogen dynamics in the stability and productivity of syntrophic communities

The experimental determination of reliable biodegradation rates for mono-aromatics towards evaluating QSBR models

Quantitative Structure Biodegradation Relationships (QSBRs) are a tool to predict the biodegradability of chemicals. The objective of this work was to generate reliable biodegradation data for mono-aromatic chemicals in order to evaluate and verify previously developed QSBRs models. A robust biodegradation test method was developed to estimate specific substrate utilization rates, which were used as a proxy for biodegradation rates of chemicals in pure culture. Five representative mono-aromatic chemicals were selected that spanned a wide range of biodegradability. Aerobic biodegradation experiments were performed for each chemical in batch reactors seeded with known degraders. Chemical removal, degrader growth and CO2 production were monitored over time. Experimental data were interpreted using a full carbon mass balance model, and Monod kinetic parameters (Y, Ks, qmax and μmax) for each chemical were determined. In addition, stoichiometric equations for aerobic mineralization of the test chemicals were developed. The theoretically estimated biomass and CO2 yields were similar to those experimentally observed; 35 (s.d ± 8) of the recovered substrate carbon was converted to biomass, and 65 (s.d ± 8) was mineralised to CO2. Significant correlations were observed between the experimentally determined specific substrate utilization rates, as represented by qmax and qmax/Ks, at high and low substrate concentrations, respectively, and the first order biodegradation rate constants predicted by a previous QSBR study. Similarly, the correlation between qmax and selected molecular descriptors characterizing the chemicals structure in a previous QSBR study was also significant. These results suggest that QSBR models can be reliable and robust in prioritising chemical half-lives for regulatory screening purposes

Embedded Large-Scale Handwritten Chinese Character Recognition

As handwriting input becomes more prevalent, the large symbol inventory required to support Chinese handwriting recognition poses unique challenges. This paper describes how the Apple deep learning recognition system can accurately handle up to 30,000 Chinese characters while running in real-time across a range of mobile devices. To achieve acceptable accuracy, we paid particular attention to data collection conditions, representativeness of writing styles, and training regimen. We found that, with proper care, even larger inventories are within reach. Our experiments show that accuracy only degrades slowly as the inventory increases, as long as we use training data of sufficient quality and in sufficient quantity.Comment: 5 pages, 7 figure

Mogelijkheden tot vermindering van emissie van lachgas uit landbouwgronden bij toepassing van verschillende mestsoorten en nitrificatieremmers; laboratoriumproeven en aanbevelingen voor veldexperimenten

Landbouwgronden zijn een belangrijke bron van het broeikasgas lachgas (N2O). In het kader van het Reductieplan Overige Broeikasgassen (ROB) worden via onderzoek maatregelen verkend die kunnen leiden tot een vermindering van emissie van de overige broeikasgassen methaan en lachgas. Het gebruik van nieuwe (kunst)meststoffen is een van die maatregelen. Ze zijn ontwikkeld om de verliezen van stikstof via nitraatuitspoeling en denitrificatie ter beperken, en hun eventuele gebruik komt dus tegemoet aan het streven naar een meer duurzame landbouw. Bij het begrip nieuwe meststoffen moet gedacht worden aan meststoffen waaraan nitrificatieremmers zijn toegevoegd, of die gecoat zijn om de nutriëntenafgifte te vertragen. Het valt te verwachten dat deze eigenschappen ook de emissie van lachgas beïnvloeden. In het voorliggende onderzoek is door Alterra in een reeks laboratoriumexperimenten nagegaan wat de effecten zijn van het gebruik van aantal van dergelijke nieuwe mestsoorten op de emissie van lachgas uit een zand- en een kleigrond. De nieuwe mestsoorten zoals zijn niet beter dan de traditionele mestsoorten als ze worden beoordeeld op de het verminderen van de emissie van lachgas. De toevoeging van denitrificatieremmers resulteert in zand vaker in minder lachgas dan in klei. In klei leidt de toevoeging van denitrificatieremmers ook tot verhoging van de emissie van lachgas. Op grond van de resultaten van dit laboratorium-onderzoek worden aanbevelingen gedaan en keuzes gemaakt voor een nog uit te voeren veld-onderzoek