Microbial Toluene Removal in Hypoxic Model Constructed Wetlands Occurs Predominantly via the Ring Monooxygenation Pathway.

In the present study, microbial toluene degradation in controlled constructed wetland model systems, planted fixed-bed reactors (PFRs), was queried with DNA-based methods in combination with stable isotope fractionation analysis and characterization of toluene-degrading microbial isolates. Two PFR replicates were operated with toluene as the sole external carbon and electron source for 2 years. The bulk redox conditions in these systems were hypoxic to anoxic. The autochthonous bacterial communities, as analyzed by Illumina sequencing of 16S rRNA gene amplicons, were mainly comprised of the families Xanthomonadaceae, Comamonadaceae, and Burkholderiaceae, plus Rhodospirillaceae in one of the PFR replicates. DNA microarray analyses of the catabolic potentials for aromatic compound degradation suggested the presence of the ring monooxygenation pathway in both systems, as well as the anaerobic toluene pathway in the PFR replicate with a high abundance of Rhodospirillaceae. The presence of catabolic genes encoding the ring monooxygenation pathway was verified by quantitative PCR analysis, utilizing the obtained toluene-degrading isolates as references. Stable isotope fractionation analysis showed low-level of carbon fractionation and only minimal hydrogen fractionation in both PFRs, which matches the fractionation signatures of monooxygenation and dioxygenation. In combination with the results of the DNA-based analyses, this suggests that toluene degradation occurs predominantly via ring monooxygenation in the PFRs.

Field application of a planted fixed bed reactor (PFR) for support media and rhizosphere investigation using undisturbed samples from full-scale constructed wetlands.

This study presents a novel method for investigations on undisturbed samples from full-scale horizontal subsurface-flow constructed wetlands (HSSFCW). The planted fixed bed reactor (PFR), developed at the Helmholtz Center for Environmental Research (UFZ), is a universal test unit for planted soil filters that reproduces the operational conditions of a constructed wetland (CW) system in laboratory scale. The present research proposes modifications on the PFR original configuration in order to allow its operation in field conditions. A mobile device to obtain undisturbed samples from real-scale HSSFCW was also developed. The experimental setting is presented with two possible operational configurations. The first allows the removal and replacement of undisturbed samples in the CW bed for laboratory investigations, guaranteeing sample integrity with a mobile device. The second allows the continuous operation of the PFR and undisturbed samples as a fraction of the support media, reproducing the same environmental conditions outside the real-scale system. Investigations on the hydrodynamics of the adapted PFR were carried out with saline tracer tests, validating the proposed adaptation. Six adapted PFR units were installed next to full-scale HSSFCW beds and fed with interstitial liquid pumped from two regions of planted and unplanted support media. Fourteen points were monitored along the system, covering carbon fractions, nitrogen and sulfate. The results indicate the method as a promising tool for investigations on CW support media, rhizosphere and open space for studies on CW modeling, respirometry, kinetic parameters, microbial communities, redox potential and plant influence on HSSFCW.

Influence of the redox condition dynamics on the removal efficiency of a laboratory-scale constructed wetland.

A laboratory reactor planted with Juncus effusus treating an artificial wastewater was used to investigate the short-term and long-term variations and interactions in the redox conditions as well as the removal efficiency of C and the N turnover. The permanent circulation of the process water enabled the micro-gradient processes to be evaluated for an operating period of 20 months. Steady-state conditions were achieved throughout the operating period with high mean removal efficiencies of 92.7% total organic carbon, 82.0% ammonia and 97.6% nitrate. Daily variations in the redox state of the rhizosphere of a few hundred mV were observed, ranging from about -200 to oxidized conditions of about +200 mV and driven by daylight. Variations in pH associated with changes in light and redox were linked to the dynamics of the fates of organic and inorganic carbon species. The ammonia removal processes were found to be firmly established, including for moderately reduced redox conditions with high efficiencies for E(h)>-50 mV. The enrichment of ammonia (up to 13 mg l(-1)) closely linked to the light, particularly during summertime, indicates the existence of hitherto unconsidered additional N turnover pathways in the rhizoplane involving N(2) produced by microbes or released by plants. C turnover was strongly related to the seasonal variation in illumination with minimum efficiencies during the dark season. In addition, it was characterized by oscillation with periods of approximately 1 month. The relationships found are dominant for biofilms on the rhizoplane and decisive for the removal efficiency of especially simple constructed and natural wetlands. The results highlight the importance of helophytes and their physiological specifics for removal processes.

Sulphate reduction and the removal of carbon and ammonia in a laboratory-scale constructed wetland.

Sulphate is a normal constituent of domestic wastewater and reduced sulphur compounds are known to be potent inhibitors of plant growth and certain microbial activities. However, the knowledge about sulphate reduction and the effect on the removal of C and N in constructed wetlands is still limited. Investigations in laboratory-scale constructed wetland reactors were performed to evaluate the interrelation of carbon and nitrogen removal with the sulphate reduction by use of artificial domestic wastewater. Carbon removal was found to be only slightly affected and remained at high levels of efficiency (75-90%). Only at sulphate reduction intensities above 75 mgl(-1) (50% removal), a decrease of carbon removal of up to 20% was observed. A highly contrary behaviour of ammonia removal was found in general, which decreased exponentially from 75% to 35% related to a linear increase of sulphate reduction up to 75 mgl(-1) (50% removal). Since sulphate removal is considered to be dependant on the load of electron donors, the carbon load of the system was varied. Variation of the load changed the intensities of sulphate reduction immediately, but did not influence the carbon removal effectiveness. Doubling of the carbon concentration of 200 mgl(-1) BOD(5) for domestic wastewater usually led to sulphate reduction of up to 150 mgl(-1) (100% removal). The findings show that, particularly in constructed wetland systems, the sulphur cycle in the rhizosphere is of high importance for performance of the waste water treatment and may initiate a reconsideration of the amount of sulphate present in the tap water systems.

Effects of plants and microorganisms in constructed wetlands for wastewater treatment.

Constructed wetlands are a natural alternative to technical methods of wastewater treatment. However, our understanding of the complex processes caused by the plants, microorganisms, soil matrix and substances in the wastewater, and how they all interact with each other, is still rather incomplete. In this article, a closer look will be taken at the mechanisms of both plants in constructed wetlands and the microorganisms in the root zone which come into play when they remove contaminants from wastewater. The supply of oxygen plays a crucial role in the activity and type of metabolism performed by microorganisms in the root zone. Plants' involvement in the input of oxygen into the root zone, in the uptake of nutrients and in the direct degradation of pollutants as well as the role of microorganisms are all examined in more detail. The ways in which these processes act to treat wastewater are dealt with in the following order: Technological aspects; The effect of root growth on the soil matrix; Gas transport in helophytes and the release of oxygen into the rhizosphere; The uptake of inorganic compounds by plants; The uptake of organic pollutants by plants and their metabolism; The release of carbon compounds by plants; Factors affecting the elimination of pathogenic germs.

Annual cycle of nitrogen removal by a pilot-scale subsurface horizontal flow in a constructed wetland under moderate climate.

The annual course of nitrogen removal in a stable operating subsurface horizontal flow constructed wetland (SSF) in a moderate climate was evaluated using a large pool of data from 4 years of operation. In spring and autumn removal efficiencies were found to depend on the nitrogen load in a linear mode. The efficiencies in winter and summer differed extremely (mean removal rates of 0.15/0.7 g m(-2) d(-1) (11%/53%) in January/August) and were independent of the nitrogen load (0.7-1.7 g m(-2) d(-1)) in principle. Oscillations of the removal rates in spring, forming several maxima, suggest seasonal specific effects caused by the dynamics of the plant-physiology finally determining the nitrification efficiency, i.e. via O(2)-supply. Nitrification is limited by temperature during all seasons and surprisingly in midsummer additionally restricted by other seasonal aspects forming a clear-cut relative nitrification minimum (mean rate of 0.43 g m(-2) d(-1) (32%)) in July. The importance and the effect of the plants' gas exchange and oxygen input into the rhizosphere are discussed. Denitrification was nearly complete in midsummer and was clearly restricted at seasonal temperatures below 15 degrees C.

Response of ammonium removal to growth and transpiration of Juncus effusus during the treatment of artificial sewage in laboratory-scale wetlands.

The correlation between nitrogen removal and the role of the plants in the rhizosphere of constructed wetlands are the subject of continuous discussion, but knowledge is still insufficient. Since the influence of plant growth and physiological activity on ammonium removal has not been well characterized in constructed wetlands so far, this aspect is investigated in more detail in model wetlands under defined laboratory conditions using Juncus effusus for treating an artificial sewage. Growth and physiological activity, such as plant transpiration, have been found to correlate with both the efficiency of ammonium removal within the rhizosphere of J. effusus and the methane formation. The uptake of ammonium by growing plant stocks is within in a range of 45.5%, but under conditions of plant growth stagnation, a further nearly complete removal of the ammonium load points to the likely existence of additional nitrogen removal processes. In this way, a linear correlation between the ammonium concentration inside the rhizosphere and the transpiration of the plant stocks implies that an influence of plant physiological activity on the efficiency of N-removal exists. Furthermore, a linear correlation between methane concentration and plant transpiration has been estimated. The findings indicate a fast response of redox processes to plant activities. Accordingly, not only the influence of plant transpiration activity on the plant-internal convective gas transport, the radial oxygen loss by the plant roots and the efficiency of nitrification within the rhizosphere, but also the nitrogen gas released by phytovolatilization are discussed. The results achieved by using an unplanted control system are different in principle and characterized by a low efficiency of ammonium removal and a high methane enrichment of up to a maximum of 72.7% saturation.

Characterizing a stable methane-utilizing mixed culture used in the synthesis of a high-quality biopolymer in an open system.

AIMS: To characterize a methane-utilizing poly-beta-hydroxybutyrate (PHB)-producing microbial community. METHODS AND RESULTS: Three different approaches based on microbiology, analytical chemistry and molecular biology were used to determine the composition of the mixed culture. The dominant species, Methylocystis sp. GB25, represents more than 86% of the total biomass. Seven accompanying bacterial species are present in the mixed culture of which two are methylotrophic bacteria and five are utilizers of complex carbon sources. Both these groups were found to be present at the same ratio with respect to each other. Results of fatty acid analysis and PCR-DGGE fingerprints reflect the stability of the mixed-culture composition in the open system during multiple continuous growth and polymer formation processes throughout a period of 29 months. The consistently high quality of the accumulated polymer further corroborates this finding. CONCLUSION: The methane-utilizing mixed culture has the potential of self-regulation resulting in a stable composition even under non-aseptic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Avoiding the necessity of sterile conditions, as demonstrated in this paper, is an important step towards the development of a viable large-scale process for the production of PHB using cheap substrates like methane from natural or renewable sources. This is the first report characterizing a bacterial mixed culture being used for the biotechnological production of a high-value product in an open system.