Per Halkjær Nielsen

Extraction of extracellular polymers from activated sludge using a cation exchange resin

Abstract The extraction of water soluble extracellular polymeric substances (EPS) from activated sludge was investigated. The extraction procedure was based upon cation exchange using a cation exchange resin (CER). Activated sludge from two different types of treatment plants responded very similarly to the extraction procedure. The EPS yield was enhanced by increasing the stirring intensity, the amounts of CER added and by increasing the extraction time. For the chosen extraction procedure the yield was twice as high as other commonly used procedures. The extract consisted mainly of protein but also humic compounds, carbohydrates, uronic acids and DNA were found in significant amounts. The extracted amounts and relative fraction of the individual compounds strongly depended on how the extraction was performed. The ratio between protein and carbohydrate was found in the range 3.9–5.1 depending on the extraction time. Humic compounds and DNA were the compounds most easily extracted. HPSEC investigation of the extract revealed that the extraction did not significantly degrade the EPS. Some cell lysis was identified during the extraction for extraction times greater than 1–2 h by observing a decrease in cell number (stained by DAPI, CTC and acridine orange). The lysis was not considered a significant problem for contaminating the EPS. Measurements of the cell number and cell size distribution in the sludge suggested that the cell mass did not account for more than approximately 10–15% of the total organic fraction of the investigated sludge. Two extraction strategies were formulated. One for a very gentle and one for a very effective method. Analytical methods for analysis of sludge and EPS extracts were compared and discussed. A corrected Lowry method for analyzing protein as well as humic compounds was implemented and found suitable.

Enzymatic activity in the activated-sludge floc matrix

The enzymatic activity of activated sludge was investigated with special emphasis on the localization of the enzymes in the sludge floc matrix. Activated sludge from an advanced activated-sludge treatment plant, performing biological N and P removal, was used. An enzymatic fingerprint was established using a panel of six different enzymes. The fingerprint revealed peptidase as the most dominating specific enzyme tested. By monitoring sludge bulk enzymatic activity over a 3-month period using fluorescein diacetate as an enzyme substrate, considerable variations in activity were observed even over short periods (a few days). The variation in esterase activity was to some extent correlated to the presence of humic compounds in the sludge, but not to the sludge protein content. Comparison of full sludge enzyme activity to the activity of a batch-grown sludge culture indicated that enzymes accumulated in sludge flocs. A large proportion of the exoenzymes were immobilized in the sludge by adsorption in the extracellular polymeric substances (EPS) matrix. This was demonstrated by extraction of EPS from the activated sludge using cation exchange. Contemporary to the release of EPS a very large fraction of the exoenzymes was released into the water. This showed that the exoenzymes should be considered to be an integrated part of the EPS matrix rather than as direct indicators of the microbial activity or biomass.

Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes

Reference genomes are required to understand the diverse roles of microorganisms in ecology, evolution, human and animal health, but most species remain uncultured. Here we present a sequence composition–independent approach to recover high-quality microbial genomes from deeply sequenced metagenomes. Multiple metagenomes of the same community, which differ in relative population abundances, were used to assemble 31 bacterial genomes, including rare (<1% relative abundance) species, from an activated sludge bioreactor. Twelve genomes were assembled into complete or near-complete chromosomes. Four belong to the candidate bacterial phylum TM7 and represent the most complete genomes for this phylum to date (relative abundances, 0.06–1.58%). Reanalysis of published metagenomes reveals that differential coverage binning facilitates recovery of more complete and higher fidelity genome bins than other currently used methods, which are primarily based on sequence composition. This approach will be an important addition to the standard metagenome toolbox and greatly improve access to genomes of uncultured microorganisms.

In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants.

ABSTRACT Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of theNitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genusNitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates ofNitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospiramicrocolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources byNitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, theNitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO3− or as CO2) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by theNitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.

Complete nitrification by Nitrospira bacteria

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira-contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.

Measurement of Pools of Protein: Carbohydrate and Lipid in Domestic Wastewater

A more detailed characterization of organic matter in domestic wastewater than given by the bulk parameters BOD, COD and TOC is needed to increase the knowledge about transformations in sewer systems and the quality of wastewater as input to the wastewater treatment processes. In this paper results of various selected methods optimized for analysing pools of carbohydrate, protein and lipid in domestic wastewater were described and discussed. The most suitable method for measurement of protein was the method of Lowry et al. (1951), for carbohydrate it was the anthrone method originally introduced by Dreywood (1946) and for lipid it was an infrared method modified from Standard Methods (APHA et al., 1985). The modified methods showed high precision and compounds in the wastewater did not seem to interfere. The results of the methods were, however, slightly influenced by the choice of standard. A case study was performed using the methods for characterization of wastewater from catchment areas of different characteristics. The fractions of total protein, carbohydrate and lipid of total COD were, on average, 28, 18 and 31% with standard deviations of 4, 6 and 10%, respectively. The dissolved carbohydrate fraction varied considerably—10% as an average of dissolved COD, and 7% as standard deviation, presumably due to degradation in the sewers influenced by differences in residence times.

Complete nitrification by a single microorganism

Nitrification is a two-step process where ammonia is first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite-oxidizing bacteria. Already described by Winogradsky in 1890, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle. Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible, and it was postulated that this process could occur under conditions selecting for species with lower growth rates but higher growth yields than canonical ammonia-oxidizing microorganisms. Still, organisms catalysing this process have not yet been discovered. Here we report the enrichment and initial characterization of two Nitrospira species that encode all the enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding of the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.

On the stability of activated sludge flocs with implications to dewatering

Abstract It was shown that Ca2+ can be extracted from activated sludge from a plant with biological nitrogen and phosphorus removal either by an ion exchange process, where H+, Na+, K+ or Mg2+ served as counter ions, or by chelation by EGTA. The extraction of Ca2+ led to an increase in the number of small particles and subsequently an increase in the specific resistance to filtration. It was argued that approx. half of the total Ca2+ pool was associated with the exopolymers and thereby formed an alginate-like gel, which constituted the backbone of the floc structure. This notion was further emphasized by the observation that addition of Cu2+ which, along with releasing Ca2+, improved the dewaterability—probably because of the Cu2+ ions ability to maintain the three-dimensional structure of the exopolymers.

Role of Sulfate-reducing Bacteria in Corrosion of Mild Steel: a Review

The influence of sulfate‐reducing bacteria on corrosion of mild steel is reviewed, with special emphasis on the effects of biofilm structure and function, medium composition (dissolved oxygen and ferrous ion concentrations) and the physical and chemical properties of iron sulfides. A summary of different corrosion mechanisms is critically discussed, based on electrochemical and rate process analyses. A mechanism is proposed which explains the high corrosion rates observed in the field.

Thaumarchaeotes abundant in refinery nitrifying sludges express amoA but are not obligate autotrophic ammonia oxidizers

Nitrification is a core process in the global nitrogen cycle that is essential for the functioning of many ecosystems. The discovery of autotrophic ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota has changed our perception of the microbiology of nitrification, in particular since their numerical dominance over ammonia-oxidizing bacteria (AOB) in many environments has been revealed. These and other data have led to a widely held assumption that all amoA-encoding members of the Thaumarchaeota (AEA) are autotrophic nitrifiers. In this study, 52 municipal and industrial wastewater treatment plants were screened for the presence of AEA and AOB. Thaumarchaeota carrying amoA were detected in high abundance only in four industrial plants. In one plant, thaumarchaeotes closely related to soil group I.1b outnumbered AOB up to 10,000-fold, and their numbers, which can only be explained by active growth in this continuous culture system, were two to three orders of magnitude higher than could be sustained by autotrophic ammonia oxidation. Consistently, 14CO2 fixation could only be detected in AOB but not in AEA in actively nitrifying sludge from this plant via FISH combined with microautoradiography. Furthermore, in situ transcription of archaeal amoA, and very weak in situ labeling of crenarchaeol after addition of 13CO2, was independent of the addition of ammonium. These data demonstrate that some amoA-carrying group I.1b Thaumarchaeota are not obligate chemolithoautotrophs.