Frederik Hammes

KEY ROLES OF PH AND CALCIUM METABOLISM IN MICROBIAL CARBONATE PRECIPITATION

This paper reviews the general mechanismsof microbial carbonate precipitation and offersan alternative view on the role of calciummetabolism in this process, as well as on theoccurrence of species- and environment-specificcalcification.

Strain-specific ureolytic microbial calcium carbonate precipitation

ABSTRACT During a study of ureolytic microbial calcium carbonate (CaCO3) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO3 crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected. Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (Km) and maximum hydrolysis rates (Vmax) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium.

A novel approach to calcium removal from calcium-rich industrial wastewater

Calcium-rich wastewater is a problem for industries due to calcification during downstream processing. The potential for Ca2+ removal from industrial wastewater through ureolytic microbiological carbonate precipitation was investigated for the first time. Batch experiments were used to determine feasible urea concentrations and hydraulic retention times. These results were applied in a semi-continuous reactor system, where the emphasis was placed on the development of a calcifying sludge. Calcium removal in excess of 90% was achieved throughout the experimental period, while the effluent pH remained at a reasonable level.

Evaluating the Growth Potential of Pathogenic Bacteria in Water

ABSTRACT The degree to which a water sample can potentially support the growth of human pathogens was evaluated. For this purpose, a pathogen growth potential (PGP) bioassay was developed based on the principles of conventional assimilable organic carbon (AOC) determination, but using pure cultures of selected pathogenic bacteria (Escherichia coli O157, Vibrio cholerae, or Pseudomonas aeruginosa) as the inoculum. We evaluated 19 water samples collected after different treatment steps from two drinking water production plants and a wastewater treatment plant and from ozone-treated river water. Each pathogen was batch grown to stationary phase in sterile water samples, and the concentration of cells produced was measured using flow cytometry. In addition, the fraction of AOC consumed by each pathogen was estimated. Pathogen growth did not correlate with dissolved organic carbon (DOC) concentration and correlated only weakly with the concentration of AOC. Furthermore, the three pathogens never grew to the same final concentration in any water sample, and the relative ratio of the cultures to each other was unique in each sample. These results suggest that the extent of pathogen growth is affected not only by the concentration but also by the composition of AOC. Through this bioassay, PGP can be included as a parameter in water treatment system design, control, and operation. Additionally, a multilevel concept that integrates the results from the bioassay into the bigger framework of pathogen growth in water is discussed. The proposed approach provides a first step for including pathogen growth into microbial risk assessment.

Critical Evaluation of the Volumetric “Bottle Effect” on Microbial Batch Growth

ABSTRACT We have analyzed the impact of surface-to-volume ratio on final bacterial concentrations after batch growth. We examined six bottle sizes (20 to 1,000 ml) using three independent enumeration methods to quantify growth. We found no evidence of a so-called volumetric bottle effect, thus contradicting numerous previous reports.

Absolute quantification of microbial taxon abundances

High-throughput amplicon sequencing has become a well-established approach for microbial community profiling. Correlating shifts in the relative abundances of bacterial taxa with environmental gradients is the goal of many microbiome surveys. As the abundances generated by this technology are semi-quantitative by definition, the observed dynamics may not accurately reflect those of the actual taxon densities. We combined the sequencing approach (16S rRNA gene) with robust single-cell enumeration technologies (flow cytometry) to quantify the absolute taxon abundances. A detailed longitudinal analysis of the absolute abundances resulted in distinct abundance profiles that were less ambiguous and expressed in units that can be directly compared across studies. We further provide evidence that the enrichment of taxa (increase in relative abundance) does not necessarily relate to the outgrowth of taxa (increase in absolute abundance). Our results highlight that both relative and absolute abundances should be considered for a comprehensive biological interpretation of microbiome surveys.

Effect of a water extract of Moringa oleifera seeds on the hydrolytic microbial species diversity of a UASB reactor treating domestic wastewater.

The effect of a continuous supply of a water extract of Moringa oleifera seeds (WEMOS) on the hydrolytic microbial population of biomass grown in mesophilic upflow anaerobic sludge blanket reactors treating domestic wastewater was investigated. The WEMOS‐treated sludge had seemingly a wider diversity, with enterobacter and klebsiella as dominant hydrolytic bacteria, compared with the control sludge. Additional tests indicated that various hydrolytic bacteria could degrade WEMOS. It appeared that a continuous supply of WEMOS to an anaerobic digester, treating domestic wastewater, increased the diversity of hydrolytic bacteria and therefore enhanced the biological start‐up of the reactor.

Measurement of Microbial Colonisation of Two Types of Stainless Steel

The biological colonisation of stainless steel surfaces (AISI 304 resp. 316L) was investigated by monitoring the development of natural biofilms, axenic biofilms with selected laboratory strains, and axenic biofilms with a natural biofilm isolate. Digital image analysis of epifluorescence microscopic photographs for quantification of biofilm formation was applied. The strains producing extracellular polymeric substances (EPS) were faster at colonising of steel surface than a non-EPS producing reference strain. However, by far the most rapid colonisation was achieved by the xenic microbiota present in surface water. A distinct preferential colonisation of grain boundaries of the steel surface was noticeable. This is probably related to minor physical or chemical differences in surface characteristics. These (micro) heterogeneities on the steel surface are considered to be related to subsequent anode/cathode formation or to corrosion of the steel. Finally, the steel grade 304 was slightly more susceptible to colonisation than 316L as observed by digital image analysis, most probably due to the presence of molybdenum in the case of 316L.

Chlorine-susceptible and chlorine-resistant type 021N bacteria occurring in bulking activated sludges

ABSTRACT Two filamentous bacteria causing bulking in two activated sludges were examined. Investigations using morphological features, staining techniques, and fluorescent in situ hybridization identified both filaments as type 021N. However, an examination of the effect of chlorine on the sludges revealed a chlorine-susceptible type 021N in one sludge and a chlorine-resistant type 021N in the other.

A uniform bacterial growth potential assay for different water types

The bacterial growth potential is important to understand and manage bacterial regrowth-related water quality concerns. Bacterial growth potential depends on growth promoting/limiting compounds, therefore, nutrient availability is the key factor governing bacterial growth potential. Selecting proper tools for bacterial growth measurement is essential for routine implementation of the growth potential measurement. This study proposes a growth potential assay that is universal and can be used for different water types and soil extract without restrictions of pure culture or cultivability of the bacterial strain. The proposed assay measures the sample bacterial growth potential by using the indigenous community as inocula. Flow cytometry (FCM) and adenosine tri-phosphate (ATP) were used to evaluate the growth potential of six different microbial communities indigenous to the sample being analyzed, with increasing carbon concentrations. Bottled mineral water, non-chlorinated tap water, seawater, river water, wastewater effluent and a soil organic carbon extract were analyzed. Results showed that indigenous bacterial communities followed normal batch growth kinetics when grown on naturally present organic carbon. Indigenous bacterial growth could detect spiked organic carbon concentrations as low as 10 μg/L. The indigenous community in all samples responded proportionally to the increase in acetate-carbon and proportional growth could be measured with both FCM and ATP. Bacterial growth was proportional to the carbon concentration but not the same proportion factor for the different water samples tested. The effect of inoculating the same water with different indigenous microbial communities on the growth potential was also examined. The FCM results showed that the highest increase in total bacterial cell concentration was obtained with bacteria indigenous to the water sample. The growth potential assay using indigenous bacterial community revealed consistent results of bacterial growth in all the different samples tested and therefore providing a fast, more stable, and accurate approach for monitoring the biological stability of waters compared to the previously developed assays. The growth potential assay can be used to aid in detecting growth limitations by compounds other than organic carbon.