Evaluation of two rapid ultrafiltration-based methods for SARS-CoV-2 concentration from wastewater.

Quantitative measurements of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in raw wastewater have been implemented worldwide since the beginning of the pandemic. Recent efforts are being made to evaluate different viral concentration methodologies to overcome supplier shortages during lockdowns. A set of 22-wastewater samples seeded with murine hepatitis virus (MHV), a member of the Coronaviridae family, and the bacteriophage MS2, were used to characterize and compare two ultrafiltration-based methods: a centrifugal ultrafiltration device (Centricon® Plus-70) and the automated concentrating pipette CP-Select™. Based on the recovery efficiencies, significant differences were observed for MHV, with Centricon® Plus-70 (24%) being the most efficient method. Nevertheless, concentrations of naturally occurring SARS-CoV-2, Human adenoviruses and JC polyomaviruses in these samples did not result in significant differences between methods suggesting that testing naturally occurring viruses may complement the evaluation of viral concentration methodologies. Based on the virus adsorption to solids and the necessity of a pre-centrifugation step to remove larger particles and avoid clogging when using ultrafiltration methods, we assessed the percentage of viruses not quantified after ultrafiltration. Around 23% of the detected SARS-CoV-2 would be discarded during the debris removal step. The CP-Select™ provided the highest concentration factor (up to 333×) and the lowest LoD (6.19 × 103 GC/l) for MHV and proved to be fast, automatic, highly reproducible and suitable to work under BSL-2 measures.

Human exposure assessment to antibiotic-resistant Escherichia coli through drinking water.

Antibiotic-resistant bacteria (ARB) are a potential threat to human health through drinking water with strong evidence of ARB presence in post treated tap water around the world. This study examines potential human exposure to antibiotic-resistant (AR) Escherichia coli (E. coli) through drinking water, the effect of different drinking water treatments on AR E. coli and the concentration of AR E. coli required in the source water for the EU Drinking Water Directive (DWD) (Council Directive 98/83/EC, 0CFU/100ml of E. coli in drinking water) to be exceeded. A number of scenarios were evaluated to examine different water treatment combinations and to reflect site specific conditions at a study site in Europe. A literature search was carried out to collate data on the effect of environmental conditions on AR E. coli, the effect of different water treatments on AR E. coli and typical human consumption levels of tap water. A human exposure assessment model was developed with probability distributions used to characterise uncertainty and variability in the input data. Overall results show the mean adult human exposure to AR E. coli from tap water consumption ranged between 3.44×10-7 and 2.95×10-1cfu/day for the scenarios tested and varied depending on the water treatments used. The level of AR E. coli required in the source water pre-treatment to exceed the DWD varied between 1 and 5logcfu/ml, depending on the water treatments used. This can be used to set possible monitoring criteria in pre-treated water for potential ARB exposure in drinking water.

Changes in Microbial Biofilm Communities during Colonization of Sewer Systems.

The coexistence of sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) in anaerobic biofilms developed in sewer inner pipe surfaces favors the accumulation of sulfide (H2S) and methane (CH4) as metabolic end products, causing severe impacts on sewerage systems. In this study, we investigated the time course of H2S and CH4 production and emission rates during different stages of biofilm development in relation to changes in the composition of microbial biofilm communities. The study was carried out in a laboratory sewer pilot plant that mimics a full-scale anaerobic rising sewer using a combination of process data and molecular techniques (e.g., quantitative PCR [qPCR], denaturing gradient gel electrophoresis [DGGE], and 16S rRNA gene pyrotag sequencing). After 2 weeks of biofilm growth, H2S emission was notably high (290.7±72.3 mg S-H2S liter(-1) day(-1)), whereas emissions of CH4 remained low (17.9±15.9 mg COD-CH4 liter(-1) day(-1)). This contrasting trend coincided with a stable SRB community and an archaeal community composed solely of methanogens derived from the human gut (i.e., Methanobrevibacter and Methanosphaera). In turn, CH4 emissions increased after 1 year of biofilm growth (327.6±16.6 mg COD-CH4 liter(-1) day(-1)), coinciding with the replacement of methanogenic colonizers by species more adapted to sewer conditions (i.e., Methanosaeta spp.). Our study provides data that confirm the capacity of our laboratory experimental system to mimic the functioning of full-scale sewers both microbiologically and operationally in terms of sulfide and methane production, gaining insight into the complex dynamics of key microbial groups during biofilm development.

New phylotypes of mesophilic filamentous anoxygenic phototrophic bacteria enriched from sulfide-containing environments.

Agar-based solid media with increasing concentrations of organic matter were used to isolate new members of the Chloroflexaceae (phylum Chloroflexi) from mesophilic environments containing sulfide. Inorganic media yielded less than 10% positive enrichments, which were not able to be maintained after repetitive inoculations in fresh medium. The use of casaminoacids and complex organic acid mixtures increased the number of positive enrichments (up to 45%) from both water and sediment samples. Two different green filamentous bacteria, SisoF2 and SalF, could be stably maintained as co-cultures for long periods and their phylogeny inferred from the analysis of complete sequences of the 16S rRNA gene. Ribotype SalF showed a high homology (95-98%) to previously isolated Oscillochloris trichoides strains. The 16S rRNA gene sequence retrieved from culture SisoF2 was largely divergent (< 92% similarity) from any sequence derived from either cultured representatives or environmental samples, suggesting that ribotype SisoF2 may constitute a new genus within the phylum. The presence of the new morphotypes in the environment from where they were enriched was analysed by high-resolution phylogenetic fingerprinting.

Light responses in the green sulfur bacterium Prosthecochloris aestuarii: changes in prosthecae length, ultrastructure, and antenna pigment composition.

The morphology (mainly prosthecae length), ultrastructure, and antenna pigment composition of the green sulfur bacterium Prosthecochloris aestuarii changed when grown under different light intensities. At light intensities of 0.5 and 5 micromol quanta m(-2) s(-1), the cells had a star-like morphology. Prosthecae, the characteristic appendages of the genus Prosthecochloris, were 232 nm and 194 nm long, respectively. In contrast, when grown at 100 micromol quanta m(-2) s(-1), these appendages were shorter (98 nm) and the cells appeared more rod-shaped. Transmission electron microscopy revealed a significant decrease in the cell perimeter to area ratio and in the number of chlorosomes per linear microm of membrane as light intensity increased. In addition to these morphological and ultrastructural responses, Prosthecochloris aestuarii exhibited changes in its pigment composition as a function of light regime. Lower specific pigment content and synthesis rates were found in cultures grown at light intensities above 5 micromol quanta m(-2) s(-1). A blue shift in the bacteriochlorophyll (BChl) c Q(y) absorption maximum of up to 17.5 nm was observed under saturating light conditions (100 micromol quanta m(-2) s(-1)). This displacement was accompanied by changes in the composition of BChl c homologs and by a very low carotenoid content. The morphological, ultrastructural and functional changes exhibited by Prosthecochloris aestuarii revealed the strong light-response capacity of this bacterium to both high and low photon-flux densities.

Effect of carotenoid deficiency on cells and chlorosomes of Chlorobium phaeobacteroides.

The effects of inhibition of carotenoid biosynthesis by 2-hydroxybiphenyl on the photosynthetic growth, pigment composition and chlorosome structure of Chlorobium phaeobacteroides strain CL1401 were examined. At a concentration of 20 micrograms 2-hydroxybiphenyl .ml-1, carotenoid synthesis was largely inhibited (85%), but the photosynthetic growth rate was almost unaffected (mu control = 0.00525 +/- 0.00007 h-1 and mu HBP-treated = 0.00505 +/- 0.0005 h-1). Cells grown in the presence of the inhibitor were 5 microns-70 microns long, while control cells were between 2-5 microns long. Moreover, 2-hydroxybiphenyl-treated cells contained fewer, unevenly distributed chlorosomes per micron of cytoplasmic membrane with an irregular arrangement (2.5 +/- 1.5 vs of 9.1 +/- 1.9). This was concomitant to the 83% decrease in the content of bacteriochlorophyll (BChl) e in 2-hydroxybiphenyl-treated cells. Electron microscopy revealed that the shape of carotenoid-depleted chlorosomes changed from ellipsoidal to spherical, although the mean volume was similar to that of control chlorosomes. SDS-PAGE analysis of the chlorosome polypeptide composition showed that the amount of CsmA protein decreased by 60% in carotenoid-depleted chlorosomes. This was paralleled by a decrease in the baseplate BChl a content. The data suggest that carotenoids are close to the chlorosomal baseplate, where they carry out both structural and photoprotective functions.

Identification of the bacteriochlorophyll homologues of Chlorobium phaeobacteroides strain UdG6053 grown at low light intensity.

Detailed APCI LC-MS/MS analysis using an improved HPLC separation reveals the green sulphur bacterium Chlorobium phaeobacteroides strain UdG6053 to contain a wider range of distinct bacteriochlorophyll homologues than has been previously recognised in Chlorobiaceae. The diversity in the homologue distribution is confirmed as arising from differences in the extent of alkylation of the macrocycle and variation in the nature of the esterifying alcohol and a novel series of bacteriochlorophyll structures has been recognised. Homologues containing esterifying alcohols other than farnesol, a number of which have not previously been reported in Chlorobiaceae, are present in high relative abundance. Confirmation of the structures of the esterifying alcohols has been obtained by hydrolysis and analysis by GC-MS.

Nanosecond laser photolysis studies of chlorosomes and artificial aggregates containing bacteriochlorophyll e: evidence for the proximity of carotenoids and bacteriochlorophyll a in chlorosomes from Chlorobium phaeobacteroides strain CL1401.

Time-resolved, laser-induced changes in absorbance, delta A(lambda; t), have been recorded with a view to probing pigment-pigment interactions in chlorosomes (control as well as carotenoid-depleted) and artificial aggregates of bacteriochlorophyll e (BChle). Control chlorosomes were isolated from Chlorobium phaeobacteroides strain CL1401, whose chromophores comprise BChle, bacteriochlorophyll a (BChla) and several carotenoid (Car) pigments; Car-depleted chlorosomes, from cells grown in cultures containing 2-hydroxybiphenyl. Artificial aggregates were prepared by dispersing BChle in aqueous phase in the presence of monogalactosyl diglyceride. In chlorosomes delta A(lambda; t) shows, besides a signal attributable to triplet Car (with a half-life of about 4 microseconds), signals in the Qy regions of both BChl. The BChla signal decays at the same rate as the Car signal, which is explained by postulating that some Car are in intimate contact with some baseplate BChla pigments, and that when a ground-state Car changes into a triplet Car, the absorption spectrum of its BChla neighbors undergoes a concomitant change (termed transient environment-induced perturbation). The signal in the Qy-region of BChle behaves differently: its amplitude falls, under reducing conditions, by more than a factor of two during the first 0.5 microsecond (a period during which the Car signal suffers negligible diminution), and is much smaller under nonreducing conditions. The BChle signal is also attributed to transient environment-induced perturbation, but in this case the perturber is a BChle photoproduct (probably a triplet or a radical ion). The absence of long-lived BChle triplets in all three systems, and of long-lived BChla triplets in chlorosomes, indicates that BChle in densely packed assemblies is less vulnerable to photodamage than monomeric BChle and that, in chlorosome, BChla rather than BChle needs, and receives, photoprotection from an adjacent Car.

Effect of carotenoid biosynthesis inhibition on the chlorosome organization in Chlorobium phaeobacteroides strain CL1401.

We have studied the effect of the absence of carotenoids on the organization of bacteriochlorophylls (BChls) in chlorosomes of Chlorobium (Chl.) phaeobacteroides strain CL1401. Carotenoid-depleted chlorosomes were obtained by means of 2-hydroxybiphenyl-supplemented cultures. In the presence of the inhibitor, isorenieratene (Isr) and beta-Isr biosynthesis were inhibited to more than 95%, leading to an accumulation of the colorless precursor phytoene inside the chlorosomes. In addition, there was a 30-40% decrease in the baseplate BChl a content. The absorption spectrum of the carotenoid-depleted chlorosomes showed a 10 nm blue shift in the BChl e Qy absorption peak. Under reducing conditions, a decrease in the BChl a/BChl e fluorescence emission ratio was observed in carotenoid-depleted chlorosomes relative to that in control chlorosomes, caused mainly by the decrease in the BChl a content. The steady-state fluorescence emission anisotropy in the BChl e region dropped from approximately 0.24 for native chlorosomes to approximately 0.14 for carotenoid-depleted ones, indicating reorganization of BChl e. The circular dichroism (CD) signal of the carotenoid-depleted chlorosomes was increased two times in the BChl e Qy region. A simple model based on the structure proposed was used to explain the observed effects. Carotenoids might affect the angle between the direction of the BChl e Qy transition and the axis of the rod. The orientation of BChl a in the baseplate remains unchanged in carotenoid-depleted chlorosomes, although there is a partial loss of BChl a as a consequence of a decrease in the baseplate size. The carotenoids are most likely rather close to the BChls and appear to be important for the aggregate structure in Chl. phaeobacteroides.

Fast energy transfer between BChl d and BChl c in chlorosomes of the green sulfur bacterium Chlorobium limicola.

We have studied energy transfer in chlorosomes of Chlorobium limicola UdG6040 containing a mixture of about 50% bacteriochlorophyll (BChl) c and BChl d each. BChl d-depleted chlorosomes were obtained by acid treatment. The energy transfer between the different pigment pools was studied using both steady-state and time-resolved fluorescence spectroscopy at room temperature and low temperature. The steady-state emission of the intact chlorosome originated mainly from BChl c, as judged by comparison of fluorescence emission spectra of intact and BChl d-depleted chlorosomes. This indicated that efficient energy transfer from BChl d to BChl c takes place. At room temperature BChl c/d to BChl a excitation energy transfer (EET) was characterized by two components of 27 and 74 ps. At low temperature we could also observe EET from BChl d to BChl c with a time constant of approximately 4 ps. Kinetic modeling of the low temperature data indicated heterogeneous fluorescence kinetics and suggested the presence of an additional BChl c pool, E790, which is more or less decoupled from the baseplate BChl a. This E790 pool is either a low-lying exciton state of BChl c which acts as a trap at low temperature or alternatively represents the red edge of a broad inhomogeneous absorption band of BChl c. We present a refined model for the organization of the spatially separated pigment pools in chlorosomes of Cb. limicola UdG6040 in which BChl d is situated distal and BChl c proximal with respect to the baseplate.

Rearrangement of light harvesting bacteriochlorophyll homologues as a response of green sulfur bacteria to low light intensities.

The pigment composition of two species of green-colored BChl c-containing green sulfur bacteria (Chlorobium limicola and C. chlorovibrioides) and two species of brown-colored BChl e-containing ones (C. phaeobacteroides and C. phaeovibrioides) incubated at different light intensities have been studied. All species responded to the reduction of light intensity from 50 to 1 µEinstein(E) m(-2) s(-1) by an increase in the specific content of light harvesting pigments, bacteriochlorophylls and carotenoids. At critical light intensities (0.5 to 0.1 µE m(-2) s(-1)) only brown-colored chlorobia were able to grow, though at low specific rates (0.002 days(-1) mg prot(-1)). High variations in the relative content of farnesyl-bacteriochlorophyll homologues were found, in particular BChl e 1 and BChl e 4, which were tentatively identified as [M, E] and [I, E] BChlF e, respectively. The former was almost completely lost upon reduction of light intensity from 50 to 0.1 µE m(-2) s(-1), whereas the latter increased from 7.2 to 38.4% and from 13.6 to 42.0% in C. phaeobacteroides and C. phaeovibrioides, respectively. This increase in the content of highly alkylated pigment molecules inside the chlorosomes of brown species is interpreted as a physiological mechanism to improve the efficiency of energy transfer towards the reaction center. This study provides some clues for understanding the physiological basis of the adaptation of brown species to extremely low light intensities.

Separation of bacteriochlorophyll homologues from green photosynthetic sulfur bacteria by reversed-phase HPLC.

A reversed-phase High Performance Liquid Cromatography (HPLC) method has been developed to accurately separate bacteriochlorophyllsc, d ande homologues in a reasonably short run time of 60 minutes. By using this method, two well-defined groups of bacteriochlorophyll homologue peaks can be discriminated. The first one consists of 4 peaks (min 24 to 30), which corresponds to the four main farnesyl homologues. The second peak subset is formed by a cluster of up to 10 minor peaks (min 33 to 40). These peaks can be related with series of several alcohol esters of the different chlorosome chlorophylls. The number of homologues was, however, quite variable depending on both, the bacteriochlorophyll and the bacterial species. The method hereby described, also provides a good separation of other photosynthetic pigments, either bacterial (Bacteriochlorophylla, chlorobactene, isorenieratene and okenone) or algal ones (Chlorophylla, Pheophytina and ß-carotene). A preliminary screening of the homologue composition of several green photosynthetic bacterial species and isolates, has revealed different relative quantitative patterns. These differences seem to be related to physiological aspects rather than to taxonomic ones. The application of the method to the study of natural populations avoids the typical drawbacks on the pigment identification of overlapping eukaryotic and prokaryotic phototrophic microorganisms, giving further information about their physiological status.