Yehuda Cohen

Dynamics of biofilm formation under different nutrient levels and the effect on biofouling of a reverse osmosis membrane system

Pseudomonas aeruginosa PAO1 wild type and a mucoid derivative (FRD1) which over produces alginate were used to foul reverse osmosis (RO) membranes. When operated at a constant flux, biofilm formation on the RO membrane resulted in a slow rise in transmembrane pressure (TMP) of 22% for the initial four days of operation, followed by a sharp increase of 159% over the following two days. The initial slow increase in TMP was probably due to the formation of a biofilm on the membrane surface, which then accelerated the rate of biofouling through the effect of concentration polarization. At later stages of operation, most of the bacterial biomass consisted of dead cells. The amount of extracellular polymeric substances appeared to correlate positively with the number of dead cells. The results indicate that prolonging the initial stage of slow TMP increase and avoiding the latter stage of accelerated TMP increase would provide a sustainable operation of the RO system. These results suggest that nutrient limitation could reduce biofilm accumulation and delay the increase in TMP.

Elevated level of the second messenger c-di-GMP in Comamonas testosteroni enhances biofilm formation and biofilm-based biodegradation of 3-chloroaniline.

The bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that determines bacterial lifestyle between the planktonic and biofilm modes of life. Although the role of c-di-GMP signaling in biofilm development and dispersal has been extensively studied, how c-di-GMP signaling influences environmental bioprocess activities such as biodegradation remains unexplored. To elucidate the impacts of elevating c-di-GMP level on environmental bioprocesses, we constructed a Comamonas testosteroni strain constitutively expressing a c-di-GMP synthase YedQ from Escherichia coli and examined its capability in biofilm formation and biodegradation of 3-chloroaniline (3-CA). The high c-di-GMP strain exhibited an increased binding to Congo red dye, a decreased motility, and an enhanced biofilm formation capability. In planktonic cultures, the strain with an elevated c-di-GMP concentration and the wild type could degrade 3-CA comparably well. However, under batch growth conditions with a high surface to volume ratio, an elevated c-di-GMP concentration in C. testosteroni significantly increased the contribution of biofilms in 3-CA biodegradation. In continuous submerged biofilm reactors, C. testosteroni with an elevated c-di-GMP level exhibited an enhanced 3-CA biodegradation and a decreased cell detachment rate. Taken together, this study provides a novel strategy to enhance biofilm-based biodegradation of toxic xenobiotic compounds through manipulating bacterial c-di-GMP signaling.

The correlation between biofilm biopolymer composition and membrane fouling in submerged membrane bioreactors

Biofouling, the combined effect of microorganism and biopolymer accumulation, significantly reduces the process efficiency of membrane bioreactors (MBRs). Here, four biofilm components, alpha-polysaccharides, beta-polysaccharides, proteins and microorganisms, were quantified in MBRs. The biomass of each component was positively correlated with the transmembrane pressure increase in MBRs. Proteins were the most abundant biopolymer in biofilms and showed the fastest rate of increase. The spatial distribution and co-localization analysis of the biofouling components indicated at least 60% of the extracellular polysaccharide (EPS) components were associated with the microbial cells when the transmembrane pressure (TMP) entered the jump phase, suggesting that the EPS components were either secreted by the biofilm cells or that the deposition of these components facilitated biofilm formation. It is suggested that biofilm formation and the accumulation of EPS are intrinsically coupled, resulting in biofouling and loss of system performance. Therefore, strategies that control biofilm formation on membranes may result in a significant improvement of MBR performance.

The application of nitric oxide to control biofouling of membrane bioreactors

A novel strategy to control membrane bioreactor (MBR) biofouling using the nitric oxide (NO) donor compound PROLI NONOate was examined. When the biofilm was pre‐established on membranes at transmembrane pressure (TMP) of 88–90u2009kPa, backwashing of the membrane module with 80u2009μM PROLI NONOate for 45u2009min once daily for 37 days reduced the fouling resistance (Rf) by 56%. Similarly, a daily, 1u2009h exposure of the membrane to 80u2009μM PROLI NONOate from the commencement of MBR operation for 85 days resulted in reduction of the TMP and Rf by 32.3% and 28.2%. The microbial community in the control MBR was observed to change from days 71 to 85, which correlates with the rapid TMP increase. Interestingly, NO‐treated biofilms at 85 days had a higher similarity with the control biofilms at 71 days relative to the control biofilms at 85 days, indicating that the NO treatment delayed the development of biofilm bacterial community. Despite this difference, sequence analysis indicated that NO treatment did not result in a significant shift in the dominant fouling species. Confocal microscopy revealed that the biomass of biopolymers and microorganisms in biofilms were all reduced on the PROLI NONOate‐treated membranes, where there were reductions of 37.7% for proteins and 66.7% for microbial cells, which correlates with the reduction in TMP. These results suggest that NO treatment could be a promising strategy to control biofouling in MBRs.

Characterization of the archaeal community fouling a membrane bioreactor

Biofilm formation, one of the primary causes of biofouling, results in reduced membrane flux or increased transmembrane pressure and thus represents a major impediment to the wider implementation of membrane bioreactor (MBR) technologies for water purification. Most studies have focused on the role of bacteria in membrane fouling as they are the most dominant and best studied organisms present in the MBR. In contrast, there is limited information on the role of the archaeal community in biofilm formation in MBRs. This study investigated the composition of the archaeal community during the process of biofouling in an MBR. The archaeal community was observed to have lower richness and diversity in the biofilm than the sludge during the establishment of biofilms at low transmembrane pressure (TMP). Clustering of the communities based on the Bray-Curtis similarity matrix indicated that a subset of the sludge archaeal community formed the initial biofilms. The archaeal community in the biofilm was mainly composed of Thermoprotei, Thermoplasmata, Thermococci, Methanopyri, Methanomicrobia and Halobacteria. Among them, the Thermoprotei and Thermoplasmata were present at higher relative proportions in the biofilms than they were in the sludge. Additionally, the Thermoprotei, Thermoplasmata and Thermococci were the dominant organisms detected in the initial biofilms at low TMP, while as the TMP increased, the Methanopyri, Methanomicrobia, Aciduliprofundum and Halobacteria were present at higher abundances in the biofilms at high TMP.

Probing the internal micromechanical properties of Pseudomonas aeruginosa biofilms by Brillouin imaging

Biofilms are organised aggregates of bacteria that adhere to each other or surfaces. The matrix of extracellular polymeric substances that holds the cells together provides the mechanical stability of the biofilm. In this study, we have applied Brillouin microscopy, a technique that is capable of measuring mechanical properties of specimens on a micrometre scale based on the shift in frequency of light incident upon a sample due to thermal fluctuations, to investigate the micromechanical properties of an active, live Pseudomonas aeruginosa biofilm. Using this non-contact and label-free technique, we have extracted information about the internal stiffness of biofilms under continuous flow. No correlation with colony size was found when comparing the averages of Brillouin shifts of two-dimensional cross-sections of randomly selected colonies. However, when focusing on single colonies, we observed two distinct spatial patterns: in smaller colonies, stiffness increased towards their interior, indicating a more compact structure of the centre of the colony, whereas, larger (over 45u2009μm) colonies were found to have less stiff interiors.Biofilm structure: Shining a light on stiffnessA specialized microscopy technique can monitor biofilm stiffness in a non-destructive manner, yielding insights into biofilm structure and development. The technique, called Brillouin imaging, uses changes in the frequency of light interacting with a substance to reveal fine detail about the material’s mechanical properties. Peter Török and colleagues at Imperial College London, with co-workers in Singapore, used Brillouin imaging to study biofilms of Pseudomonas aeruginosa bacteria at different stages in their life cycle. In young colonies, stiffness increased towards the interior of the biofilm, while mature colonies had less stiff interiors. The older biofilms may therefore have hollow interiors or may have been moving towards a phase of bacterial dispersal from the biofilm state. This non-disruptive method to study mechanical variations within and between living biofilms may help efforts to combat biofilms in clinical, environmental and industrial situations.

Isolation of Bdellovibrio bacteriovorus from a tropical wastewater treatment plant and predation of mixed species biofilms assembled by the native community members

It is reported here that a predatory bacterium belonging to the Genus Bdellovibrio, was isolated from activated sludge at the Ulu Pandan Water Reclamation Plant, Singapore. 16S rDNA gene sequencing analysis revealed that this isolate was 99% identical to Bdellovibrio bacteriovorus strain Tiberius and hence is designated as Bdellovibrio bacteriovorus UP. Using a novel approach based on fluorescence in situ hybridization (FISH), a prey cell density-dependent growth pattern of B. bacteriovorus UP was established. B. bacteriovorus UP preyed upon a broad range of bacterial species (60 species) isolated from the activated sludge. Except for Ochrobactrum anthropi, all Gram-negative species were sensitive to predation by B. bacteriovorus UP irrespective of the mode of growth (planktonic or biofilm). Similarly, the predation-sensitive species were not protected by the predation-resistant species, O. anthropi, as determined in multiple dual-species planktonic and biofilm consortia. Given the broad prey spectrum, B. bacteriovorus UP may impact functional community members, which are largely members of the Proteobacteria. Thus, these results provide an important insight to the role of predatory bacteria in shaping of community structure and function in both natural and engineered ecosystems.

Draft Genome Sequence of the Model Naphthalene-Utilizing Organism Pseudomonas putida OUS82

ABSTRACT Pseudomonas putida OUS82 was isolated from petrol- and oil-contaminated soil in 1992, and ever since, it has been used as a model organism to study the microbial assimilation of naphthalene and phenanthrene. Here, we report the 6.7-Mb draft genome sequence of P. putida OUS82 and analyze its featured pathways for biodegradation.

Predation by Bdellovibrio bacteriovorus significantly reduces viability and alters the microbial community composition of activated sludge flocs and granules

We recently isolated and characterised a predatory Bdellovibrio bacteriovorus strain from activated sludge (Ulu Pandan Water Reclamation Plant, Singapore), and this strain, B. bacteriovorus UP, was able to prey upon a broad spectrum of bacterial isolates from the activated sludge when grown as planktonic cells or as biofilms. Here, we have tested the effect of Bdellovibrio predation on floccular and granular sludge to determine if the spatial organisation, loosely or tightly aggregated communities, was protective from predation. The effect of predation was assessed using a combination of biomass quantification, cellular activity measurement and microscopic image analysis to determine community viability. Additionally, changes in the microbial communities due to predation by B. bacteriovorus UP were analysed through total RNA sequencing. Predation led to a significant reduction in microbial activity and total biomass for both floccular and granular sludge communities. Predation was also associated with significant changes in the microbial community composition in both communities, with >90% of the community members reduced in relative abundance after 24 h. Of those community members, the dominant organisms, such as Proteobacteria and Bacteroidetes, were the most affected phylotypes. This suggests that predatory bacteria, which display indiscriminant feeding, could significantly shift the species composition and thus, may disturb the operational performance of wastewater treatment systems.