Periodic Reporting for period 1 - FULLREMOVAL (Feasibility of ULtrafiLtration membranes for REMOVAL of antibiotic resistant bacteria and antibiotic resistance genes from secondary effluents from wastewater treatment plants)
Reporting period: 2021-10-01 to 2023-09-30
Antibiotic resistance in bacterial pathogens is responsible for thousands of deaths each year as diseases caused by antibiotic resistant bacteria (ARB) are no longer treatable with standard antibiotics. Although wastewater treatment plants (WWTPs) are expected to return water to the environment in safe conditions, the increase in antibiotic consumption has made these facilities hotspots for the development of ARB and the spread of antibiotic resistance genes (ARGs), for which a mitigation strategy is required.
Although membrane technology is widely extended for wastewater treatment, the use of low-pressure membranes is mainly limited to secondary treatment with membrane bioreactors (MBR). MBR systems outperform conventional activated sludge (CAS) treatment in terms of effluent quality, but conventional plants are still dominant in most European countries. Upgrading existing plants to MBR would be expensive and does not seem realistic in the short term. However, the addition of ultrafiltration post-treatment may be a feasible solution for existing WWTPs.
The main objective of FULLREMOVAL was to evaluate the use of ultrafiltration membranes as a post-treatment of secondary effluents from conventional WWTPs in terms of removal efficiency of both ARB and ARGs. In addition, the effect of some variables such as DNA characteristics or the degree of membrane fouling on the removal efficiencies was also considered. According to the results of the FULLREMOVAL project, ultrafiltration membranes are highly efficient for the removal of ARB and ARGs in different scenarios. In addition, they do not require high working pressures, for which they are considered a promising tool to mitigate the current spread of antibiotic resistance in the WWTP effluent receiving environments.
Although membrane technology is widely extended for wastewater treatment, the use of low-pressure membranes is mainly limited to secondary treatment with membrane bioreactors (MBR). MBR systems outperform conventional activated sludge (CAS) treatment in terms of effluent quality, but conventional plants are still dominant in most European countries. Upgrading existing plants to MBR would be expensive and does not seem realistic in the short term. However, the addition of ultrafiltration post-treatment may be a feasible solution for existing WWTPs.
The main objective of FULLREMOVAL was to evaluate the use of ultrafiltration membranes as a post-treatment of secondary effluents from conventional WWTPs in terms of removal efficiency of both ARB and ARGs. In addition, the effect of some variables such as DNA characteristics or the degree of membrane fouling on the removal efficiencies was also considered. According to the results of the FULLREMOVAL project, ultrafiltration membranes are highly efficient for the removal of ARB and ARGs in different scenarios. In addition, they do not require high working pressures, for which they are considered a promising tool to mitigate the current spread of antibiotic resistance in the WWTP effluent receiving environments.
FULLREMOVAL project studied the potential of using ultrafiltration membranes as post-treatment for the removal of antibiotic resistant bacteria (ARB) and extracellular antibiotic resistance genes (eARGs) from secondary effluents to avoid their dissemination in the receiving water environments.
The tasks performed during this MSCA fellowship were included in five specific work packages (WP), three of which were related to purely scientific tasks, and the other two were dedicated to dissemination, training and project management activities. The main scientific results are summarized in the next bullet points:
WP2: ARB and eARGs monitorization in local WWTP and evaluation of the transforming ability of eARGs: Transformation experiments performed with two bacterial species commonly found in wastewater, E. coli and P. putida, allowed to evaluate the risk of horizontal gene transfer by transformation entailed by extracellular antibiotic resistance genes present in the secondary effluents discharge. Both strains were incubated together with three different plasmids conferring resistance to ampicillin and streptomycin antibiotics. However, neither E. coli nor P. putida showed growth of bacteria in the presence of those antibiotics, meaning natural transformation did not occur. Our conclusion was that several conditions are required for natural transformation occurrence, such as natural competence for transformation of the strain of interest, certain plasmids characteristics as well as experimental conditions and all these requisites are not always fulfilled simultaneously, for which it can be more difficult than initially thought. However, further experiments will be performed in this direction in the future with different strains and plasmids.
WP3: Evaluation of ARB and eARGs removal efficiency of ultrafiltration membranes: A tailored lab-scale ultrafiltration facility was built to evaluate the removal efficiency of ultrafiltration membranes for ARB and eARGs in WWTP secondary effluents. The experimental facility comprised basically a cross-flow membrane cassette connected to a feed water bottle by a peristaltic pump. This lab-scale plant intended to mimic a tertiary treatment facility for which the feed water was always a synthetic effluent matrix to which ARB or eARGs were added separately so that the final concentrations were comparative to those in real secondary effluents. The experiments with model ARB (P. putida KT2440) were performed in first place and the results allowed concluding that ultrafiltration membranes with 100 kDa MWCO were able to remove more than 99.99% of the bacteria present in the feed water. The experiments with model eARG (supercoiled R388 plasmids) were performed with the same lab-scale facility and identical conditions for a new membrane cassette, obtaining an average removal efficiency of 99.2%. In addition, the effect of certain parameters such as plasmid length and conformation in the removal efficiency was also evaluated, concluding that plasmid size is not a relevant fact for the retention by ultrafiltration membranes. On the contrary, the plasmid conformation did make a difference and linear plasmids could permeate more easily than supercoiled, probably because of their less branched structure.
WP4:Verification of ARB and eARGs presence inside membrane biofouling layer and conjugation occurrence: The presence of ARB within the membrane biofouling layer was verified with Scanning Electron Microscopy (SEM) as well as Helium Ion Microscopy (HIM). In addition, the presence of resistance plasmids in the membrane fouling layer was successfully verified with Direct-gene fluorescence in situ hybridization or Direct-GeneFISH, a technique with great potential for the detection of genes but not used before for the detection of plasmids deposition in ultrafiltration membrane fouling layers. The verification of conjugation occurrence was replaced by experiments evaluating the effect of fouling in the removal efficiency of ARB and eARGs by ultrafiltration membranes, concluding that the presence of a fouling layer can slightly diminish the retention of eARGs by ultrafiltration membranes.
Scientific papers, communications to conferences, outreach initiatives such as Science is wonderful or Researchers night as well as other knowledge transfer activities, assured the exploitation and dissemination of the results, and their accessibility for further research activities related to FULLREMOVAL.
The tasks performed during this MSCA fellowship were included in five specific work packages (WP), three of which were related to purely scientific tasks, and the other two were dedicated to dissemination, training and project management activities. The main scientific results are summarized in the next bullet points:
WP2: ARB and eARGs monitorization in local WWTP and evaluation of the transforming ability of eARGs: Transformation experiments performed with two bacterial species commonly found in wastewater, E. coli and P. putida, allowed to evaluate the risk of horizontal gene transfer by transformation entailed by extracellular antibiotic resistance genes present in the secondary effluents discharge. Both strains were incubated together with three different plasmids conferring resistance to ampicillin and streptomycin antibiotics. However, neither E. coli nor P. putida showed growth of bacteria in the presence of those antibiotics, meaning natural transformation did not occur. Our conclusion was that several conditions are required for natural transformation occurrence, such as natural competence for transformation of the strain of interest, certain plasmids characteristics as well as experimental conditions and all these requisites are not always fulfilled simultaneously, for which it can be more difficult than initially thought. However, further experiments will be performed in this direction in the future with different strains and plasmids.
WP3: Evaluation of ARB and eARGs removal efficiency of ultrafiltration membranes: A tailored lab-scale ultrafiltration facility was built to evaluate the removal efficiency of ultrafiltration membranes for ARB and eARGs in WWTP secondary effluents. The experimental facility comprised basically a cross-flow membrane cassette connected to a feed water bottle by a peristaltic pump. This lab-scale plant intended to mimic a tertiary treatment facility for which the feed water was always a synthetic effluent matrix to which ARB or eARGs were added separately so that the final concentrations were comparative to those in real secondary effluents. The experiments with model ARB (P. putida KT2440) were performed in first place and the results allowed concluding that ultrafiltration membranes with 100 kDa MWCO were able to remove more than 99.99% of the bacteria present in the feed water. The experiments with model eARG (supercoiled R388 plasmids) were performed with the same lab-scale facility and identical conditions for a new membrane cassette, obtaining an average removal efficiency of 99.2%. In addition, the effect of certain parameters such as plasmid length and conformation in the removal efficiency was also evaluated, concluding that plasmid size is not a relevant fact for the retention by ultrafiltration membranes. On the contrary, the plasmid conformation did make a difference and linear plasmids could permeate more easily than supercoiled, probably because of their less branched structure.
WP4:Verification of ARB and eARGs presence inside membrane biofouling layer and conjugation occurrence: The presence of ARB within the membrane biofouling layer was verified with Scanning Electron Microscopy (SEM) as well as Helium Ion Microscopy (HIM). In addition, the presence of resistance plasmids in the membrane fouling layer was successfully verified with Direct-gene fluorescence in situ hybridization or Direct-GeneFISH, a technique with great potential for the detection of genes but not used before for the detection of plasmids deposition in ultrafiltration membrane fouling layers. The verification of conjugation occurrence was replaced by experiments evaluating the effect of fouling in the removal efficiency of ARB and eARGs by ultrafiltration membranes, concluding that the presence of a fouling layer can slightly diminish the retention of eARGs by ultrafiltration membranes.
Scientific papers, communications to conferences, outreach initiatives such as Science is wonderful or Researchers night as well as other knowledge transfer activities, assured the exploitation and dissemination of the results, and their accessibility for further research activities related to FULLREMOVAL.
The results of FULLREMOVAL will be of high value for European research, as they may contribute to the definition of a mitigation strategy for conventional wastewater treatment plants currently in use, whose limited capacity to retain antibiotic resistance has been widely reported. In addition, the project results will shed light on the use of low-pressure membranes to remove not only bacteria, but also extracellular resistance genes, as most of the studies available to date have been in the field of DNA purification, but scarce in a wastewater context. On the other hand, this project will make it possible to consider ultrafiltration as a promising option for tertiary treatment, which is often neglected in favour of high-pressure processes such as nanofiltration or reverse osmosis, with the consequent higher energy consumption.