Stimulatory effects on bacteria induced by chemical cleaning cause severe biofouling of membranes

Chemical cleaning with hypochlorite is routinely used in membrane-based processes. However, a high-transient cleaning ef ﬁ ciency does not guarantee a low biofouling rate when ﬁ ltration is restarted, with the physiological mechanisms largely remaining unknown. Herein, we investigated the microbial regrowth and surface colonization on membrane surfaces after NaOCl cleaning had been completed. Results of this study showed that the regrowth of model bacteria, Pseudomonas aeruginosa , was initially subject to inhibition due to the damage of key enzymes ’ activity and the accumulation of intracellular reactive oxygen species although the oxidative stress induced by NaOCl had been removed. However, with the resuscitation ongoing, the stimulatory effects became obvious, which was associated with the enhanced production of N -acyl homoserine lactones and the secretion of eDNA that ultimately led to more severe biofouling on the membrane surface. This study elucidates the inhibition – stimulation mechanisms involved in bio ﬁ lm reformation (membrane biofouling) after membrane chemical cleaning, which is of particular signi ﬁ cance to the improvement of cleaning ef ﬁ ciency and application of membrane technologies.


INTRODUCTION
Sodium hypochlorite (NaOCl) is one of the most intensively used cleaning reagents (Wang et al. ; Han et al. ), largely ascribed to its low cost and high efficacy in disinfection (Wang et al. ). NaOCl damages the microbial integrity and causes inactivation through the induced This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited generation of oxidative stress and thus inhibition of the activities of key enzymes (Han et al. , ). Furthermore, NaOCl also solubilizes the organics by increasing their hydrophilicity, which is expected to facilitate the detachment of the biofilm from membranes (Wang et  The apparent contradictory effects of NaOCl exposure prompt us to investigate the microbial behaviors relating to inhibition (e.g., cell death and biofouling alleviation) and resuscitation (e.g., biofilm reformation) after membrane chemical cleaning. In particular, we hypothesize that the stimulatory effects on the QS system of bacteria although the oxidative stress induced by NaOCl has been removed, if any, could cause more severe biofouling of membranes.
As aforementioned, QS is known to influence a wide range of bacterial processes including biofilm formation through the production of and response to signal molecules called autoinducers. Even though NaOCl exposure can cause an initial inhibition of the QS system (Dukan et al. ; Waters & Bassler ; Kim et al. ), its behavior during the resuscitation stage when the oxidative stress is removed in the ongoing operation, which is likely to be very different, has not been systematically studied yet.
In this work, we elucidated the microbial response and colonization behavior on the membrane surface after membrane cleaning had been completed, with the response of QS and the excretion of extracellular DNA (eDNA) during resuscitation clarified. Note that consideration has been largely given to the physiological step of biofouling, i.e., a microbe-driven process at minimum pressure, though there is no avoiding that the pressure applied in membrane filtration plays a vital role in the ongoing formation of cake layer. Our study provides an in-depth understanding of the inhibition-stimulation mechanisms involved in the biofilm reformation (membrane biofouling) after membrane chemical cleaning, which offers new insights into improving the cleaning efficiency for membrane-based processes.
NaOCl exposure and bacteria resuscitation P. aeruginosa was used as the model strain, with pure colonies incubated in a Luria-Bertani (LB) medium at 37 C for 8 h. The colonies were then harvested by centrifugation (4,000 g and 4 C for 10 min), with said cells resuspended in a dilute LB medium at the cell density (∼10 8 cells/mL) similar to an oligotrophic environment (3 × 10 7 ∼ 8 × 10 7 cells/mL) when biofilm is formed (Han et al. ).
NaOCl was initially introduced into the suspensions at different concentrations (0, 5, 10, 20, 50 and 100 ng/10 5 cells) that were chosen based on the levels to which microorganisms are potentially exposed during chemical cleaning (1) where N(t) is the number of live cells (OD 600 in this study), and P d (u) and P m (u) [or P d (t) and P m (t)] are the division and mortality probability functions, respectively. P d (t i )Δt during the next Δt units represents the probability of division, and P m (t i )Δt during the next Δt units indicates the probability of mortality. The probability of inactivation (remaining alive but undivided) is expressed by 1 À P d (t i )Δt-P m (t i )Δt.
Details of the Horowitz's probabilistic model can be found in Supporting Information (SI) Section S1.
Bioassay for the detection of QS signal molecules N-acyl homoserine lactone (AHL), a major class of autoinducers in P. aeruginosa's QS system, was chosen as a model QS signal molecule in the current study due to its sig- (1.0 mL) were mixed in a tube, which was then vortexed for 60 s. After vortexing, the hexadecane and aqueous phases were allowed to separate for 30 min. The absorbance of the aqueous phase was then measured at 400 nm using a multi-mode microplate reader (Synergy 4, Bio-Tek, USA).
Hydrophobicity is expressed as the ratio of hexadecane bound cells to total cells, which is calculated using the following: where A 0 is the absorbance of cell suspension without hexadecane at 400 nm, A is the absorbance of aqueous phase after the addition of hexadecane and %Hex is the relative hydrophobicity of bacterial cells.
For zeta potential test, the cell samples were washed twice and resuspended to OD ¼ 0. All experiments were carried out at least in triplicate with average values and standard deviations provided.

RESULTS AND DISCUSSION
Regrowth of P. aeruginosa after exposure to NaOCl The growth curves of P. aeruginosa exhibit a lag phase followed by an exponential phase and eventually a stationary phase [ Figure 1(a) and SI Figure S1]. For example, significant inhibition on P. aeruginosa division was observed for the initial several hours after disinfection, which has been reported widely in membrane chemical cleaning (Shi et al. We further monitored the production of AHL, a major class of autoinducers in the QS system of P. aeruginosa  Figure S2).

Biofilm formation
Consideration was then given to the effects of pre-exposure to NaOCl (at levels of 0, 20 and 50 ng/10 5 cells) on biofilm formation by using CLSM (Figure 3). According to the In the stimulatory stage (24 h), a significantly higher |Δf| (67.9 ± 1.3 Hz, p < 0.01) was observed for the bacteria sample that had been subject to the oxidative stress of 20 ng NaOCl/10 5 cells compared to the control test (48.8 ± 1.1 Hz) and 50 ng NaOCl/10 5 cells (45.1 ± 0.5 Hz).
Regarding the growth curves of P. aeruginosa [ Figure 1(a)], the aqueous cell density of the sample exposed to 20 ng NaOCl/10 5 cells was roughly increased by ∼100% from 5 to 24 h; however, much pronounced surface colonization Therefore, in this study, we further evaluated P. aeruginosa's intracellular response after exposure to NaOCl. As shown in Figure 5(a), the intracellular ROS contents for P. aeruginosa exposed to 20 and 50 ng NaOCl/10 5 cells are initially higher than the control test (One-tailed, p < 0.01), suggesting that the disinfection process (chemical cleaning) leads to the accumulation of ROS (such as peroxide, superoxide and hydroxyl radicals) (Puspitasari et al. ), with the intracellular stress remaining for, at least, 5 hours after removal of the extracellular stress. Changes in the activities of the key enzymes responsible for the detoxification of ROS [e.g., SOD and catalase (CAT)] are provided in Figures 5(b) and 5(c). It can be seen that the activities of CAT and SOD of P. aeruginosa exposed to NaOCl are significantly lower than the control test (one-tailed, p < 0.01), indicating that NaOCl-mediated membrane cleaning is capable of dete- Following the exponential growth (∼15 hours), the eDNA content in the sample exposed to 20 ng NaOCl/10 5 cells was significantly increased compared to the others, and severe membrane biofouling has been observed [ Figure 3(e)].  NaOCl dosages of up to 20, 50 and 50 ng NaOCl/10 5 cells, respectively (SI Figures S5, S6 and S12). Nevertheless, E. coli, a microorganism without AHL production, exhibited a similar stimulatory effect under NaOCl exposure. Therefore, the role of the QS system, including other signal molecules, needs to be further investigated.
Although overdosage can largely avoid the adverse stimulatory effects (Figure 3