Bioremediation on a chip: A portable microfluidic device for efficient screening of bacterial biofilm with polycyclic aromatic hydrocarbon removal capacity

Highlights

• A microfluidic device was developed to grow biofilms of Pseudomonas and Gordonia.

• The microfluidic system was constructed based on computational fluid dynamics.

• The microchip enables to screen PAHs' degrading capability at continuous flow.

• Continuous flow and high shear stress in the microchip enhance biofilms formation.

• Pseudomonas sp. P26 biofilm in the microdevice shows the highest PAHs removal.

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are pollutants of critical environmental and public health concern and their elimination from contaminated sites is significant for the environment. Biodegradation studies have demonstrated the ability of bacteria in biofilm conformation to enhance the biodegradation of pollutants. In this study, we used our newly developed microfluidic platform to explore biofilm development, properties, and applications of fluid flow, as a new technique for screening PAHs-degrading biofilms. The optimization and evaluation of the flow condition in the microchannels were performed through computational fluid dynamics (CFD). The formation of biofilms by PAHs-degrading bacteria Pseudomonas sp. P26 and Gordonia sp. H19, as pure cultures and co-culture, was obtained in the developed microchips. The removal efficiencies of acenaphthene, fluoranthene and pyrene were determined by HPLC. All the biofilms formed in the microchips removed all tested PAHs, with the higher removal percentages observed with the Pseudomonas sp. P26 biofilm (57.4% of acenaphthene, 40.9% of fluoranthene, and 28.9% of pyrene). Pseudomonas sp. P26 biofilm removed these compounds more efficiently than planktonic cultures. This work proved that the conformation of biofilms enhances the removal rate. It also provided a new tool to rapid and low-cost screen for effective pollutant-degrading biofilms.

Introduction

Oil pollution is a persistent and virtually chronic ecological problem globally, especially as a result of industrialized oil activities (Nuñal et al., 2014). Among the hydrocarbons derived from petroleum are the polycyclic aromatic hydrocarbons (PAHs) that contain condensed rings of benzene and are toxic, mutagenic, and carcinogenic (“IARC Monographs on the Evaluation of Carcinogenic Risks to Humans,” 2010). Although bacterial communities are able to metabolize persistent organic compounds, the low relative abundance of microorganisms, the limited access, and the shortage of nutrients in the environment make the rate of microbial activities extremely slow (Krell et al., 2013). Several PAHs, which are classified as low molecular weight (LMW) PAHs (two or three benzene rings) and high molecular weight (HMW) PAHs (four or more benzene rings), have been described as degraded by different bacteria (Sakshi and Haritash, 2020). LMW PAHs (such as naphthalene, fluorene, acenaphthene, phenanthrene, anthracene and fluoranthene) and HMW PAHs (such as pyrene and benzo[a]pyrene), alone or in a hydrocarbon mix, were efficiently degraded by Pseudomonas, Bacillus, Streptomyces, Rhodococcus, Gordonia, Amycolatopsis, Stenotrophomonas, Raoultella, Serratia, and Aeromonas, among other bacterial genera (Alegbeleye et al., 2017; Alessandrello et al., 2017; Bourguignon et al., 2014; Chaudhary et al., 2011; Isaac et al., 2013; Zhu et al., 2016). On the other hand, the ability of microorganisms to congregate in biofilms generates many advantages compared to planktonic cultures, such as greater protection against the external environment, developed abilities for cellular communication and exchange of genetic material, higher availability of nutrients, and persistence in different metabolic states (Zhang et al., 2019b). The tolerance of biofilms to toxic compounds is of biotechnological interest for their application in bioremediation and biotransformation protocols, even when the concentrations of these substrates are much higher than those that microorganisms could support in a planktonic state (Isaac et al., 2017).

The microfluidics devices are identified as Lab On a Chip (LOC), and they consist of a network of microchannels that incorporate different sections, chambers, columns, and reservoirs (Whitesides, 2006). The LOC application for the study of microorganisms is an emerging field, where the benefits of miniaturization of the system offer new opportunities. In particular, the combination of high-resolution images and precise spatiotemporal control over the microenvironments of organisms provide an accurate view of biological events at the micron level. This new tool opens alternatives of recreating biological environments more realistically than traditional macroscopic cultures in dishes, well plates or flasks (Pérez-Rodríguez et al., 2021). Microfluidic approaches allow to accurately simulate the complex natural environment and mimic variations in environmental conditions (Stanley et al., 2016).

Since the use of microdevices of the LOC type is relatively new in the field of molecular biology, there are few studies on the use of these microdevices for the cultivation of microorganisms and their application to different processes. Currently, there is an increase in the research of LOC for studies on bacterial behavior in response to external stimulus, including taxis processes (Miller et al., 2019; Pérez-Rodríguez et al., 2021; Roggo et al., 2018; Stricker et al., 2020), drug screening (Anutrakunchai et al., 2018; Bourguignon et al., 2019; Matsumoto et al., 2016) and biofilm formation (Kim et al., 2012; Liu et al., 2019a; Valiei et al., 2012; Zhang et al., 2019a, 2019b). A LOC system based on a miniaturized chip is the best method to study the initial biofilm formation and the effects on the biofilms imposed by environmental conditions, including nutrient concentration, surface properties, shear stress, quorum sensing (Zhang et al., 2019a, 2019b). Researchers have recently advanced the development of different detection modes coupled to the microfluidic system to study biofilms (Funari et al., 2018; Liu et al., 2019a). With the focus of environmental applications, microfluidic systems were used to evaluate the tolerance of heavy metals by actinobacteria strains (Cao et al., 2013), and the intake of benzo[a]pyrene by fungi (Baranger et al., 2021).

One of the limitations of current bioremediation techniques is to understand microbial processes, and one part of these bioprocesses consists of laboratory tests showing that microorganisms from the samples have the potential to transform the contaminants, while bioaugmentation or biostimulation is necessary. Nowadays, all these laboratory tests are done at macroscale with standard microbiologic methodologies, and which take long periods of time to obtain results and high cost due to the supplies and reagents. Additionally, there is a need to find a technique to effectively monitor and control bioremediation processes in situ in contaminated soils, to guide the success of bioprocesses. (Azubuike et al., 2016).

The development of LOC will improve the identification of bacterial populations biofilms capable of degrading oil derivative compounds which will have positive impacts on the bioremediation processes with effective control and monitoring. Moreover, the novel LOC technology, as a miniaturized system, provides advantages of interaction between bacterial biofilms and the hydrodynamic environment condition (for example, shear stress) (Kim et al., 2012) and could also be used in the generation of a portable and easy to use biosensor to analyze effluents or contaminated soil samples. In this study, we proposed a new pipeline to screen for PAHs-degrading bacterial biofilms with high throughput using microfluidic techniques (Fig. 1A). A newly developed technique was used to increase the efficiency of the screening process: a microreactor device (Bourguignon et al., 2018a) for biofilm-forming bacteria selection and high-throughput/fast PAHs-degrading biofilm screening (Fig. 1). The aim of the present work was to validate the implementation of microfluidic systems created to optimize the formation of bacterial biofilms in order to evaluate the capacity of bacteria to carry out the biodegradation of pollutants in a physiological biofilm state. The study of this biological process is significant for future optimizations of PAH bioremediation protocols using bacterial biofilms and biostimulation approaches.