Enrichment of phosphate-accumulating organisms (PAOs) in a microfluidic model biofilm system by mimicking a typical aerobic granular sludge feast/famine regime

Wastewater treatment using aerobic granular sludge has gained increasing interest due to its advantages compared to conventional activated sludge. The technology allows simultaneous removal of organic carbon, nitrogen, and phosphorus in a single reactor system and is independent of space-intensive settling tanks. However, due to the microscale, an analysis of processes and microbial population along the radius of granules is challenging. Here, we introduce a model system for aerobic granular sludge on a small scale by using a machine-assisted microfluidic cultivation platform. With an implemented logic module that controls solenoid valves, we realized alternating oxic hunger and anoxic feeding phases for the biofilms growing within. Sampling during ongoing anoxic cultivation directly from the cultivation channel was achieved with a robotic sampling device. Analysis of the biofilms was conducted using optical coherence tomography, fluorescence in situ hybridization, and amplicon sequencing. Using this setup, it was possible to significantly enrich the percentage of polyphosphate-accumulating organisms (PAO) belonging to the family Rhodocyclaceae in the community compared to the starting inoculum. With the aid of this miniature model system, it is now possible to investigate the influence of a multitude of process parameters in a highly parallel way to understand and efficiently optimize aerobic granular sludge-based wastewater treatment systems.Key points• Development of a microfluidic model to study EBPR.• Feast-famine regime enriches polyphosphate-accumulating organisms (PAOs).• Microfluidics replace sequencing batch reactors for aerobic granular sludge research.

In situ quantification of poly(3-hydroxybutyrate) and biomass in Cupriavidus necator by a fluorescence spectroscopic assay

Fluorescence spectroscopy offers a cheap, simple, and fast approach to monitor poly(3-hydroxybutyrate) (PHB) formation, a biodegradable polymer belonging to the biodegradable polyester class polyhydroxyalkanoates. In the present study, a fluorescence and side scatter-based spectroscopic setup was developed to monitor in situ biomass, and PHB formation of biotechnological applied Cupriavidus necator strain. To establish PHB quantification of C. necator, the dyes 2,2-difluoro-4,6,8,10,12-pentamethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaene (BODIPY493/503), ethyl 5-methoxy-1,2-bis(3-methylbut-2-enyl)-3-oxoindole-2-carboxylate (LipidGreen2), and 9-(diethylamino)benzo[a]phenoxazin-5-one (Nile red) were compared with each other. Fluorescence staining efficacy was obtained through 3D-excitation-emission matrix and design of experiments. The coefficients of determination were ≥ 0.98 for all three dyes and linear to the high-pressure liquid chromatography obtained PHB content, and the side scatter to the biomass concentration. The fluorescence correlation models were further improved by the incorporation of the biomass-related side scatter. Afterward, the resulting regression fluorescence models were successfully applied to nitrogen-deficit, phosphor-deficit, and NaCl-stressed C. necator cultures. The highest transferability of the regression models was shown by using LipidGreen2. The novel approach opens a tailor-made way for a fast and simultaneous detection of the crucial biotechnological parameters biomass and PHB content during fermentation. Key points • Intracellular quantification of PHB and biomass using fluorescence spectroscopy. • Optimizing fluorescence staining conditions and 3D-excitation-emission matrix. • PHB was best obtained by LipidGreen2, followed by BODIPDY493/503 and Nile red. Graphical abstract

An adaptable microreactor to investigate the influence of interfaces on Pseudomonas aeruginosa biofilm growth

Biofilms are ubiquitous and notoriously difficult to eradicate and control, complicating human infections and industrial and agricultural biofouling. However, most of the study had used the biofilm model that attached to solid surface and developed in liquid submerged environments which generally have neglected the impact of interfaces. In our study, a reusable dual-chamber microreactor with interchangeable porous membranes was developed to establish multiple growth interfaces for biofilm culture and test. Protocol for culturing Pseudomonas aeruginosa (PAO1) on the air–liquid interface (ALI) and liquid–liquid interface (LLI) under static environmental conditions for 48 h was optimized using this novel device. This study shows that LLI model biofilms are more susceptible to physical disruption compared to ALI model biofilm. SEM images revealed a unique “dome-shaped” microcolonies morphological feature, which is more distinct on ALI biofilms than LLI. Furthermore, the study showed that ALI and LLI biofilms produced a similar amount of extracellular polymeric substances (EPS). As differences in biofilm structure and properties may lead to different outcomes when using the same eradication approaches, the antimicrobial effect of an antibiotic, ciprofloxacin (CIP), was chosen to test the susceptibility of a 48-h-old P. aeruginosa biofilms grown on ALI and LLI. Our results show that the minimum biofilm eradication concentration (MBEC) of 6-h CIP exposure for ALI and LLI biofilms is significantly different, which are 400 μg/mL and 200 μg/mL, respectively. These results highlight the importance of growth interface when developing more targeted biofilm management strategies, and our novel device provides a promising tool that enables manipulation of realistic biofilm growth. Key points • A novel dual-chamber microreactor device that enables the establishment of different interfaces for biofilm culture has been developed. • ALI model biofilms and LLI model biofilms show differences in resistance to physical disruption and antibiotic susceptibility.