High-throughput screening of environmental polysaccharide-degrading bacteria using biomass containment and complex insoluble substrates

Abstract

Carbohydrate degradation by microbes plays an important role in global nutrient cycling, human nutrition, and biotechnological applications. Studies that focus on the degradation of complex recalcitrant polysaccharides are challenging because of the insolubility of these substrates as found in their natural contexts. Specifically, current methods to examine carbohydrate-based biomass degradation using bacterial strains or purified enzymes are not compatible with high-throughput screening using complex insoluble materials. In this report, we developed a small 3D printed filter device that fits inside a microplate well that allows for the free movement of bacterial cells, media, and enzymes while containing insoluble biomass. These devices do not interfere with standard microplate readers and can be used for both short- (24-48 h) and long-duration (> 100 h) experiments using complex insoluble substrates. These devices were used to quantitatively screen in a high-throughput manner environmental isolates for their ability to grow using lignocellulose or rice grains as a sole nutrient source. Additionally, we determined that the microplate-based containment devices are compatible with existing enzymatic assays to measure activity against insoluble biomass. Overall, these microplate containment devices provide a platform to study the degradation of complex insoluble materials in a high-throughput manner and have the potential to help uncover ecologically important aspects of bacterial metabolism as well as to accelerate biotechnological innovation.

Keywords: 3D printing; Cellulose; Cellvibrio japonicus; Chitin; Lignocellulose; Polysaccharide.

Fig. 1

Assessment of three mBCD prototypes for continuous measurements of bacterial growth in a microplate reader. Diagram of Mk-1 m prototype (a). Frontal view photograph (a1) and schematic (a2). Top view photograph (a3) and schematic (a4). Diagram of the Mk-2 m prototype (b). Frontal view photograph (b1) and schematic (b2). Top view photograph (b3) and schematic (b4). Diagram of the Mk-3 m prototype (c). Frontal view photograph (c1) and schematic (c2). Top view photograph (c3) and schematic (c4). In panels (a–c), the yellow bar at the bottom of each image corresponds to 5 mm. The inset (d) shows the mBCDs placement inside of a single microplate well. Growth analysis of Cellvibrio japonicus on minimal media supplemented with 0.5% glucose (Glc) to evaluate growth dynamics (e). All experiments were done in biological triplicates using wild-type C. japonicus at 30 °C in a microplate with a constant level of aeration. Error bars indicate standard deviation, but at times are too small to be depicted

Fig. 2

Growth analysis of wild-type C. japonicus (circles) and a Δgsp deletion mutant (squares) using mBCDs for short-term experiments with insoluble carbon sources. The experiments were performed in a minimal media with (a) 5% β-chitin, (b) 10% glutinous rice, (c) 5% fungal biomass, or (d) 5% mealworm cuticle as the only source of carbon. All growth experiments were performed in biological triplicate at 30 °C with high levels of aeration. Error bars indicate standard deviation, but at times are too small to be observed

Fig. 3

Growth analysis of C. japonicus wild-type (circles) and a Δgsp deletion mutant (squares) using mBCDs for long-term experiments with insoluble carbon source. The experiments were designed to compare microplate BCDs (a-c) with test tube BCDs (D-F). All growth experiments were performed in a minimal media with (a, d) 5% α-chitin, (b, e) 5% filter paper, or (c, f) 10% crab shell as the only source of carbon. Growth analyses were done at 30 °C with high levels of aeration in biological triplicate. Error bars show standard deviation, but are often too small to be depicted

Fig. 4

Using mBCDs to screen environmental isolates capacity to degrade insoluble substrates. Either (a) corn stover or (b) glutinous rice was used as the only source of carbon. The tested strains were Cellvibrio japonicus (Cj WT), Cellvibrio japonicus Δgsp (Cj Δgsp), Escherichia coli K-12 (K-12), Arthrobacter nicotianae (WW33), Klebsiella oxytoca (WW55), Enterobacter spp. (WW64), and Bacillus firmus (LZ66). All growth analysis experiments were done in biological triplicate at 30 °C with high levels of aeration. Error bars indicate standard deviation

Fig. 5

Activity assay of WW33 supernatants against glutinous rice using biomass containment. Glucose released from degradation of the starches in glutinous rice by the hydrolytic activity of enzymes collected from the growth medium of Arthrobacter nicotianae (WW33) was determined at 0, 4, 8, and 24 h. Each column represents an average of triplicate measurements, with the error bars depicting standard deviation