Custom fabrication of biomass containment devices using 3-D printing enables bacterial growth analyses with complex insoluble substrates

Abstract

Physiological studies of recalcitrant polysaccharide degradation are challenging for several reasons, one of which is the difficulty in obtaining a reproducibly accurate real-time measurement of bacterial growth using insoluble substrates. Current methods suffer from several problems including (i) high background noise due to the insoluble material interspersed with cells, (ii) high consumable and reagent cost and (iii) significant time delay between sampling and data acquisition. A customizable substrate and cell separation device would provide an option to study bacterial growth using optical density measurements. To test this hypothesis we used 3-D printing to create biomass containment devices that allow interaction between insoluble substrates and microbial cells but do not interfere with spectrophotometer measurements. Evaluation of materials available for 3-D printing indicated that UV-cured acrylic plastic was the best material, being superior to nylon or stainless steel when examined for heat tolerance, reactivity, and ability to be sterilized. Cost analysis of the 3-D printed devices indicated they are a competitive way to quantitate bacterial growth compared to viable cell counting or protein measurements, and experimental conditions were scalable over a 100-fold range. The presence of the devices did not alter growth phenotypes when using either soluble substrates or insoluble substrates. We applied biomass containment to characterize growth of Cellvibrio japonicus on authentic lignocellulose (non-pretreated corn stover), and found physiological evidence that xylan is a significant nutritional source despite an abundance of cellulose present.

Comprehensive functional characterization of the glycoside hydrolase family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification

“…We hypothesized that the ability of the Δ4βG mutant to grow on XyG was due in part to xylose utilization arising from the functional α‐xylosidase Xyl31A encoded by the xyloglucan utilization locus (XyGUL) of C. japonicus (Larsbrink et al ., ). To test this hypothesis, we deleted the xylose isomerase gene xylA , from the Δ4βG mutant strain, as it was previously shown that a C. japonicus Δ xylA mutant was unable to utilize xylose as a carbon source (Nelson et al ., ). The quintuple mutant had a reduced growth rate compared to the Δ4βG mutant and achieved threefold lower maximum density (Supporting Information Fig.…”

The complex physiology of Cellvibrio japonicus xylan degradation relies on a single cytoplasmic β‐xylosidase for xylo‐oligosaccharide utilization

“…The combination of C. japonicus mutational analysis and E. coli heterologous expression indicated that the gly43F gene product was a cytoplasmic β‐xylosidase that constituted the final step of heteroxylan breakdown. As expected, a previously constructed Δ xylA mutant strain (Nelson et al ., ), which encodes xylose isomerase, was unable to grow using xylo‐oligosaccharides or xylose. This Δ xylA mutant is a strong negative control strain because xylose isomerase is an essential component for conversion of xylose to xyulose‐5‐phosphate, which is the entry point into the pentose phosphate pathway (van Maris et al ., ).…”

Systems analysis of the glycoside hydrolase family 18 enzymes from Cellvibrio japonicus characterizes essential chitin degradation functions

“…All cultures were incubated at 30 °C with an aeration of 225 RPM. For insoluble substrates growth was measured as a function of the optical density at 600 nm (OD 600 ) with a Spec20D+ spectrophotometer (Thermo Scientific) using biomass containment as needed (56). In growth experiments with glucose or Nacetylglucosamine growth was monitored using a Tecan M200Pro microplate reader at 600 nm (OD 600 ) (Tecan Trading AG, Switzerland).…”

“…To assay for chitin degradation, 10 µL of overnight cultures of the C. japonicus strains to be analyzed were spotted onto the chitin plate and incubated for 4 d at 30 °C. Then, plates were stained with a 0.1% (w/v) Congo red solution for 10 min followed by 10 min destaining with a 1M NaCl solution, as described previously for detection of degradation of carboxyl methyl cellulose (27,45,56).…”