Multiplexed chemostat system for quantification of biodiversity and ecosystem functioning in anaerobic digestion

<div><p>Continuous cultures in chemostats have proven their value in microbiology, microbial ecology, systems biology and bioprocess engineering, among others. In these systems, microbial growth and ecosystem performance can be quantified under stable and defined environmental conditions. This is essential when linking microbial diversity to ecosystem function. Here, a new system to test this link in anaerobic, methanogenic microbial communities is introduced. Rigorously replicated experiments or a suitable experimental design typically require operating several chemostats in parallel. However, this is labor intensive, especially when measuring biogas production. Commercial solutions for multiplying reactors performing continuous anaerobic digestion exist but are expensive and use comparably large reactor volumes, requiring the preparation of substantial amounts of media. Here, a flexible system of Lab-scale Automated and Multiplexed Anaerobic Chemostat system (LAMACs) with a working volume of 200 mL is introduced. Sterile feeding, biomass wasting and pressure monitoring are automated. One module containing six reactors fits the typical dimensions of a lab bench. Thanks to automation, time required for reactor operation and maintenance are reduced compared to traditional lab-scale systems. Several modules can be used together, and so far the parallel operation of 30 reactors was demonstrated. The chemostats are autoclavable. Parameters like reactor volume, flow rates and operating temperature can be freely set. The robustness of the system was tested in a two-month long experiment in which three inocula in four replicates, i.e., twelve continuous digesters were monitored. Statistically significant differences in the biogas production between inocula were observed. In anaerobic digestion, biogas production and consequently pressure development in a closed environment is a proxy for ecosystem performance. The precision of the pressure measurement is thus crucial. The measured maximum and minimum rates of gas production could be determined at the same precision. The LAMACs is a tool that enables us to put in practice the often-demanded need for replication and rigorous testing in microbial ecology as well as bioprocess engineering.</p></div

Detailed prices of major components required for the construction of one LAMACs module containing six reactors.

<p>Detailed references for these items can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193748#pone.0193748.s006" target="_blank">S1 Table</a>.</p

Linear regression of biogas accumulation over time.

<p>Linear regression of biogas accumulation over time.</p

Biogas production of four anaerobic digesters measured by the LAMACs.

<p>A) Actual accumulated biogas over time (red curves) and linear regressions (blue lines) B) Biogas production rates derived from linear regressions.</p

General view of the LAMACs and of components of one chemostat in the LAMACs module.

<p>(A) 3D view of a LAMACs module, i.e., six chemostats. The electric box containing controlling cards is on the top of the module and above any source of liquids to prevent electric failure in case of leakage. A waterproof stirring plate is underneath the heating block containing the six reactor vessels. Three peristaltic pumps are aligned above each vessel. The upper range of bags is for biogas collection; the lower range serves as substrate reservoirs. The dimensions of one module are 50 cm width × 52 cm length × 100 cm height. (B) Schematic view of a chemostat detailing the use of the three-hose connector. One port is used as inlet for feeding, and two ports as outlet for biomass wasting and degassing. One port for manual sampling is sealed with a rubber stopper. A 0.45 μm pore-size filter is placed in the feedline ahead of the reactor to prevent contamination of the medium.</p

Stable temperature of the LAMACs during operation.

<p>Average temperature and standard deviation over three days of operation at three different positions in the heating block. Data points were recorded every 15 minutes, with 280 total data points.</p

Development of average weekly biogas production rates over time for anaerobic digesters with three different inocula.

<p>Four replicated anaerobic digesters were operated per condition. White boxes stand for the INOC A condition, light gray boxes for INOC B and dark-gray boxes for INOC C. The Kruskal Wallis test was significant in the first eight weeks and the Dunn post hoc test for pairwise multiple comparison displays inocula differences with ‘*’ symbols. In weeks 4 to 6, the simple substrate was used instead of the complex substrate.</p

Dynamics of biomass concentration for twelve anaerobic digesters over nine weeks.

<p>Biomass concentration is expressed as volatile solids. A color code was applied by inoculum origin; Light gray stands for INOC A replicates, dark-gray stands for INOC B replicates and black stands for INOC C replicates. During weeks 4 to 6, a simple substrate with halved loading rate in terms of COD was applied to the reactors.</p

Experimental and analytical set-up of the reactor system.

<p>(A) The bubble column reactors: Aeration of the reactors served the purpose of providing oxygen, homogenizing the water column and applying a constant shear stress on the biofilm surface. (B) Monochloramine was added daily or weekly to two of the three reactors. (C) Biofilms were sampled by removing individual polyethylene coupons that were glued onto stainless steel rings. The rings were reinserted into the bubble column. On each coupon, biofilm morphology was analyzed by automated quantitative image analysis. From the same coupon, DNA was extracted and bacterial community dynamics were assessed using molecular community fingerprinting.</p

Morphology and community development with experimental time for the three reactors in the first experiment.

<p>The data for the second experiment can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080692#pone.0080692.s006" target="_blank">Figure S6</a>. Treatment periods were shaded where appropriate (panels C-F). Each monochloramine pulse injection is indicated by an arrow. Vertical gray lines mark Mondays. By performing Principal Component and Principal Coordinate Analyses, most of the variability in the data sets could be summarized in the first Principal Component (PC1) for morphology data and the first Principal Co-ordinate Axis (PCoA1) for the community data. More negative values on PC1 (note inverted axis labels) were related to more complex biofilm morphology. Similarly, the higher the value on PCoA1, the more complex was the community structure. Note the inverted axis in panels <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080692#pone-0080692-g003" target="_blank">Figure <b>3</b></a> and S6A, C and E.</p