Fig 1.
Hardware configuration and schematic depiction of culture-refreshing steps.
(A) Three-dimensional representation of the versatile continuous cultivation device (VCCD). The system consists of three independently controlled continuous culture units supported by a plexiglass acrylic structure. For each unit, transmittance is measured through the culture chamber by a light-emitting diode coupled to a photo receiver and then reported to a user interface control system. The culture refreshing capability is provided by computer-controlled pinch valves that manage air and liquid flows inside each culture unit. (B) First step of a culture refresh cycle (culture dilution). Upon pinch valve activation, two tubes of the culture unit get pinched, and the air flow is diverted to the medium bottle resulting in the addition of medium into the culture tube. (C) Second step of a culture refresh cycle (excess culture removal). By returning the pinch valve to its original position, two tubes of the culture unit are pinched, which then redirects the air flow to the culture tube and causes the excess of volume to be evacuated into the trash bottle.
Fig 2.
Typical transmittance curves associated with bacterial growth.
(A) Increasing opacity observed when monitoring transmittance of an E. coli culture in LB broth, where the growing cells increasingly block the incident light generated by a light emitting diode. (B) In some cases, growth can alternatively be measured by adding phenol red to the growth medium as a pH indicator. This method is especially employed to follow the growth of M. florum in ATCC 1161 medium, where medium acidification caused by metabolic activity is reported by an increase in the 560nm transmittance.
Fig 3.
Calibration of the Versatile continuous culture device (VCCD) using batch cultures.
(A) Comparison of 560nm transmittance measured by the VCCD and 600nm absorbance measured by a conventional spectrophotometer of an E. coli culture in LB broth. (B) Relationship between relative 560nm transmittance measured by the VCCD and cell density of an E. coli culture grown in LB broth. Red squares are excluded from the correlation determination because they are not part of the exponential growth phase. (C) Relationship between relative 560nm transmittance measured by the VCCD and cell density of S. cerevisae growing in YPAD 2% glucose medium. (D) Relative 560nm transmittance of ATCC 1161 medium through different pH values generally observed during M. florum growth. (E) Cell density of M. florum growing in ATCC 1161 medium through pH decrease. (F) Relationship between relative 560nm transmittance measured by the VCCD and cell density of an M. florum culture grown in ATCC 1161 medium.
Fig 4.
Illustration of available continuous culture modes used to maintain cell growth.
(A) Under its present software configuration, the versatile continuous cultivation device (VCCD) can behave like a turbidostat or a chemostat, or simply be used to measure the transmittance of a batch culture without performing any culture refresh. In the turbidostat mode, the culture is refreshed when a desired transmittance value is detected until: 1) a second value is reached [Real-time feedback loop] or 2) a specified refresh time has elapsed [Threshold-activated]. Alternatively, the chemostat behavior uses a Time interval mode to refresh the culture with a constant specified dilution rate. (B) In the Time interval mode, additional options are available to choose if the interval timer starts at a specified value or at a specified time, and to decide if refreshes are stopped in a time-dependent manner or using a transmittance threshold.
Fig 5.
Establishment of continuous cultures using the versatile continuous culture device (VCCD).
Example of transmittance curves monitored by the VCCD of an E. coli culture growing in LB broth (A) and a M. florum culture growing in ATCC 1161 medium (B) maintained for several hours at 40% and 16% of transmittance, respectively. Culture refreshes were performed using the Real-time feedback loop mode. Cell concentrations measured throughout continuous culture experiments carried out on M. florum (C), E. coli (E), and S. cerevisae cultures (G) using different culture refresh modes, as well as box and whiskers plots of the whole experiments (D), (F), and (H) respectively.