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Temperature as competitive strategy determining factor in pulse-fed aerobic bioreactors.

ABSTRACT: Exposing a microbial community to alternating absence and presence of carbon substrate in aerobic conditions is an effective strategy for enrichment of storage polymers (polyhydroxybutyrate, PHB) producing microorganisms. In this work we investigate to which extent intermediate storage polymer production is a temperature independent microbial competition determining factor. Eight parallel bioreactors were operated in the temperature range of 20-40?°C, but intermediate storage polymer production was only obtained at 25-35?°C. Besides PHB production and consumption, cell decay and subsequent cryptic growth on lysis products was found to determine process properties and the microbial community structure at all operational temperatures. At 40?°C decay processes cannot be overcome with additional energy from storage polymers, and fast-growing microorganisms dominate the system. At 20?°C, highly competitive communities with ambiguous storage properties were enriched. The results described here demonstrate that a rigorous experimental approach could aid in the understanding of competitive strategies in microbial communities.

SUBMITTER: Stouten GR

PROVIDER: S-EPMC6864080 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Temperature as competitive strategy determining factor in pulse-fed aerobic bioreactors.

Stouten Gerben Roelandt GR Hogendoorn Carmen C Douwenga Sieze S Kilias Estelle Silvia ES Muyzer Gerard G Kleerebezem Robbert R

The ISME journal 20190902 12

Exposing a microbial community to alternating absence and presence of carbon substrate in aerobic conditions is an effective strategy for enrichment of storage polymers (polyhydroxybutyrate, PHB) producing microorganisms. In this work we investigate to which extent intermediate storage polymer production is a temperature independent microbial competition determining factor. Eight parallel bioreactors were operated in the temperature range of 20-40 °C, but intermediate storage polymer production ...[more]

PMID: 31477820

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Project description:Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 106 cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2-10 × 106 cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22-34 × 106 cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3-6 × 106 cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.

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Temperature as competitive strategy determining factor in pulse-fed aerobic bioreactors.

Publications

Temperature as competitive strategy determining factor in pulse-fed aerobic bioreactors.

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