Engineering self‐flocculating Halomonas campaniensis for wastewaterless open and continuous fermentation

Abstract

Halomonas has been developed as a platform for the next generation industrial biotechnology allowing open and nonsterile growth without microbial contamination under a high-salt concentration and alkali pH. To reduce downstream cost associated with continuous centrifugation and salt containing wastewater treatment, Halomonas campaniensis strain LS21 was engineered to become self-flocculating by knocking out an etf operon encoding two subunits of an electron transferring flavoprotein in the predicted electron transfer chain. Self-flocculation could be attributed to the decrease of the surface charge and increase of the cellular hydrophobicity resulted from deleted etf. A wastewaterless fermentation strategy based on the self-flocculating H. campaniensis was developed for growth and the production of poly-3-hydroxybutyrate (PHB) as an example. Most microbial cells flocculated and precipitated to the bottom of the bioreactor within 1 min after stopping the aeration and agitation. The supernatant can be used again without sterilization or inoculation for the growth of the next batch after collecting the precipitated cell mass. The wastewaterless process was conducted for four runs without generating wastewater. PHB accumulation by the self-flocculent strain was enhanced via promoter and ribosome binding site optimizations, the productivities of cell dry weight and PHB were increased from 0.45 and 0.18 g·L −1 ·hr −1 for the batch process compared to 0.82 and 0.33 g·L −1 ·hr −1 for the wastewaterless continuous process, respectively. This has clearly demonstrated the advantages of the wastewaterless process in that it not only reduces wastewater but also increases cell growth and product formation efficiency in a given period of time. K E Y W O R D S continuous process, etf, flocculation, Halomonas campaniensis, next generation industry biotechnology, NGIB, PHB

From Organic Wastes to Bioplastics: Feasibility of Nonsterile Poly(3-hydroxybutyrate) Production by Zobellella denitrificans ZD1

“… 59 Ling et al reported that glucose-grown Halomonas campaniensis LS21 could accumulate 45% PHB, producing approximately 2.7 g/L PHB. 60 This PHB production is lower than those observed in this study when strain ZD1 was grown on crude glycerol or on high-strength saline wastewater. Additionally, these extremophiles require high salinity (150–200 g/L, i.e., 15–20% NaCl) in the growth medium, 59 which is considered to be the major bottleneck in their potential application for the industrial production of PHB.…”

In vivo and Post-synthesis Strategies to Enhance the Properties of PHB-Based Materials: A Review

“…Cell shape is also an important factor influencing further cell separation from the culture broth. A convenient downstream process has been designed in engineered Halomonas campaniensis LS21,by deleting an etf operon encoding two subunits of an electron transferring flavoprotein, causing self-flocculation and an easy and rapid recovery at the bottom of the bioreactor (Ling et al, 2019;Shen et al, 2019).…”

Current developments on polyhydroxyalkanoates synthesis by using halophiles as a promising cell factory

“…Thus, controlling cell morphology efficiently enhanced PHB accumulation in H. campaniensis LS21. Furthermore, a self-flocculating H. campaniensis strain LSKO was obtained by knocking out a nonessential etf operon encoding the α and β subunits of an electron transfer flavoprotein [139]. LSKO accumulated up to 45% (wt) PHB.…”