Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB), is one of the most valuable biopolymers because of its flexible mechanical properties. In this study, the goal is to establish a scaled-up process of low cost P(3HB-co-4HB) from a 7.5-L fermentor to 1- and 5-m industrial bioreactors, respectively, using Halomonas bluephagenesis TD40 grown on glucose, γ-butyrolactone, and waste corn steep liquor (CSL) as substrates, under open non-sterile and fed-batch or continuous conditions. The non-sterile process enables the energy reduction for less steam consumption. Moreover, waste gluconate is successfully utilized to replace glucose as a carbon source for cell growth and PHA accumulation in 7.5-L fermentor, which opens the possibility of 60% of raw material cost reduction for recycling the waste resources. A mathematical model and rational calculation is established to help guide the feeding strategy and scale-up, respectively, leading to 100 g L cell dry weight (CDW) containing 60.4% P(3HB-co-mol 13.5% 4HB) after 36 h of growth in the 5 m vessel. An even higher P(3HB-co-4HB) content of 74% is achieved by decreasing the use of waste CSL. A stable and continuous open process for efficient low-cost production of P(3HB-co-4HB) is successfully developed coupling fermentation with the downstream extraction processing.

Promoters for the expression of heterologous genes in Halomonas bluephagenesis are quite limited, and many heterologous promoters function abnormally in this strain. P, a promoter of the strongest expressed protein porin in H. bluephagenesis, is one of the few promoters available for heterologous expression in H. bluephagenesis, yet it has a fixed transcriptional activity that cannot be tuned. A stable promoter library with a wide range of activities is urgently needed. This study reports an approach to construct a promoter library based on the P core region, namely, from the -35 box to the transcription start site, a spacer and an insulator. Saturation mutagenesis was conducted inside the promoter core region to significantly increase the diversity within the promoter library. The promoter library worked in both E. coli and H. bluephagenesis, covering a wide range of relative transcriptional strengths from 40 to 140 000. The library is therefore suitable for the transcription of many different heterologous genes, serving as a platform for protein expression and fine-tuned metabolic engineering of H. bluephagenesis TD01 and its derivative strains. H. bluephagenesis strains harboring the orfZ gene encoding 4HB-CoA transferase driven by selected promoters from the library were constructed, the best one produced over 100 g/L cell dry weight containing 80% poly(3-hydroxybutyrate- co-11 mol % 4-hydroxybutyrate) with a productivity of 1.59 g/L/h after 50 h growth under nonsterile fed-batch conditions. This strain was found the best for P(3HB- co-4HB) production in the laboratory scale.

Capillary flows inside microchannels with patterned-surfaces are investigated theoretically and numerically. The surface energy method is used to derive an equivalent contact angle (ECA) model for small capillary number flows. The SIMPLE algorithm using a volume of fluid (VOF) method is adopted to investigate the flows in those microchannels. The flow characteristics such as the liquid front shapes and the evolution of the liquid lengths are obtained. The numerical results reveal that capillary flows in a patterned-surface microchannel still follow the traditional capillary theories. The ECA model is confirmed by the numerical results. It indicates that the capillary flows inside the patterned-surface microchannels can be estimated by means of the homogeneous-surface microchannels with the equivalent contact angle. The ECA model provides a good criterion for the total wettability of a patterned-surface microchannel, as well.

Ectoine, a compatible solute synthesized by many halophiles for hypersalinity resistance, has been successfully produced by metabolically engineered Halomonas bluephagenesis, which is a bioplastic poly(3-hydroxybutyrate) producer allowing open unsterile and continuous conditions. Here we report a de novo synthesis pathway for ectoine constructed into the chromosome of H. bluephagenesis utilizing two inducible systems, which serve to fine-tune the transcription levels of three clusters related to ectoine synthesis, including ectABC, lysC and asd based on a GFP-mediated transcriptional tuning approach. Combined with bypasses deletion, the resulting recombinant H. bluephagenesis TD-ADEL-58 is able to produce 28 g L −1 ectoine during a 28 h fed-batch growth process. Co-production of ectoine and PHB is achieved to 8 g L −1 ectoine and 32 g L −1 dry cell mass containing 75% PHB after a 44 h growth. H. bluephagenesis demonstrates to be a suitable co-production chassis for polyhydroxyalkanoates and non-polymer chemicals such as ectoine.

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