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Significance This report describes an opportunity for the bacterial cellulose nanofiber (BCNF) membrane, which has recently been attracting much attention due to its sustainability and high thermal stability, as a “single-layer,” good alternative membrane to the existing chemical-derived one in batteries, and thus allows us to find possible solutions to the environmental and safety issues. We achieved highest BCNF yield ever reported so far and fabricated a cylindrical lithium ion battery (LIB) by roll-to-roll process to realize remarkable cycle stability with 80% capacity retention even after 1,000 cycles, which is comparable to those of commercialized batteries. Finally, we introduced a Lewis base to improve thermal safety of the battery to a level comparable to that of a commercial ceramic-coated separator.

Lignocellulosic biomass was pretreated with lime, and used for the production of VFA (volatile fatty acid) through batch anaerobic digestion. About 0.34 g VFA yield was obtained using 10 g/L reed, after 3 days of fermentation with lime treatment; however, a higher VFA yield (more than 0.5 g/g biomass) was achieved with a modified lime treatment. Overall, our study showed that that the modified lime treatment is better suited for VFA production. VFAs can be widely used in platforms for fuels and chemicals from biomass.

Algae biomass is a potential raw material for the production of biofuels and other chemicals. In this study, biomass of the marine algae, Ulva lactuca, Gelidium amansii,Laminaria japonica, and Sargassum fulvellum, was treated with acid and commercially available hydrolytic enzymes. The hydrolysates contained glucose, mannose, galactose, and mannitol, among other sugars, at different ratios. The Laminaria japonica hydrolysate contained up to 30.5% mannitol and 6.98% glucose in the hydrolysate solids. Ethanogenic recombinant Escherichia coli KO11 was able to utilize both mannitol and glucose and produced 0.4g ethanol per g of carbohydrate when cultured in L. japonica hydrolysate supplemented with Luria-Bertani medium and hydrolytic enzymes. The strategy of acid hydrolysis followed by simultaneous enzyme treatment and inoculation with E. coli KO11 could be a viable strategy to produce ethanol from marine alga biomass.

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.

The use of high purity oxygen as a feeding gas in microbial fermentation has recent gained huge attention in industrial scale process due to its advantageous effect to increase viable cell density as well as target metabolite productivity. However, usage of highly pure oxygen is currently too expensive for industrialization. To overcome this limitation, off-gas recycle pressure swing adsorption (PSA) was employed which assist in continuous production of high purity oxygen during fermentation process. The off-gas from fermentation process contained about 70 ∼ 80% oxygen which was re-utilized as the input gas in PSA, whereas conventional PSA process utilized air with 21% oxygen content. This difference in the oxygen content of the input gas decreased the size of PSA, resulting in reduction of equipment costs. Using the characteristics of off-gas recycle PSA, we analyzed and compared the economic feasibility of a highly pure oxygen supplying strategy for the production of poly(3-hydroxybutyrate) (PHB) by Ralstonia eutropha based on previously reported data, using both air and highly pure oxygen. To achieve annual production of 3,630 tonnes of PHB the aforementioned method was adopted, by doing so the production cost of PHB was significantly reduced to 4.2 $/kg. While, the conventional process (utilizing air as the feeding gas) resulted in the highest cost (5.9 $/kg). The low cost of off-gas recycle PSA was due to reduction of fixed capital cost, which was about 3 times lower than that of the conventional fermentation process. Therefore, the production cost of fermentation was considerably decreased by using a highly pure oxygen supplying strategy based on off-gas recycle PSA.

H2S in biogas was removed by sludge-loaded biofiltration, rendering the biogas suitable for catalytic reforming into a mixture of CO and H2 syngas that was then applied for the generation of electricity using a solid oxide fuel cell or for the chemical synthesis of methanol. The biogas was anaerobically produced in a 2 m3 bioreactor at 35°C for 2 years using restaurant food waste from Korea Advanced Institute of Science and Technology (KAIST), and the concentration of H2S in the biogas ranged from 612 to 1,500 ppmv (Avg. 1,060 ppmv). Two immobilized cell bioreactors 0.2 and 8.5 L in volume were loaded with aerobic sludge and used to study characteristics of H2S removal from biogas. At a retention time of 400 sec, the removal efficiency of H2S was over 99% following initial stabilization for 7 days in the 8.5 L bioreactor installed at the on-site biogas facility. The maximum rate of H2S removal in this study was 359 g-H2S/m3/h with an average mass loading rate of 14.7 g-H2S/m3/h (kinetic analysis: Vm = 842.6 g-H2S/m3/h and Ks = 2.2 mg/L). Therefore, purified biogas with a negligible concentration H2S was efficiently reformed to syngas. This study demonstrates the feasibility of biogas purification as a part of high-quality syngas production.

The typical biorefinery platforms are sugar, thermochemical (syngas), carbon-rich chains, and biogas platforms, each offering unique advantages and disadvantages. The sugar platform uses hexose and pentose sugars extracted or converted from plant body mass. The thermochemical (syngas) platform entails a chemical or biological conversion process using pyrolysis or gasification of plants to produce biofuels. The carbon-rich chains platform is used to produce biodiesel from long-chain fatty acids or glycerides. In the present work, we suggest a new platform using volatile fatty acids (VFAs) for the production of biofuels and biochemicals production. The VFAs are short-chain fatty acids composed mainly of acetate and butyrate in the anaerobic digestion (AD) process, which does not need sterilization, additional hydrolysis enzymes (cellulose or xylanase), or a high cost pretreatment step. VFAs are easily produced from almost all kinds of biomass with low lignin content (terrestrial, aquatic, and marine biomass) by the AD process. Considering that raw material alone constitutes 40∼80% of biofuel production costs, biofuels made from VFAs derived from waste organic biomass potentially offer significant economical advantage.

Succinic acid was produced by continuous fermentation of Actinobacillus succinogenes sp. 130Z in an external membrane cell recycle reactor to improve viable cell concentration and productivity. Using this system, cell concentration increased to 16.4 g/l at the dilution rate 0.2 h-1, up to 3 times higher than that of batch culture, and the volumetric productivity of succinic acid increased up to 6.63 g/l/h at the dilution rate 0.5 h-1, 5 times higher than that of batch fermentation. However, in the continuous culture using a high dilution rate, operational problems including severe membrane fouling and contamination by lactic acid producer were observed. Another succinic acid producer, Mannheimia succiniciproducens MBEL55E, was also utilized in this system, and the cell concentration and productivity of succinic acid at the dilution rate of 0.3 h-1 were found to be above 3 and 2.3 times higher, respectively, compared with those obtained at the dilution rate of 0.1 h-1. These observations give a deep insight into the process design for a continuous succinic acid production by microorganisms.

Bermudagrass, reed and rapeseed were pretreated with phosphoric acid-acetone and used for ethanol production by means of simultaneous saccharification and fermentation (SSF) with a batch and fed-batch mode. When the batch SSF experiments were conducted in a 3% low effective cellulose, about 16 g/L of ethanol were obtained after 96 h of fermentation. When batch SSF experiments were conducted with a higher cellulose content (10% effective cellulose for reed and bermudagrass and 5% for rapeseed), higher ethanol concentrations and yields (of more than 93%) were obtained. The fed-batch SSF strategy was adopted to increase the ethanol concentration further. When a higher water-insoluble solid (up to 36%) was applied, the ethanol concentration reached 56 g/L of an inhibitory concentration of the yeast strain used in this study at 38 degrees C. The results show that the pretreated materials can be used as good feedstocks for bioethanol production, and that the phosphoric acid-acetone pretreatment can effectively yield a higher ethanol concentration.

Recombinant Escherichia coli has been studied as a main host for recombinant protein productions, but it is still difficult to cultivate E. coli in a large industrial-scale process due to the oxygen supply limitation. In this study, E. coli BL(21) harboring a new constructed plasmid (pEHUb-hGH) was used for producing recombinant human growth hormone (r-hGH) in 5-L and 30-L scale fermentors by supplying air and high purity oxygen, respectively, where the high purity oxygen was produced from a vacuum pressure swing adsorption (PSA). The impact of oxygen supply modes, i.e., air and high purity oxygen, on cell growth and r-hGH production was investigated in different scale fermentors. In the case of high purity oxygen supply, the final cell density and r-hGH concentrations were 63.0 and 4.8 g/L in the 5-L fermentor, 51.6 and 4.0 g/L in the 30-L fermentor, respectively. In addition, the productivity of r-hGH was doubled in the 5-L fermentor, and increased 4-fold in the 30-L fermentor, compared to the results obtained in the case of the air supply. The supply of high purity oxygen eliminated the oxygen limitation and acetate formation effectively, and apparently, did not affect the degradation of r-hGH. This shows that the recombinant E. coli cultivation with high purity oxygen produced from PSA may provide an effective method for large-scale industrial production of recombinant proteins.

An upflow packed-bed cell recycle bioreactor (IUPCRB) is proposed for obtaining a high cell density. The system is comprised of a stirred tank bioreactor in which cells are retained partially by a packed-bed. A 1.3 cm (ID) × 48 cm long packed-bed was installed inside a 2 L bioreactor (working volume 1 L). Continuous ethanol fermentation was carried out using a 100 g/L glucose solution containing Saccharomyces cerevisiae (ATCC 24858). Cell retention characteristics were investigated by varying the void fraction (VF) of the packed bed by packing it with particles of 0.8∼2.0 mm sized stone, cut hollow fiber pieces, ceramic, and activated carbon particles. The best results were obtained using an activated carbon bed with a VF of 30∼35%. The IUPCRB yielded a maximum cell density of 87 g/L, an ethanol concentration of 42 g/L, and a productivity of 21 g/L/h when a 0.5 h−1 dilution rate was used. A natural bleeding of cells from the filter bed occurred intermittently. This cell loss consisted of an average of 5% of the cell concentration in the bioreactor when a high cell concentration (approximately 80 g/L) was being maintained.

Volatile fatty acids (VFAs) derived from organic waste, were used as a low cost carbon source for high bioreactor productivity and titer. A multi-stage continuous high cell density culture (MSC-HCDC) process was employed for economic assessment of microbial lipids for biodiesel production. In a simulation study we used a lipid yield of 0.3 g/g-VFAs, cell mass yield of 0.5 g/g-glucose or wood hydrolyzates, and employed process variables including lipid contents from 10–90% of cell mass, bioreactor productivity of 0.5–48 g/L/h, and plant capacity of 20000–1000000 metric ton (MT)/year. A production cost of USD 1.048/kg-lipid was predicted with raw material costs of USD 0.2/kg for wood hydrolyzates and USD 0.15/kg for VFAs; 9 g/L/h bioreactor productivity; 100, 000 MT/year production capacity; and 75% lipids content. The variables having the highest impact on microbial lipid production costs were the cost of VFAs and lipid yield, followed by lipid content, fermenter cost, and lipid productivity. The cost of raw materials accounted for 66.25% of total operating costs. This study shows that biodiesel from microbial lipids has the potential to become competitive with diesels from other sources.

Because algal cells are so efficient at absorbing incoming light energy, providing more light energy to photobioreactors would simply decrease energy conversion efficiency. Furthermore, the algal biomass productivity in photobioreactor is always proportional to the total photosynthetic rate. In order to optimize the productivity of algal photobioreactors (PBRs), the oxygen production rate should be estimated. Based on a simple model of light penetration depth and algal photosynthesis, the oxygen production rate in high-density microalgal cultures could be calculated. The estimated values and profiles of oxygen production rate by this model were found to be in accordance with the experimental data. Optimal parameters for PBR operations were also calculated using the model.

A cell-based quantitative assay system for Hcy has been developed by utilizing two Escherichia coli auxotrophs that grow in the presence of methionine (Met) and either homocysteine (Hcy) or Met, respectively. A bioluminescent reporter gene, which produces luminescence as cells grow, was inserted into the auxotrophs, so that cell growth can be readily determined. When the relative luminescence unit (RLU) values from the two auxotrophs immobilized within agarose gels arrayed on a well plate were measured, the amount of Hcy was quantitatively determined on the basis of differences between two RLU values corresponding to cell growth of two auxotrophs with excellent levels of precision and reproducibility. Finally, the diagnostic utility of this assay system was verified by its employment in reliably determining different stages of hyperhomocysteinemia in human plasma samples providing CVs of within and between assays that are less than 2.9% and 7.1%, respectively, and recovery rates of within and between assays that are in the range of 99.1-103.5% and 97.5-105.5%, respectively. In contrast to existing conventional methods, the new system developed in this effort is simple, rapid, and cost-effective. As a result, it has great potential to serve as a viable alternative for Hcy quantification in the diagnosis of hyperhomocysteinemia.

The use of volatile fatty acids (VFAs) for microbial lipid accumulation was investigated in flask cultures of Cryptococcus albidus. The optimum culture temperature and pH were 25°C and pH 6.0, respectively, and the highest lipid content (27.8%) was obtained with ammonia chloride as a nitrogen source. The lipid yield coefficient on VFAs was 0.167 g/g of C. albidus with a VFAs (acetic, propionic, butyric acids) ratio of 8:1:1, which was in good agreement with a theoretically predicted lipid yield coefficient of the VFAs as a carbon source. The major fatty acids of the lipids accumulated by C. albidus were similar to those of soybean oil and jatropha oil. A preliminary cost analysis shows that VFAs-based biodiesel production is competitive with current palm and soybean based biodiesels. Further process development for lower aeration cost and higher lipid yield will make this process more economical.

Dissolved oxygen (DO)-controlled nar promoter expression vectors were constructed, and their expression efficiency was compared with that of the T7 promoter pET22 expression vector by expressing human growth hormone (hGH), enhanced green fluorescence protein (EGFP), and β-tyrosinase in Escherichia coli cells. The nar promoter expression vector pRBS, which was engineered with a 5′-untranslated region and ribosomal binding site for the T7 promoter, expressed hGH at a rate of up to 32% of the total cellular proteins (TCP) in E. coli W3110narL − . The expression level of hGH was further enhanced, up to ∼42% of the TCP, by adding the N-terminal peptide tag of β-galactosidase to hGH, which was comparable to the expression of ∼43% of the TCP in pET-lac:hGH/BL21(DE3). A further engineered expression vector, pRBS(fnr), which coexpressed fumarate/nitrate reductase ( fnr ), expressed more EGFP than pET22 in BL21(DE3). In addition, recombinant β-tyrosinase was successfully expressed at a rate of up to ∼45% of the TCP in pRBS(fnr) in W3110narL − . From these results, the DO-controlled nar promoter system developed in this study can be considered a reliable and cost-effective expression system for protein production, especially in large-scale fermentation, as an alternative to the pET/BL(DE3) system.