Develop a hybrid machine learning model for promoting microbe biomass production.

Since the cultivation condition of microbe biomass production (mycelia yield) involves a variety of factors, it's a laborious process to obtain the optimal cultivation condition of Antrodia cinnamomea (A. cinnamomea). This study proposed a hybrid machine learning approach (i.e., ANFIS-NM) to identify the potent factors and optimize the cultivation conditions of A. cinnamomea based on a 32 fractional factorial design with seven factors. The results indicate that the ANFIS-NM approach successfully identified three key factors (i.e., glucose, potato dextrose broth, and agar) and significantly boosted mycelia yield. The interpretability of ANFIS rules made the cultivation conditions visually interpretable. Subsequently, a three-factor five-level central composite design was used to probe the optimal yield. This study demonstrates the proposed hybrid machine learning approach could significantly reduce the time consumption in laboratory cultivation and increase mycelia yield that meets SDGs 7 and 12, hitting a new milestone for biomass production.

Exploring the high lipid production potential of a thermotolerant microalga using statistical optimization and semi-continuous cultivation.

A recently isolated thermotolerant microalga Desmodesmus sp. F2 has the traits of becoming potential biodiesel feedstock, such as high growth rate, high lipid content, and quick precipitation. Its overall lipid productivity was 113 mg/L/d when grown under non-optimal conditions using batch cultivation. A two-step response surface methodology was adopted to optimize its cultivation conditions. The overall lipid productivity was increased to 263 mg/L/d when the cells were grown under the optimized conditions of 6.6mM initial nitrogen level and 6 days nitrogen depletion treatment in 700 µmol/m(2)/s light intensity at 35°C using batch cultivation. Fed-batch and semi-continuous cultivations were employed to further increase its lipid productivity to 213 and 302 mg/L/d, respectively. The 302 mg/L/d is the highest overall lipid productivity of microalgae ever reported in the literature. This study provides the information required for the design and operation of photobioreactors for large scale outdoor cultivation of this species.

Photobioreactor strategies for improving the CO2 fixation efficiency of indigenous Scenedesmus obliquus CNW-N: statistical optimization of CO2 feeding, illumination, and operation mode.

Statistical experimental design and bioreactor strategies were applied to enhance CO(2) fixation ability of microalga Scenedesmus obliquus CNW-N. Four operating parameters strongly influencing microalgal CO(2) fixation efficiency (namely, CO(2) concentration, CO(2) flow rate, magnesium concentration, and light intensity) were optimized with response surface methodology. The optimal range of parameters achieving the best overall performance of specific growth rate and CO(2) fixation rate was determined with overlay counter plot techniques. Optimal ranges of CO(2) concentration, CO(2) flow rate, magnesium concentration and light intensity were 2.0-2.5%, 0.3-0.5 vvm, 1.7-2.7 mM and 180-250 µmol m(-2) s(-1), respectively, achieving a specific growth rate of >1.22 d(-1) and CO(2) fixation rate of >800 mg L(-1) d(-1). Semi-batch operations further enhanced the biomass productivity, photosynthesis efficiency, and CO(2) fixation rate to 1030 mg L(-1) d(-1), 10.5%, and 1782 mg L(-1) d(-1), respectively. This performance is better than the results reported by most related studies.

Rhamnolipid production with indigenous Pseudomonas aeruginosa EM1 isolated from oil-contaminated site.

Rhamnolipid is one of the most effective and commonly used biosurfactant with wide industrial applications. Systematic strategies were applied to improve rhamnolipid (RL) production with a newly isolated indigenous strain Pseudomonas aeruginosa EM1 originating from an oil-contaminated site located in southern Taiwan. Seven carbon substrates and four nitrogen sources were examined for their effects on RL production. In addition, the effect of carbon to nitrogen (C/N) ratio on RL production was also studied. Single-factor experiments show that the most favorable carbon sources for RL production were glucose and glycerol (both at 40 g/L), giving a RL yield of 7.5 and 4.9 g/L, respectively. Meanwhile, sodium nitrate appeared to be the preferable nitrogen source, resulting in a RL production of 8.6g/L. Using NaNO(3) as the nitrogen source, an optimal C/N ratio of 26 and 52 was obtained for glucose- and glycerol-based culture, respectively. To further optimize the composition of fermentation medium, twenty experiments were designed by response surface methodology (RSM) to explore the favorable concentration of three critical components in the medium (i.e., glucose, glycerol, and NaNO(3)). The RSM analysis gave an optimal concentration of 30.5, 18.1, and 4.9 g/L for glucose, glycerol, and NaNO(3), respectively, predicting a maximum RL yield of 12.6 g/L, which is 47% higher than the best yield (8.6 g/L) obtained from preliminary selection tests and single factor experiments (glucose and NaNO(3) as the carbon and nitrogen source). The NMR and mass spectrometry analysis show that the purified RL product contained L-rhamnosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate (RL1) and L-rhamnosyl L-rhamnosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate (RL2). Meanwhile, HPLC analysis indicates that the molar ratio of RL1 and RL2 in the purified rhamnolipid product was ca. 1:1.

Improved phototrophic H2 production with Rhodopseudomonas palustris WP3-5 using acetate and butyrate as dual carbon substrates.

An indigenous purple nonsulfur bacterium Rhodopseudomonas palustris WP3-5 was used to produce hydrogen phototrophically from acetate (HAc) and butyrate (HBu), which are the major soluble products from acidogenic dark fermentation. Statistical experimental design methodology was applied to identify optimal composition of the two carbon substrates in the medium, leading to better H2 production performance of R. palustris WP3-5. Three performance indexes were used to assess the effectiveness of the phototrophic H2 production; they were H2 yield (Y H2), maximum H2 production rate (Rmax) and maximum cumulative H2 evolution (Hmax). An overlay contour plot was used to determine the optimal concentration range of HAc and HBu, taking into account all three performance indexes (i.e., Rmax, Hmax, and Y H2) simultaneously. With the response surface analysis, R. palustris WP3-5 could produce H2 efficiently with the best Rmax, Hmax, and Y H2 of 39.5 ml/h, 2738 ml, and 51.6%, respectively. This performance is superior to most reported values in the literature, indicating that the statistical experimental design is an effective tool to improve phototrophic H2 production with R. palustris WP3-5.

Exploring multi-metal biosorption by indigenous metal-hyperresistant Enterobacter sp. J1 using experimental design methodologies.

A novel experimental design, combining mixture design and response surface methodology (RSM), was developed to investigate the competitive adsorption behavior of lead, copper and cadmium by an indigenous isolate Enterobacter sp. J1 able to tolerate high concentrations of a variety of heavy metals. Using the proposed combinative experimental design, two different experiment designs in a ternary metal biosorption system can be integrated to a succinct experiment and the number of experimental trials was markedly reduced from 38 to 26 by reusing the mutual experimental data. Triangular contour diagrams and triangular three-dimensional surface plots were generated to describe the ternary metal biosorption equilibrium data in mixture design systems. The results show that the preference of metal sorption of Enterobacter sp. J1 decreased in the order of Pb(2+)>Cu(2+)>Cd(2+). The presence of other metals resulted in a competitive effect. The influence of the other two metals in ternary metal biosorption system can be easily determined by comparing the stray distance from the single metal biosorption. The behavior of competitive biosorption was successfully described and predicted using a combined Langmuir-Freundlich model along with new three-dimensional contour-surface plots.

Improved production of biosurfactant with newly isolated Pseudomonas aeruginosa S2.

An indigenous strain Pseudomonas aeruginosa S2 (P. aeruginosa S2), isolated from diesel-contaminated soil, produced extracellular surface-active material identified as rhamnolipid. Due to its excellent surface activity, rhamnolipid is known to be well-suited for stimulating the bioremediation efficiency of oil contaminated sites. To improve production yield of rhamnolipid with P. aeruginosa S2, various carbon and nitrogen sources were screened to select favorable ones leading to better biosurfactant production yield. It was found that using 4% glucose could attain better rhamnolipid yield, while 50 mM NH4NO3 appeared to be the most preferable nitrogen source. Meanwhile, the effect of carbon to nitrogen ratio (C/N ratio) on rhamnolipid yield was also investigated, and the optimal C/N ratio was identified as approximately 11.4. Moreover, response surface methodology (RSM) was applied to optimize the trace element concentration for rhamnolipid production. Results from two-level design indicate that concentrations of MgSO4 and FeSO4 were the most significant factors affecting rhamnolipid production. Using steepest ascent method and RSM analysis, an optimal medium composition was determined, giving a rhamnolipid production yield of 2.37 g/L in 100 h at 37 degrees C and 200 rpm agitation. Scale-up production of rhamnolipid in a well-controlled 5 L jar fermentor using the optimal medium and operating condition (at 37 degrees C and pH 6.8) further elevated the biosurfactant production yield to 5.31 g/L (in 97 h), which is over 2-fold higher than the best results obtained from shake-flask tests.

Use of active consortia of constructed ternary bacterial cultures via mixture design for azo-dye decolorization enhancement.

This first-attempt study used constructed bacterial consortia containing Escherichia coli DH5alpha (a weak decolorizer) and its UV-irradiated mutants (E. coli UVT1 and UV68; strong decolorizers) via equilateral triangle diagram and mixture experimental design to assess color removal during species evolution. The results showed that although strain DH5alpha was not an effective decolorizer, its presence might still played a significant role in affecting optimal color removal capabilities of mixed consortia (e.g., E. coli DH5alpha, UVT1 and UV68) for two model azo dyes; namely, reactive red 22 (RR22) and reactive black 5 (RB5). Contour analysis of ternary systems also clearly showed that decolorization of RR22 and RB5 by DH5alpha-containing active mixed consortia was more effective than mono-cultures of the stronger decolorizer alone (e.g., UVT1). The optimal composition of the mixed consortium (UV68, UVT1, DH5alpha) achieving the highest specific decolorization rate was (13%:58%:29%) and (0%:74%:26%) for decolorization of RR22 and RB5, respectively, with initial total cell density fixed at OD(600)=3.5+/-0.28.

Biosorption of lead, copper and cadmium by an indigenous isolate Enterobacter sp. J1 possessing high heavy-metal resistance.

This study was undertaken to investigate biosorption kinetics and equilibria of lead (Pb), copper (Cu) and cadmium (Cd) ions using the biomass of Enterobacter sp. J1 isolated from a local industry wastewater treatment plant. Efficiency of metal ion recovery from metal-loaded biomass to regenerate the biosorbent was also determined. The results show that Enterobacter sp. J1 was able to uptake over 50mg of Pb per gram of dry cell, while having equilibrium adsorption capacities of 32.5 and 46.2mg/g dry cell for Cu and Cd, respectively. In general, Langmuir and Freundlich models were able to describe biosorption isotherm fairly well, except that prediction of Pb adsorption was relatively poor with Langmuir model, suggesting a different mechanism for Pb biosorption. Adjusting the pH value to 3.0 led to nearly complete desorption of Cd from metal-loaded biomass, while over 90% recovery of Pb and Cu ions was obtained at pH

Observer-based online compensation of inner filter effect in monitoring fluorescence of GFP-expressing plant cell cultures.

The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria is a very useful reporter for real-time bioprocess sensing. GFP culture fluorescence is a composite signal that can be influenced by factors such as culture autofluorescence, inner filter effect (IFE), and photobleaching. These factors complicate accurate estimation of GFP concentrations from the culture fluorescence. IFE is especially problematic when using GFP in monitoring transgenic plant cell suspension cultures, due to the aggregated nature of the cells and the high biomass concentration in these culture systems. Reported approaches for online compensation of IFE in monitoring culture NADH fluorescence or bioluminescence require online measurement of biomass density or culture turbidity/optical density, in addition to fluorescence/bioluminescence measurement. In this study, culture GFP fluorescence was used successfully to estimate GFP concentration and other important states in bioreactor culture of transgenic tobacco cells, while the influences of IFE and culture autofluorescence were rectified without the need for an additional biomass sensor. This was achieved by setting up a novel model-based state observer. First, we developed an improved model for a backscatter fluorescence probe that takes into account the influence of IFE and autofluorescence on reporting culture GFP concentration from online fluorescence. The state observer was then established using the extended Kalman filter (EKF), based on the fluorescence probe model, a dynamic state model of the plant cell bioreactor, and online GFP fluorescence measurement. Several versions of the observer were introduced to address practical requirements associated with monitoring GFP fluorescence of plant cell cultures. The proposed approach offers an effective means for online compensation of IFE to enable quantitative interpretation of the culture fluorescence signals for accurate reporting of GFP or GFP-fusion protein expression.