Advanced fed-batch microbioreactors mitigate scale up risks and more closely mimic industrial cultivation practices. Recently, high throughput microscale feeding strategies have been developed which improve the accessibility of microscale fed-batch cultivation irrespective of experimental budget. This review explores such technologies and their role in accelerating bioprocess development. Diffusion- and enzyme-controlled feeding achieve a continuous supply of substrate while being simple and affordable. More complex feed profiles and greater process control require additional hardware. Automated liquid handling robots may be programmed to predefined feed profiles and have the sensitivity to respond to deviations in process parameters. Microfluidic technologies have been shown to facilitate both continuous and precise feeding. Holistic approaches, which integrate automated high-throughput fed-batch cultivation with strategic design of experiments and model-based optimisation, dramatically enhance process understanding whilst minimising experimental burden. The incorporation of real-time data for online optimisation of feed conditions can further refine screening. Although the technologies discussed in this review hold promise for efficient, low-risk bioprocess development, the expense and complexity of automated cultivation platforms limit their widespread application. Future attention should be directed towards the development of open-source software and reducing the exclusivity of hardware.
Lactic acid bacteria are industrially important microorganisms recognized for their fermentative ability mostly in their probiotic benefits as well as lactic acid production for various applications. Nevertheless, lactic acid fermentation often suffers end-product inhibition which decreases the cell growth rate. The inhibition of lactic acid is due to the solubility of the undissociated lactic acid within the cytoplasmic membrane and insolubility of dissociated lactate, which causes acidification of cytoplasm and failure of proton motive forces. This phenomenon influences the transmembrane pH gradient and decreases the amount of energy available for cell growth. In general, the restriction imposed by lactic acid on its fermentation can be avoided by extractive fermentation techniques, which can also be exploited for product recovery.
The production of lipids from oleaginous yeasts involves several stages starting from cultivation and lipid accumulation, biomass harvesting and finally lipids extraction. However, the complex and relatively resistant cell wall of yeasts limits the full recovery of intracellular lipids and usually solvent extraction is not sufficient to effectively extract the lipid bodies. A pretreatment or cell disruption method is hence a prerequisite prior to solvent extraction. In general, there are no recovery methods that are equally efficient for different species of oleaginous yeasts. Each method adopts different mechanisms to disrupt cells and extract the lipids, thus a systematic evaluation is essential before choosing a particular method. In this review, mechanical (bead mill, ultrasonication, homogenization and microwave) and nonmechanical (enzyme, acid, base digestions and osmotic shock) methods that are currently used for the disruption or permeabilization of oleaginous yeasts are discussed based on their principle, application and feasibility, including their effects on the lipid yield. The attempts of using conventional and "green" solvents to selectively extract lipids are compared. Other emerging methods such as automated pressurized liquid extraction, supercritical fluid extraction and simultaneous in situ lipid recovery using capturing agents are also reviewed to facilitate the choice of more effective lipid recovery methods.
Fermentation employing lactic acid bacteria (LAB) often suffers end-product inhibition which reduces the cell growth rate and the production of metabolite. The utility of adsorbent resins for in situ lactic acid removal to enhance the cultivation performance of probiotic, Pediococcus acidilactici was studied. Weak base anion-exchange resin, Amberlite IRA 67 gave the highest maximum uptake capacity of lactic acid based on Langmuir adsorption isotherm (0.996 g lactic acid/g wet resin) compared to the other tested anion-exchange resins (Amberlite IRA 410, Amberlite IRA 400, Duolite A7 and Bowex MSA). The application of Amberlite IRA 67 improved the growth of P. acidilactici about 67 times compared to the control fermentation without resin addition. Nevertheless, the in situ addition of dispersed resin in the culture created shear stress by resins collision and caused direct shear force to the cells. The growth of P. acidilactici in the integrated bioreactor-internal column system containing anion-exchange resin was further improved by 1.4 times over that obtained in the bioreactor containing dispersed resin. The improvement of the P. acidilactici growth indicated that extractive fermentation using solid phase is an effective approach for reducing by-product inhibition and increasing product titer.
Gamma-aminobutyric acid (GABA) is a non-protein amino acid widely distributed in nature and extensively explored for its numerous physiological functions and effects on metabolic disorders. Lactic acid bacteria (LAB) are one of the most important GABA producers, vigorously pursued due to their high GABA content and generally regarded as safe (GRAS) status that allows for direct formulation in various GABA-enriched food products. To meet the strict requirements of the food and nutraceutical industries, the biosynthesis of GABA is typically preferred over the chemical synthesis route. The production of GABA varies among various strains of LAB and is affected by different fermentation conditions. Hence, optimizing the fermentation conditions to enhance the activity of the key enzyme glutamic acid decarboxylase is essential to maximize GABA production. This paper reviews the beneficial effects of GABA on human health and its applications in fermented food products. A particular emphasis is given to the biosynthetic approach for producing GABA by various LAB species via the microbial fermentation route. Efficient strategies for enhancing GABA production through optimization of the fermentation conditions, mode of fermentation, two-step fermentation, co-culturing approach, immobilization technique and genetic engineering are discussed in detail.