The fed-batch production of mannosylerythritol lipids by Ustilago maydis DSM 4500 from hydrophilic carbon sources.

Glycolipids are a class of widely studied biosurfactants with excellent applicability in cosmetic and pharmaceutical formulations. This class of biosurfactants includes mannosylerythritol lipids (MELs), which have gained particular interest due to their moisturizing and healing activity for dry and damaged human skin, arising from conditions such as eczema. Traditionally, MELs have been produced by growing certain basidiomycetous yeasts on vegetable oils. However, oils are a comparatively expensive substrate, which negatively affects the economic performance of MEL production. In addition to this, vegetable oils significantly complicate the downstream processing required to produce a product with the required purity for most applications. To address these challenges, this study investigated MEL-A production exclusively from hydrophilic carbon sources by Ustilago maydis DSM 4500. By implementing a fed-batch production strategy, maximum MEL-A concentration of 0.87 g/L was achieved from glucose exclusively. Also, adding micronutrients (such as MnSO4) to MEL-A production showed a 24.1% increase in the product titer, implying other metabolites are formed, favoring MEL production.

A review on the upstream production and downstream purification of mannosylerythritol lipids.

Biosurfactants are natural compounds with remarkable surface-active properties that may offer an eco-friendly alternative to conventional surfactants. Among them, mannosylerythritol lipids (MELs) stand out as an intriguing example of a glycolipid biosurfactant. MELs have been used in a variety of sectors for various applications, and are currently commercially produced. Industrially, they are used in the pharmaceutical, cosmetic, food, and agricultural industries, based on their ability to reduce surface tension and enhance emulsification. However, despite their utility, their production is comparatively limited industrially. From a bioprocessing standpoint, two areas of interest to improve the production process are upstream production and downstream (separation and purification) product recovery. The former has seen a significant amount of research, with researchers investigating several production factors: the microbial species or strain employed, the producing media composition, and the production strategy implemented. Improvement and optimization of these are key to scale-up the production of MELs. On the other hand, the latter has seen comparatively limited work presented in the literature. For the most part traditional separation techniques have been employed. This systematic review presents the production and purification methodologies used by researchers by comprehensively analyzing the current state-of-the-art with regards the production, separation, and purification of MELs. By doing so, the review presents different possible approaches, and highlights some potential areas for future work by identifying opportunities for the commercialization of MELs.

The effect of Alcanivorax borkumensis SK2, a hydrocarbon-metabolising organism, on gas holdup in a 4-phase bubble column bioprocess.

To design bioprocesses utilising hydrocarbon-metabolising organisms (HMO) as biocatalysts, the effect of the organism on the hydrodynamics of bubble column reactor (BCR), such as gas holdup, needs to be investigated. Therefore, this study investigates the first use of an HMO, Alcanivorax borkumensis SK2, as a solid phase in the operation and hydrodynamics of a BCR. The study investigated the gas holdup in 3-phase and 4-phase systems in a BCR under ranges of superficial gas velocities (UG) from 1 to 3 cm/s, hydrocarbon (chain length C13-21) concentrations (HC) of 0, 5, and 10% v/v and microbial concentrations (MC) of 0, 0.35, 0.6 g/l. The results indicated that UG was the most significant parameter, as gas holdup increases linearly with increasing UG from 1 to 3 cm/s. Furthermore, the addition of hydrocarbons into the air-deionized water -SK2 system showed the highest increase in the gas holdup, particularly at high UG (above 2 cm/s). The solids (yeast, cornflour, and SK2) phases had differing effects on gas holdup, potentially due to the difference in surface activity. In this work, SK2 addition caused a reduction in the fluid surface tension in the bioprocess which therefore resulted in an increase in the gas holdup in BCR. This work builds upon previous investigations in optimising the hydrodynamics for bubble column hydrocarbon bioprocesses for the application of alkane bioactivation.

The liquid-liquid extractive fermentation of L-lactic acid in a novel semi-partition bioreactor (SPB).

Fermentation technology is commonly used as a mature process to produce numerous products with the help of micro-organisms. However, these organisms are sometimes inhibited by the accumulation of these products or their by-products. One route to circumvent this is via extractive fermentation, which combines the fermentation process with extraction. To facilitate this, novel bioreactor designs are required, such as the semi-partition bioreactor (SPB) which has been recently proposed for in-situ extractive fermentation. The latter combines a fermentation and an extraction unit into a single vessel using a mixer-settler principle. Where the bioproduct is produced in the mixer and removed continuous in the settler. As the SPB functionality is a subject of interest, this study builds on demonstrating different process conditions in the production of a sample bioprocess (lactic acid (LA)) which is susceptible to product inhibition. The results showed a 34.5 g/L LA concentration was obtained in the pH-controlled condition. While LA production can suffer from product inhibition, neutralizing agents can be easily used to curb inhibitory problems, however, the LA fermentation is a simple (and well-studied) example, which can demonstrate an alternative route to avoiding product inhibition (for systems which cannot be rescued using pH control). Hence, to replicate a scenario of product inhibition, two different process conditions were investigated, no pH control with no extraction (non-integrated), and no pH control with integrated extractive fermentation. Key findings showed higher LA concentration in integrated (25.10 g/L) as compared to the non-integrated (14.94 g/L) case with improved yield (0.75 gg-1 (integrated) versus 0.60 gg-1 (non-integrated)) and overall productivity (0.35 gL-1h-1(integrated) versus 0.20 gL-1h-1(non-integrated)) likewise. This is the first demonstration of an SP bioreactor, and shows how the reactor can be applied to improve productivity. Based on these results, the SPB design can be applied to produce any product liable to product inhibition.

Process Development in Biosurfactant Production.

Biosurfactants encompass a number of structurally and chemically diverse compounds, all of which demonstrate surface active properties. The potential and current application of these compounds ranges from enhanced oil recovery, through detergents, emulsifiers in foods, antifungal agents, antibiotics, and even to uses in the minerals processing industry. And while the market demand for these products is growing, the industry still is significantly smaller than the synthetic surfactants market. Part of the reason for this is that biosurfactants are currently comparatively expensive to produce. This article reviews the process consideration steps required to develop a biosurfactant bioprocess (organism selection and modification, reactor type and operation, media, downstream processing, and final use) and considers the state of the art of each process step, with an eye to considering the overall process development, and establishment of both technically and economically viable routes to production.

Investigating and modeling the effect of light intensity on Rhodopseudomonas palustris growth.

Photosynthetic bacteria can be useful biotechnological tools-they produce a variety of valuable products, including high purity hydrogen, and can simultaneously treat recalcitrant wastewaters. However, while photobioreactors have been designed and modeled for photosynthetic algae and cyanobacteria, there has been less work on understanding the effect of light in photosynthetic bacterial fermentations. To design photobioreactors, and processes using these organisms, robust models of light penetration, utilization, and conversion are needed. This stydy uses experimental data from a tubular photobioreactor designed to focus in on light intensity effects, to model the effect of light intensity on the growth of Rhodopseudomonas palustris, a model photosynthetic bacterium. The work demonstrates that growth is controlled by light intensity, and that this organism does experience photolimitation below 200 W/m2 and photoinhibition above 600 W/m2 . This has implications for outdoor applications, as there will be low growth during the periods of limited light, and growth may be inhibited during the light intensive hours of mid-day. Further, the work presents a model for light penetration in cylindrical photobioreactors, which tends to be the most common geometry. The model developed showed good fit to the experimental data for each light intensity investigated, with high R2 values and NRMSE values all below 20%. The work extends the modeling tools for these organisms, and will allow for better photobioreactor design, and the integration of modeling tools in designing processes which use photosynthetic bacteria.

Expression of Alternative Nitrogenases in Rhodopseudomonas palustris Is Enhanced Using an Optimized Genetic Toolset for Rapid, Markerless Modifications.

The phototrophic bacterium Rhodopseudomonas palustris is emerging as a promising biotechnological chassis organism, due to its resilience to a range of harsh conditions, a wide metabolic repertoire, and the ability to quickly regenerate ATP using light. However, realization of this promise is impeded by a lack of efficient, rapid methods for genetic modification. Here, we present optimized tools for generating chromosomal insertions and deletions employing electroporation as a means of transformation. Generation of markerless strains can be completed in 12 days, approximately half the time for previous conjugation-based methods. This system was used for overexpression of alternative nitrogenase isozymes with the aim of improving biohydrogen productivity. Insertion of the pucBa promoter upstream of vnf and anf nitrogenase operons drove robust overexpression up to 4000-fold higher than wild-type. Transcript quantification was facilitated by an optimized high-quality RNA extraction protocol employing lysis using detergent and heat. Overexpression resulted in increased nitrogenase protein levels, extending to superior hydrogen productivity in bioreactor studies under nongrowing conditions, where promoter-modified strains better utilized the favorable energy state created by reduced competition from cell division. Robust heterologous expression driven by the pucBa promoter is thus attractive for energy-intensive biosyntheses suited to the capabilities of R. palustris. Development of this genetic modification toolset will accelerate the advancement of R. palustris as a biotechnological chassis organism, and insights into the effects of nitrogenase overexpression will guide future efforts in engineering strains for improved hydrogen production.

Quantification of oxygen transfer coefficients in simulated hydrocarbon-based bioprocesses in a bubble column bioreactor.

This study investigates the overall volumetric oxygen transfer coefficient (KLa) in multiphase hydrocarbon-based bioprocess under a range of hydrocarbon concentrations (HC), solid loadings (deactivated yeast) (SL) and superficial gas velocities (UG) in a bubble column reactor (BCR). KLa increased with increasing UG in the air-water system; due to an increase in the number of small bubbles which enhanced gas holdup. In air-water-yeast systems, the initial addition of yeast increased KLa significantly. Further increases in SL reduced KLa, due to increases in the bubble size with increasing SL. KLa decreased when HC was added in air-water-hydrocarbon systems. However, UG, SL and HC affected KLa differently in air-water-yeast-hydrocarbon systems: an indication of the complex interactions between the yeast and hydrocarbon phases which changed the system's hydrodynamics and therefore affected KL. This work illustrates the effect of the operating conditions (SL, HC and UG) on oxygen transfer behaviour in multiphase systems.

Design and evaluation of a continuous semipartition bioreactor for in situ liquid-liquid extractive fermentation.

Extractive fermentation (or in situ product removal (ISPR)) is an operational method used to combat product inhibition in fermentations. To achieve ISPR, different separation techniques, modes of operation and physical reactor configurations have been proposed. However, the relative paucity of industrial application necessitates continued investigation into reactor systems. This article outlines a bioreactor designed to facilitate in situ product extraction and recovery, through adapting the reaction volume to include a settler and solvent extraction and recycle section. This semipartition bioreactor is proposed as a new mode of operation for continuous liquid-liquid extractive fermentation. The design is demonstrated as a modified bench-top fermentation vessel, initially analysed in terms of fluid dynamic studies, in a model two-liquid phase system. A continuous abiotic simulation of lactic acid (LA) fermentation is then demonstrated. The results show that mixing in the main reaction vessel is unaffected by the inserted settling zone, and that the size of the settling tube effects the maximum volumetric removal rate. In these tests the largest settling tube gave a potential continuous volumetric removal rate of 7.63 ml/min; sufficiently large to allow for continuous product extraction even in a highly productive fermentation. To demonstrate the applicability of the developed reactor, an abiotic simulation of a LA fermentation was performed. LA was added to reactor continuously at a rate of 33ml/h, while continuous in situ extraction removed the LA using 15% trioctylamine in oleyl alcohol. The reactor showed stable LA concentration of 1 g/L, with the balance of the LA successfully extracted and recovered using back extraction. This study demonstrates a potentially useful physical configuration for continuous in situ extraction.

Transparent polyvinyl-alcohol cryogel as immobilisation matrix for continuous biohydrogen production by phototrophic bacteria.

BACKGROUND: Phototrophic purple non-sulfur bacteria (PNSB) have gained attention for their ability to produce a valuable clean energy source in the form biohydrogen via photofermentation of a wide variety of organic wastes. For maturation of these phototrophic bioprocesses towards commercial feasibility, development of suitable immobilisation materials is required to allow continuous production from a stable pool of catalytic biomass in which energy is not diverted towards biomass accumulation, and optimal hydrogen production rates are realised. Here, the application of transparent polyvinyl-alcohol (PVA) cryogel beads to immobilisation of Rhodopseudomonas palustris for long-term hydrogen production is described. PVA cryogel properties are characterised and demonstrated to be well suited to the purpose of continuous photofermentation. Finally, analysis of the long-term biocompatibility of the material is illustrated. RESULTS: The addition of glycerol co-solvent induces favourable light transmission properties in normally opaque PVA cryogels, especially well-suited to the near-infrared light requirements of PNSB. Material characterisation showed high mechanical resilience, low resistance to diffusion of substrates and high biocompatibility of the material and immobilisation process. The glycerol co-solvent in transparent cryogels offered additional benefit by reinforcing physical interactions to the extent that only a single freeze-thaw cycle was required to form durable cryogels, extending utility beyond only phototrophic bioprocesses. In contrast, conventional PVA cryogels require multiple cycles which compromise viability of entrapped organisms. Hydrogen production studies of immobilised Rhodopseudomonas palustris in batch photobioreactors showed higher specific hydrogen production rates which continued longer than planktonic cultures. Continuous cultivation yielded hydrogen production for at least 67 days from immobilised bacteria, demonstrating the suitability of PVA cryogel immobilisation for long-term phototrophic bioprocesses. Imaged organisms immobilised in cryogels showed a monolithic structure to PVA cryogels, and demonstrated a living, stable, photofermentative population after long-term immobilisation. CONCLUSION: Transparent PVA cryogels offer ideal properties as an immobilisation matrix for phototrophic bacteria and present a low-cost photobioreactor technology for the further advancement of biohydrogen from waste as a sustainable energy source, as well as development of alternative photo-bioprocesses exploiting the unique capabilities of purple non-sulfur bacteria.

Cyanobacterial Growth in Minimally Amended Anaerobic Digestion Effluent and Flue-Gas.

Anaerobic digestion (AD) is an important industrial process, particularly in a biorefinery approach. The liquid effluent and carbon dioxide in the off-gas, can be used to produce high-value products through the cultivation of cyanobacteria. Growth on AD effluent is often limited due to substrate limitation or inhibitory compounds. This study demonstrates the successful cultivation of Synechococcus on minimally amended AD effluent, supplemented with MgSO4 and diluted with seawater. An 8 L airlift reactor illustrated growth in a pilot scale setup. Higher biomass yields were observed for cyanobacteria grown in diluted AD effluent compared to minimal medium, with 60% total nitrogen removal in the effluent. It was demonstrated that controlling the pH, increasing dissolved salt concentrations and adding MgSO4 to the effluent allowed for the successful cultivation of the cyanobacterium, circumventing the addition of clean water for effluent dilution. This could ultimately increase the feasibility of anaerobic digestion-microalgae integrated biorefineries.

The purification of crude glycerol derived from biodiesel manufacture and its use as a substrate by Rhodopseudomonas palustris to produce hydrogen.

Crude glycerol (CG) from biodiesel production is often contaminated with several compounds, including saponified fatty acids (SFAs). Photofermentative growth of Rhodopseudomonas palustris on glycerol leads to hydrogen production; however, R. palustris is inhibited by SFAs. This study examines inhibition of R. palustris by SFAs, finding that, with increasing concentration of SFA, growth rate falls, reaching zero at an SFA concentration of 0.2 mM. Methods for purifying CG were examined, namely (i) treatment with ethanol and activated carbon, (ii) pH adjustment, (iii) solvent extraction, and (iv) precipitation of the fatty acids with calcium. The rates of growth and production of hydrogen were investigated using CG treated by these methods. It was found that treatment with activated carbon, pH reduction, and calcium precipitation reduced inhibition, while solvent extraction was effective only when used in conjunction with pH adjustment. These treatments allow crude glycerol to be used for hydrogen production by R. palustris.

Photofermentation of crude glycerol from biodiesel using Rhodopseudomonas palustris: comparison with organic acids and the identification of inhibitory compounds.

The growth of the biodiesel industry, and its concurrent production of glycerol, has lowered the price of glycerol 20-fold. While many options for using this glycerol have been proposed, the size of the waste stream means that generation of fuels is likely to be the only viable route. One such fuel is hydrogen, production of which can be achieved biologically. The photofermentation of glycerol to hydrogen using Rhodopseudomonas palustris was investigated by exploring the growth rate, hydrogen production rate and hydrogen yield. R. palustris grows on glycerol at a rate of 0.074h(-1), and photoferments glycerol into 97mol% hydrogen at a conversion efficiency nearing 90% of the 7mol H(2) theoretical maximum at a rate of 34mlH(2)/g(dw)/h. Some inhibition of growth by crude glycerol was seen. This was determined to be caused by saponified fatty acids, removal of which yielded a treated crude glycerol which showed no inhibition.