Microbial astaxanthin: from bioprocessing to the market recognition.

The attractive biological properties and health benefits of natural astaxanthin (AXT), including its antioxidant and anti-carcinogenic properties, have garnered significant attention from academia and industry seeking natural alternatives to synthetic products. AXT, a red ketocarotenoid, is mainly produced by yeast, microalgae, wild or genetically engineered bacteria. Unfortunately, the large fraction of AXT available in the global market is still obtained using non-environmentally friendly petrochemical-based products. Due to the consumers concerns about synthetic AXT, the market of microbial-AXT is expected to grow exponentially in succeeding years. This review provides a detailed discussion of AXT's bioprocessing technologies and applications as a natural alternative to synthetic counterparts. Additionally, we present, for the first time, a very comprehensive segmentation of the global AXT market and suggest research directions to improve microbial production using sustainable and environmentally friendly practices. KEY POINTS: • Unlock the power of microorganisms for high value AXT production. • Discover the secrets to cost-effective microbial AXT processing. • Uncover the future opportunities in the AXT market.

Microbial torularhodin - a comprehensive review.

The demand for food, feed, cosmeceutical, and nutraceutical supplements/additives from natural sources has been rapidly increasing, with expectations for a faster expansion than the growth of the global markets in the coming years. In this framework, a particular interest is given to carotenoids due to their outstanding antioxidant activities, particularly the xanthophylls class. Torularhodin is one of these carotenoids that stands out for its multifunctional role as: antioxidant, anticancer and antimicrobial, yet its commercial potential is still unexplored. Although most xanthophylls can be naturally found in: microbial, plant and animal sources, torularhodin is only produced by microbial species, especially red oleaginous yeast. The microbial production of xanthophylls has many advantages as compared to other natural sources, such as: the need for low production area, easier extraction, high yields (at optimum operating conditions), and low (or no) seasonal, climatic, and geographic variation dependency. Due to the importance of natural products and their relevance to the market, this review provides a comprehensive overview of the: properties, characteristics and potential health benefits of torularhodin. Moreover, the most promising developments in both upstream and downstream processing to obtain this colorant from microbial sources are considered. For this purpose, the main microorganisms used for torularhodin production are firstly reviewed, including biosynthesis pathway and torularhodin properties. Following, an overall analysis of the processing aspects related with its: extraction, separation and purification is provided. Lastly, current status and future trends of torularhodin-based processes and products such as therapeutic agents or biomaterials are discussed, indicating promising directions toward biorefinery and circular economy.

Insights into using green and unconventional technologies to recover natural astaxanthin from microbial biomass.

Microorganisms such as bacteria, microalgae and fungi, are natural and rich sources of several valuable bioactive antioxidant's compounds, including carotenoids. Among the carotenoids with antioxidant properties, astaxanthin can be highlighted due to its pharmaceutical, feed, food, cosmetic and biotechnological applications. The best-known producers of astaxanthin are yeast and microalgae cells that biosynthesize this pigment intracellularly, requiring efficient and sustainable downstream procedures for its recovery. Conventional multi-step procedures usually involve the consumption of large amounts of volatile organic compounds (VOCs), which are regarded as toxic and hazardous chemicals. Considering these environmental issues, this review is focused on revealing the potential of unconventional extraction procedures [viz., Supercritical Fluid Extraction (SFE), Ultrasound-Assisted Extraction (UAE), Microwave-Assisted Extraction (MAE), High-Pressure Homogenization (HPH)] combined with alternative green solvents (biosolvents, eutectic solvents and ionic liquids) for the recovery of microbial-based astaxanthin from microalgae (such as Haematococcus pluvialis) and yeast (such as Phaffia rhodozyma) cells. The principal advances in the area, process bottlenecks, solvent selection and strategies to improve the recovery of microbial astaxanthin are emphasized. The promising recovery yields using these environmentally friendly procedures in lab-scale are good indications and directions for their effective use in biotechnological processes for the production of commercial feed and food ingredients like astaxanthin.

Advances and trends in biotechnological production of natural astaxanthin by Phaffia rhodozyma yeast.

Astaxanthin (AXT) is a natural xanthophyll with strong antioxidant, anticancer and antimicrobial activities, widely used in the food, feed, pharmaceutical and nutraceutical industries. So far, 95% of the AXT global market is produced by chemical synthesis, but growing customer preferences for natural products are currently changing the market for natural AXT, highlighting the production from microbially-based sources such as the yeast Phaffia rhodozyma. The AXT production by P. rhodozyma has been studied for a long time at a laboratory scale, but its use in industrial-scale processes is still very scarce. The optimization of growing conditions as well as an effective integration of upstream-downstream operations into P. rhodozyma-based AXT processes has not yet been fully achieved. With this critical review, we scrutinized the main approaches for producing AXT using P. rhodozyma strains, highlighting the impact of using conventional and non-conventional procedures for the extraction of AXT from yeast cells. In addition, we also pinpointed research directions, for example, the use of low-cost residues to improve the economic and environmental sustainability of the bioprocess, the use of environmentally/friendly and low-energetic integrative operations for the extraction and purification of AXT, as well as the need of further human clinical trials using yeast-based AXT.

Assessment of artificial neural networks to predict red colorant production by Talaromyces amestolkiae.

Consumer choice is typically influenced by color, leading to an increase in the use of artificial colorants by industry. However, several artificial colorants have been banned due to their harmful effects on human health and the environment, leading to increased interest in colorants from natural sources. Natural colorants can be found in plants, insects, and microorganisms. The importance of evaluating the technical and cost feasibility for the production of natural colorants are important factors for the replacement of artificial counterpart. Therefore, it is highly beneficial to predict the productivity of microbial colorants. The use of statistical methods that generate polynomial models through multiple regressions can provide information of interest about a bioprocess. However, modeling and control of biological processes require complex systems models, because they are nonlinear and non-deterministic systems. In this regard, artificial neural networks are suitable for estimating bioprocess variables with systems modeling. In this work, two different strategies were developed to predict the production of red colorants by Talaromyces amestolkiae, namely simulation by artificial neural networks (ANN) and response surface methodology (RSM). The results showed that the colorant concentration predicted by ANN is closer to the experimental data than that predicted by polynomial models fitted by multiple regression. Thus, this work suggests that the use of ANN can identify the initial conditions of the culture parameters that have the greatest influence on colorant production and can be a tool to be employed to improve the production of biotechnological products, such as microbial colorants.

Development of Biotechnological Photosensitizers for Photodynamic Therapy: Cancer Research and Treatment-From Benchtop to Clinical Practice.

Photodynamic therapy (PDT) is a noninvasive therapeutic approach that has been applied in studies for the treatment of various diseases. In this context, PDT has been suggested as a new therapy or adjuvant therapy to traditional cancer therapy. The mode of action of PDT consists of the generation of singlet oxygen (¹O2) and reactive oxygen species (ROS) through the administration of a compound called photosensitizer (PS), a light source, and molecular oxygen (3O2). This combination generates controlled photochemical reactions (photodynamic mechanisms) that produce ROS, such as singlet oxygen (¹O2), which can induce apoptosis and/or cell death induced by necrosis, degeneration of the tumor vasculature, stimulation of the antitumor immune response, and induction of inflammatory reactions in the illuminated region. However, the traditional compounds used in PDT limit its application. In this context, compounds of biotechnological origin with photosensitizing activity in association with nanotechnology are being used in PDT, aiming at its application in several types of cancer but with less toxicity toward neighboring tissues and better absorption of light for more aggressive types of cancer. In this review, we present studies involving innovatively developed PS that aimed to improve the efficiency of PDT in cancer treatment. Specifically, we focused on the clinical translation and application of PS of natural origin on cancer.

Rhodotorula sp.-based biorefinery: a source of valuable biomolecules.

The development of an effective, realistic, and sustainable microbial biorefinery depends on several factors, including as one of the key aspects an adequate selection of microbial strain. The oleaginous red yeast Rhodotorula sp. has been studied as one powerful source for a plethora of high added-value biomolecules, such as carotenoids, lipids, and enzymes. Although known for over a century, the use of Rhodotorula sp. as resource for valuable products has not yet commercialized. Current interests for Rhodotorula sp. yeast have sparked from its high nutritional versatility and ability to convert agro-food residues into added-value biomolecules, two attractive characteristics for designing new biorefineries. In addition, as for other yeast-based bioprocesses, the overall process sustainability can be maximized by a proper integration with subsequent downstream processing stages, for example, by using eco-friendly solvents for the recovery of intracellular products from yeast biomass. This review intends to reflect on the current state of the art of microbial bioprocesses using Rhodotorula species. Therefore, we will provide an analysis of bioproduction performance with some insights regarding downstream separation steps for the extraction of high added-value biomolecules (specifically using efficient and sustainable platforms), providing information regarding the potential applications of biomolecules produced by Rhodotorula sp, as well as detailing the strengths and limitations of yeast-based biorefinery approaches. Novel genetic engineering technologies are further discussed, indicating some directions on their possible use for maximizing the potential of Rhodotorula sp. as cell factories. KEY POINTS: • Rhodotorula sp. are valuable source of high value-added compounds. • Potential of employing Rhodotorula sp. in a multiple product biorefinery. • Future perspectives in the biorefining of Rhodotorula sp. were discussed.

Aliivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical.

L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275 mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes.

Xylanase Production by Talaromyces amestolkiae Valuing Agroindustrial Byproducts.

In general, agroindustrial byproducts can be easily assimilated by several microorganisms due to their composition, which is rich in carbohydrates. Therefore, they could be appropriate for use as raw materials in a sustainable refinery concept, including the production of hydrolytic enzymes with industrial applicability. In this work, xylanase production by the filamentous fungi Talaromyces amestolkiae in submerged culture was evaluated using five agroindustrial byproducts, namely, wheat bran, citrus pulp, rice bran, peanut skin, and peanut shell. Firstly, the aforementioned byproducts were characterized in terms of cellulose, xylan, lignin, and extractives. Next, production studies were performed, and wheat bran generated the highest enzymatic activity (5.4 U·mL-1), probably because of its large amount of xylan. Subsequently, a factorial design was performed to evaluate the independent variables yeast extract, wheat bran, K2HPO4, and pH, aiming to improve the variable response, xylanase activity. The condition that promoted the highest production, 13.02 U·mL-1 (141% higher than the initial condition), was 20 g·L-1 wheat bran, 2.5 g·L-1 yeast extract, 3 g·L-1 K2HPO4, and pH 7. Thus, industrial byproducts with a high content of xylan can be used as a culture medium to produce xylanase enzymes with a Talaromyces strain through an economical and sustainable approach.

Effects of cholinium-based ionic liquids on Aspergillus niger lipase: Stabilizers or inhibitors.

Lipases are well-known biocatalysts used in several industrial processes/applications. Thus, as with other enzymes, changes in their surrounding environment and/or their thermodynamic parameters can induce structural changes that can increase, decrease, or even inhibit their catalytic activity. The use of ionic compounds as solvents or additives is a common approach for adjusting reaction conditions and, consequently, for controlling the biocatalytic activity of enzymes. Herein, to elucidate the effects of ionic compounds on the structure of lipase, the stability and enzymatic activity of lipase from Aspergillus niger in aqueous solutions (at 0.05, 0.10, 0.50, and 1.00 M) of six cholinium-based ionic liquids (cholinium chloride [Ch]Cl; cholinium acetate ([Ch][Ac]); cholinium propanoate ([Ch][Prop]); cholinium butanoate ([Ch][But]); cholinium pentanoate ([Ch][Pent]); and cholinium hexanoate ([Ch][Hex])) were evaluated over 24 hr. The enzymatic activity of lipase was maintained or enhanced in the lower concentrations of all the [Ch]+ -ILs (below 0.1 M). [Ch][Ac] maintained the biocatalytic behavior of lipase, independent of the IL concentration and incubation time. However, above 0.1 M, [Ch][Pent] and [Ch][Hex] caused complete inhibition of the catalytic activity of the enzyme, demonstrating that the increase in the anionic alkyl chain length strongly affected the conformation of the lipase. The hydrophobicity and concentration of the [Ch]+ -ILs play an important role in the enzyme activity, and these parameters can be controlled by adjusting the anionic alkyl chain length. The inhibitory effects of [Ch][Pent] and [Ch][Hex] may be of great interest to the pharmaceutical industry to induce pharmacological inhibition of gastric and pancreatic lipases.

Effect of aeration and agitation on extractive fermentation of clavulanic acid by using aqueous two-phase system.

In this work, the effects of agitation and aeration rates on aqueous two-phase system (ATPS)-based extractive fermentation of clavulanic acid (CA) by Streptomyces variabilis DAUFPE 3060 were investigated through a 22 full factorial design, where oxygen transfer rate (OTR) and oxygen uptake rate (OUR) were selected as the responses. Aeration rates significantly influenced cell growth, OUR, and CA yield, while OTR was practically the same in all the runs. Under the intermediate agitation (950 rpm) and aeration conditions (3.5 vvm) of the central point runs, it was achieved OTR of 1.617 ± 0.049 mmol L-1  h-1 , OUR of 0.132 ± 0.030 mmol L-1  h-1 , maximum CA production of 434 ± 4 mg L-1 , oxygen mass transfer coefficient of 33.40 ± 2.01 s-1 , partition coefficient of 66.5 ± 1.5, CA yield in the top and bottom phases of 75% ± 2% and 19% ± 1%, respectively, mass balance of 95% ± 4% and purification factor of 3.8 ± 0.1. These results not only confirmed the paramount role of O2 supply, broth composition and operational conditions in CA ATPS-extractive fermentation, but also demonstrated the possibility of effectively using this technology as a cheap tool to simultaneously produce and recover CA. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1444-1452, 2016.