Raman spectroscopy online monitoring of biomass production, intracellular metabolites and carbon substrates during submerged fermentation of oleaginous and carotenogenic microorganisms.

BACKGROUND: Monitoring and control of both growth media and microbial biomass is extremely important for the development of economical bioprocesses. Unfortunately, process monitoring is still dependent on a limited number of standard parameters (pH, temperature, gasses etc.), while the critical process parameters, such as biomass, product and substrate concentrations, are rarely assessable in-line. Bioprocess optimization and monitoring will greatly benefit from advanced spectroscopy-based sensors that enable real-time monitoring and control. Here, Fourier transform (FT) Raman spectroscopy measurement via flow cell in a recirculatory loop, in combination with predictive data modeling, was assessed as a fast, low-cost, and highly sensitive process analytical technology (PAT) system for online monitoring of critical process parameters. To show the general applicability of the method, submerged fermentation was monitored using two different oleaginous and carotenogenic microorganisms grown on two different carbon substrates: glucose fermentation by yeast Rhodotorula toruloides and glycerol fermentation by marine thraustochytrid Schizochytrium sp. Additionally, the online FT-Raman spectroscopy approach was compared with two at-line spectroscopic methods, namely FT-Raman and FT-infrared spectroscopies in high throughput screening (HTS) setups. RESULTS: The system can provide real-time concentration data on carbon substrate (glucose and glycerol) utilization, and production of biomass, carotenoid pigments, and lipids (triglycerides and free fatty acids). Robust multivariate regression models were developed and showed high level of correlation between the online FT-Raman spectral data and reference measurements, with coefficients of determination (R2) in the 0.94-0.99 and 0.89-0.99 range for all concentration parameters of Rhodotorula and Schizochytrium fermentation, respectively. The online FT-Raman spectroscopy approach was superior to the at-line methods since the obtained information was more comprehensive, timely and provided more precise concentration profiles. CONCLUSIONS: The FT-Raman spectroscopy system with a flow measurement cell in a recirculatory loop, in combination with prediction models, can simultaneously provide real-time concentration data on carbon substrate utilization, and production of biomass, carotenoid pigments, and lipids. This data enables monitoring of dynamic behaviour of oleaginous and carotenogenic microorganisms, and thus can provide critical process parameters for process optimization and control. Overall, this study demonstrated the feasibility of using FT-Raman spectroscopy for online monitoring of fermentation processes.

Mixture Effects of Tryptophan Intestinal Microbial Metabolites on Aryl Hydrocarbon Receptor Activity.

Aryl hydrocarbon receptor (AHR) plays pivotal roles in intestinal physiology and pathophysiology. Intestinal AHR is activated by numerous dietary, endogenous, and microbial ligands. Whereas the effects of individual compounds on AHR are mostly known, the effects of real physiological mixtures occurring in the intestine have not been studied. Using reporter gene assays and RT-PCR, we evaluated the combinatorial effects (3520 combinations) of 11 microbial catabolites of tryptophan (MICTs) on AHR. We robustly (n = 30) determined the potencies and relative efficacies of single MICTs. Synergistic effects of MICT binary mixtures were observed between low- or medium-efficacy agonists, in particular for combinations of indole-3-propionate and indole-3-lactate. Combinations comprising highly efficacious agonists such as indole-3-pyruvate displayed rather antagonist effects, caused by saturation of the assay response. These synergistic effects were confirmed by RT-PCR as CYP1A1 mRNA expression. We also tested mimic multicomponent and binary mixtures of MICTs, prepared based on the metabolomic analyses of human feces and colonoscopy aspirates, respectively. In this case, AHR responsiveness did not correlate with type of diet or health status, and the indole concentrations in the mixtures were determinative of gross AHR activity. Future systematic research on the synergistic activation of AHR by microbial metabolites and other ligands is needed.

Assessment of Biotechnologically Important Filamentous Fungal Biomass by Fourier Transform Raman Spectroscopy.

Oleaginous filamentous fungi can accumulate large amount of cellular lipids and biopolymers and pigments and potentially serve as a major source of biochemicals for food, feed, chemical, pharmaceutical, and transport industries. We assessed suitability of Fourier transform (FT) Raman spectroscopy for screening and process monitoring of filamentous fungi in biotechnology. Six Mucoromycota strains were cultivated in microbioreactors under six growth conditions (three phosphate concentrations in the presence and absence of calcium). FT-Raman and FT-infrared (FTIR) spectroscopic data was assessed in respect to reference analyses of lipids, phosphorus, and carotenoids by using principal component analysis (PCA), multiblock or consensus PCA, partial least square regression (PLSR), and analysis of spectral variation due to different design factors by an ANOVA model. All main chemical biomass constituents were detected by FT-Raman spectroscopy, including lipids, proteins, cell wall carbohydrates, and polyphosphates, and carotenoids. FT-Raman spectra clearly show the effect of growth conditions on fungal biomass. PLSR models with high coefficients of determination (0.83-0.94) and low error (approximately 8%) for quantitative determination of total lipids, phosphates, and carotenoids were established. FT-Raman spectroscopy showed great potential for chemical analysis of biomass of oleaginous filamentous fungi. The study demonstrates that FT-Raman and FTIR spectroscopies provide complementary information on main fungal biomass constituents.

Production of Enriched Biomass by Carotenogenic Yeasts Cultivated on by-Products of Poultry Processing-A Screening Study.

Carotenogenic yeasts are a group of microorganisms producing valuable metabolites such as carotenoids, ergosterol, ubiquinone or fatty acids. Their exceptional adaptability allows them to grow in diverse conditions. Owing to their extracellular lipase activity, they are capable of processing many lipid-type waste substrates. This study discusses the processing of poultry waste, specifically fat and feathers by using carotenogenic yeasts. Poultry fat does not require any pre-treatment to be utilized by yeast, but hydrolytic pre-treatment is required for the utilization of the nitrogen contained in feathers. Glycerol was used as a supplementary substrate to support the culture in the early stages of growth. Seven yeast strains were used for the experiments, of which the strain Rhodotorula mucilaginosa CCY19-4-25 achieved exceptional results of biomass production: 29.5 g/L on poultry fat + 10% glycerol at C/N ratio 25 and 28.3 g/L on media containing poultry fat + 25% glycerol at C/N 50. The bioreactor cultivation of the Rhodosporidium toruloides strain in media containing glycerol and feather hydrolysate as a nitrogen substrate achieved a biomass yield of 34.92 g/L after 144 h of cultivation. The produced enriched yeast biomass can be used as a component for poultry feeding; thus, the study is performed under the biorefinery concept.

The Effect of Oil-Rich Food Waste Substrates, Used as an Alternative Carbon Source, on the Cultivation of Microalgae-A Pilot Study.

Microalgae are mostly phototrophic microorganisms present worldwide, showcasing great adaptability to their environment. They are known for producing essential metabolites such as carotenoids, chlorophylls, sterols, lipids, and many more. This study discusses the possibility of the mixotrophic abilities of microalgae in the presence of food waste oils. The utilization of food waste materials is becoming more popular as a research subject as its production grows every year, increasing the environmental burden. In this work, waste frying oil and coffee oil were tested for the first time as a nutrition source for microalgae cultivation. Waste frying oil is produced in large amounts all over the world and its simple purification is one of its greatest advantages as it only needs to be filtered from leftover food pieces. Coffee oil is extracted from waste spent coffee grounds as a by-product. The waste frying oil and coffee oil were added to the basic algal media as an alternative source of carbon. As a pilot study for further experimentation, the effect of oil in the medium, algal adaptability, and capability to survive were tested within these experiments. The growth and production characteristics of four algae and cyanobacteria strains were tested, of which the strain Desmodesmus armatus achieved exceptional results of chlorophyll (8.171 ± 0.475 mg/g) and ubiquinone (5.708 ± 0.138 mg/g) production. The strain Chlamydomonas reindhartii showed exceptional lipid accumulation in the range of 30-46% in most of the samples.

Oleaginous Yeast Extracts and Their Possible Effects on Human Health.

Four non-conventional oleaginous and pigmented yeast strains of Metschnikowia pulcherrima, Cystofilobasidium infirmominiatum, Phaffia rhodozyma, and Rhodotorula kratochvilovae were used in this study. Complex yeast extracts were prepared and tested for biological activity, safety, and effect on human health. In this paper, we measured the antioxidant activity and antimicrobial effect of yeast biomass as a whole and their extracts to compare the influence of carotenoids and other bioactive substances in the studied biomass. All yeast extracts exhibited a significant dose-dependent antimicrobial effect against both G+ and G- bacteria and had a strong antioxidant effect. No cytotoxicity in the mouse melanoma B16F1 cell line was found in concentrations up to 20% of rehydrated biomass in cell medium. All of the extracts were cytotoxic at a concentration of 5 mg of extract/g of dry biomass. All the pigmented yeast extracts showed some positive results for apoptosis of murine melanoma cell lines and are therefore strong candidates positively effect human health. Red yeast cell biomass is a prospective material with many attractive biological functions and can be used in the food industry, as a pharmaceutical material, or in the feed industry.

Conversion of Mixed Waste Food Substrates by Carotenogenic Yeasts of Rhodotorula sp. Genus.

The consequence of the massive increase in population in recent years is the enormous production of mainly industrial waste. The effort to minimize these waste products is, therefore, no longer sufficient. Biotechnologists, therefore, started looking for ways to not only reuse these waste products, but also to valorise them. This work focuses on the biotechnological use and processing of waste oils/fats and waste glycerol by carotenogenic yeasts of the genus Rhodotorula and Sporidiobolus. The results of this work show that the selected yeast strains are able to process waste glycerol as well as some oils and fats in a circular economy model and, moreover, are resistant to potential antimicrobial compounds present in the medium. The best-growing strains, Rhodotorula toruloides CCY 062-002-004 and Rhodotorula kratochvilovae CCY 020-002-026, were selected for fed-batch cultivation in a laboratory bioreactor in a medium containing a mixture of coffee oil and waste glycerol. The results show that both strains were able to produce more than 18 g of biomass per litre of media with a high content of carotenoids (10.757 ± 1.007 mg/g of CDW in R. kratochvilovae and 10.514 ± 1.520 mg/g of CDW in R. toruloides, respectively). The overall results prove that combining different waste substrates is a promising option for producing yeast biomass enriched with carotenoids, lipids, and beta-glucans.

Rhodotorula kratochvilovae CCY 20-2-26-The Source of Multifunctional Metabolites.

Multifunctional biomass is able to provide more than one valuable product, and thus, it is attractive in the field of microbial biotechnology due to its economic feasibility. Carotenogenic yeasts are effective microbial factories for the biosynthesis of a broad spectrum of biomolecules that can be used in the food and feed industry and the pharmaceutical industry, as well as a source of biofuels. In the study, we examined the effect of different nitrogen sources, carbon sources and CN ratios on the co-production of intracellular lipids, carotenoids, ß-glucans and extracellular glycolipids. Yeast strain R. kratochvilovae CCY 20-2-26 was identified as the best co-producer of lipids (66.7 ± 1.5% of DCW), exoglycolipids (2.42 ± 0.08 g/L), ß-glucan (11.33 ± 1.34% of DCW) and carotenoids (1.35 ± 0.11 mg/g), with a biomass content of 15.2 ± 0.8 g/L, by using the synthetic medium with potassium nitrate and mannose as a carbon source. It was shown that an increased C/N ratio positively affected the biomass yield and production of lipids and ß-glucans.

Use of Waste Substrates for the Lipid Production by Yeasts of the Genus Metschnikowia-Screening Study.

Oleogenic yeasts are characterized by the ability to accumulate increased amounts of lipids under certain conditions. These microbial lipids differ in their fatty acid composition, which allows them to be widely used in the biotechnology industry. The interest of biotechnologists is closely linked to the rising prices of fossil fuels in recent years. Their negative environmental impact is caused by significantly increased demand for biodiesel. The composition of microbial lipids is very similar to vegetable oils, which provides great potential for use in the production of biodiesel. In addition, some oleogenic microorganisms are capable of producing lipids with a high proportion of unsaturated fatty acids. The presented paper's main aim was to study the production of lipids and lipid substances by yeasts of the genus Metschnikowia, to cultivate crude waste animal fat to study its utilization by yeasts, and to apply the idea of circular economy in the biotechnology of Metschnikowia yeasts. The work focuses on the influence of various stress factors in the cultivation process, such as reduced temperature or nutritional stress through the use of various waste substrates, together with manipulating the ratio of carbon and nitrogen sources in the medium. Yeast production properties were monitored by several instrumental techniques, including gas chromatography and Raman spectroscopy. The amount of lipids and in particular the fatty acid composition varied depending on the strains studied and the culture conditions used. The ability of yeast to produce significant amounts of unsaturated fatty acids was also demonstrated in the work. The most suitable substrate for lipid production was a medium containing glycerol, where the amount of accumulated lipids in the yeast M. pulcherrima 1232 was up to 36%. In our work, the crude animal fat was used for the production of high-value lipids, which to the best of our knowledge is a new result. Moreover, quantitative screening of lipase enzyme activity cultivated on animal fat substrate on selected yeasts of the genus Metschnikowia was performed. We found that for the yeast utilizing glycerol, animal fat seems to be an excellent source of carbon. Therefore, the yeast conversion of crude processed animal fat to value-added products is a valuable process for the biotechnology and food industry.

Production of Enriched Sporidiobolus sp. Yeast Biomass Cultivated on Mixed Coffee Hydrolyzate and Fat/Oil Waste Materials.

One of the most addressed topics today is the transfer from a linear model of economics to a model of circular economics. It is a discipline that seeks to eliminate waste produced by various industries. The food industry generates huge amounts of waste worldwide, particularly the coffee industry, and related industries produce millions of tons of waste a year. These wastes have potential utility in biotechnology, and in the production of energy, fuels, fertilizers and nutrients, using green techniques such as anaerobic digestion, co-digestion, composting, enzymatic action, and ultrasonic and hydrothermal carbonization. This work is focused on the biotechnological use of processed spent coffee grounds (SCG) and waste fat/oil materials by some Sporidiobolus sp. carotenogenic yeasts in the model of circular economics. The results show that selected yeast strains are able to grow on SCG hydrolysate and are resistant to antimicrobial compounds present in media. The most productive strain Sporidiobolus pararoseus CCY19-9-6 was chosen for bioreactor cultivation in media with a mixture of coffee lignocellulose fraction and some fat wastes. Sporidiobolus pararoseus CCY19-9-6 was able to produce more than 22 g/L of biomass in mixture of SCG hydrolysate and both coffee oil and frying oil. The combined waste substrates induced the production of lipidic metabolites, whereby the production of carotenoids exceeded 5 mg/g of dry biomass. On media with coffee oil, this strain produced high amounts of ubiquinone (8.265 ± 1.648 mg/g) and ergosterol (13.485 ± 1.275 mg/g). Overall, the results prove that a combination of waste substrates is a promising option for the production of carotenoid- and lipid-enriched yeast biomass.

Bioreactor Co-Cultivation of High Lipid and Carotenoid Producing Yeast Rhodotorula kratochvilovae and Several Microalgae under Stress.

The co-cultivation of red yeasts and microalgae works with the idea of the natural transport of gases. The microalgae produce oxygen, which stimulates yeast growth, while CO2 produced by yeast is beneficial for algae growth. Both microorganisms can then produce lipids. The present pilot study aimed to evaluate the ability of selected microalgae and carotenogenic yeast strains to grow and metabolize in co-culture. The effect of media composition on growth and metabolic activity of red yeast strains was assessed simultaneously with microalgae mixotrophy. Cultivation was transferred from small-scale co-cultivation in Erlenmeyer flasks to aerated bottles with different inoculation ratios and, finally, to a 3L bioreactor. Among red yeasts, the strain R. kratochvilovae CCY 20-2-26 was selected because of the highest biomass production on BBM medium. Glycerol is a more suitable carbon source in the BBM medium and urea was proposed as a compromise. From the tested microalgae, Desmodesmus sp. were found as the most suitable for co-cultivations with R. kratochvilovae. In all co-cultures, linear biomass growth was found (144 h), and the yield was in the range of 8.78-11.12 g/L of dry biomass. Lipids increased to a final value of 29.62-31.61%. The FA profile was quite stable with the UFA portion at about 80%. Around 1.98-2.49 mg/g CDW of carotenoids with torularhodine as the major pigment were produced, ubiquinone production reached 5.41-6.09 mg/g, and ergosterol yield was 6.69 mg/g. Chlorophyll production was very low at 2.11 mg/g. Pilot experiments have confirmed that carotenogenic yeasts and microalgae are capable of symbiotic co-existence with a positive impact om biomass growth and lipid metabolites yields.

Revealing the Potential of Lipid and ß-Glucans Coproduction in Basidiomycetes Yeast.

Beta (ß)-glucans are polysaccharides composed of D-glucose monomers. Nowadays, ß-glucans are gaining attention due to their attractive immunomodulatory biological activities, which can be utilized in pharmaceutical or food supplementation industries. Some carotenogenic Basidiomycetes yeasts, previously explored for lipid and carotenoid coproduction, could potentially coproduce a significant amount of ß-glucans. In the present study, we screened eleven Basidiomycetes for the coproduction of lipids and ß-glucans. We examined the effect of four different C/N ratios and eight different osmolarity conditions on the coproduction of lipids and ß-glucans. A high-throughput screening approach employing microcultivation in microtiter plates, Fourier Transform Infrared (FTIR) spectroscopy and reference analysis was utilized in the study. Yeast strains C. infirmominiatum CCY 17-18-4 and R. kratochvilovae CCY 20-2-26 were identified as the best coproducers of lipids and ß-glucans. In addition, C. infirmominiatum CCY 17-18-4, R. kratochvilovae CCY 20-2-26 and P. rhodozyma CCY 77-1-1 were identified as the best alternative producers of ß-glucans. Increased C/N ratio led to increased biomass, lipid and ß-glucans production for several yeast strains. Increased osmolarity had a negative effect on biomass and lipid production while the ß-glucan production was positively affected.

Study of Metabolic Adaptation of Red Yeasts to Waste Animal Fat Substrate.

Carotenogenic yeasts are non-conventional oleaginous microorganisms capable of utilizing various waste substrates. In this work, four red yeast strains (Rhodotorula, Cystofilobasidium, and Sporobolomyces sp.) were cultivated in media containing crude, emulsified, and enzymatically hydrolyzed animal waste fat, compared with glucose and glycerol, as single C-sources. Cell morphology (cryo-SEM (cryo-scanning electron microscopy), TEM (transmission electron microscopy)), production of biomass, lipase, biosurfactants, lipids (gas chromatography/flame ionization detection, GC/FID) carotenoids, ubiquinone, and ergosterol (high performance liquid chromatography, HPLC/PDA) in yeast cells was studied depending on the medium composition, the C source, and the carbon/nitrogen (C/N) ratio. All studied strains are able to utilize solid and processed fat. Biomass production at C/N = 13 was higher on emulsified/hydrolyzed fat than on glucose/glycerol. The production of lipids and lipidic metabolites was enhanced for several times on fat; the highest yields of carotenoids (24.8 mg/L) and lipids (54.5%/CDW (cell dry weight)) were found in S. pararoseus. Simultaneous induction of lipase and biosurfactants was observed on crude fat substrate. An increased C/N ratio (13-100) led to higher biomass production in fat media. The production of total lipids increased in all strains to C/N = 50. Oppositely, the production of carotenoids, ubiquinone, and ergosterol dramatically decreased with increased C/N in all strains. Compounds accumulated in stressed red yeasts have a great application potential and can be produced efficiently during the valorization of animal waste fat under the biorefinery concept.