Production and characterization of yeasts grown on media composed of spruce-derived sugars and protein hydrolysates from chicken by-products.

BACKGROUND: A possible future shortage of feed protein will force mankind to explore alternative protein sources that can replace conventional soymeal or fishmeal. Several large industrial organic side-streams could potentially be upgraded to feed protein using a fermentation process to generate single cell protein. Yeast is the most widely accepted microorganism for production of single cell protein, because of its superior nutritional quality and acceptability among consumers. Here, we have assessed the growth of four different yeasts, Cyberlindnera jadinii, Wickerhamomyces anomalus, Blastobotrys adeninivorans and Thermosacc® Dry (Saccharomyces cerevisiae), on media composed of enzymatically saccharified sulfite-pulped spruce wood and hydrolysates of by-products from chicken, and we have characterized the resulting yeast biomass. RESULTS: Generally, the yeast grew very well on the spruce- and chicken-based medium, with typical yields amounting to 0.4-0.5 g of cell dry weight and 0.2-0.3 g of protein per g of sugar. B. adeninivorans stood out as the most versatile yeast in terms of nutrient consumption and in this case yields were as high as 0.9 g cells and 0.5 g protein per g of sugar. The next best performing yeast in terms of yield was W. anomalus with up to 0.6 g cells and 0.3 g protein per g sugar. Comparative compositional analyses of the yeasts revealed favorable amino acid profiles that were similar to the profiles of soymeal, and even more so, fish meal, especially for essential amino acids. CONCLUSIONS: The efficient conversion of industrial biomass streams to yeast biomass demonstrated in this study opens new avenues towards better valorization of these streams and development of sustainable feed ingredients. Furthermore, we conclude that production of W. anomalus or B. adeninivorans on this promising renewable medium may be potentially more efficient than production of the well-known feed ingredient C. jadinii. Further research should focus on medium optimization, development of semi-continuous and continues fermentation protocols and exploration of downstream processing methods that are beneficial for the nutritional values of the yeast for animal feed.

Spruce sugars and poultry hydrolysate as growth medium in repeated fed-batch fermentation processes for production of yeast biomass.

The production of microbial protein in the form of yeast grown on lignocellulosic sugars and nitrogen-rich industrial residues is an attractive approach for reducing dependency on animal and plant protein. Growth media composed of enzymatically saccharified sulfite-pulped spruce wood, enzymatic hydrolysates of poultry by-products and urea were used for the production of single-cell protein. Strains of three different yeast species, Cyberlindnera jadinii, Wickerhamomyces anomalus and Blastobotrys adeninivorans, were cultivated aerobically using repeated fed-batch fermentation up to 25 L scale. Wickerhamomyces anomalus was the most efficient yeast with yields of 0.6 g of cell dry weight and 0.3 g of protein per gram of glucose, with cell and protein productivities of 3.92 g/L/h and 1.87 g/L/h, respectively. Using the conditions developed here for producing W. anomalus, it would take 25 industrial (200 m3) continuously operated fermenters to replace 10% of the fish feed protein used in Norway.

Assessment of the scalability of a microtiter plate system for screening of oleaginous microorganisms.

Recent developments in molecular biology and metabolic engineering have resulted in a large increase in the number of strains that need to be tested, positioning high-throughput screening of microorganisms as an important step in bioprocess development. Scalability is crucial for performing reliable screening of microorganisms. Most of the scalability studies from microplate screening systems to controlled stirred-tank bioreactors have been performed so far with unicellular microorganisms. We have compared cultivation of industrially relevant oleaginous filamentous fungi and microalga in a Duetz-microtiter plate system to benchtop and pre-pilot bioreactors. Maximal glucose consumption rate, biomass concentration, lipid content of the biomass, biomass, and lipid yield values showed good scalability for Mucor circinelloides (less than 20% differences) and Mortierella alpina (less than 30% differences) filamentous fungi. Maximal glucose consumption and biomass production rates were identical for Crypthecodinium cohnii in microtiter plate and benchtop bioreactor. Most likely due to shear stress sensitivity of this microalga in stirred bioreactor, biomass concentration and lipid content of biomass were significantly higher in the microtiter plate system than in the benchtop bioreactor. Still, fermentation results obtained in the Duetz-microtiter plate system for Crypthecodinium cohnii are encouraging compared to what has been reported in literature. Good reproducibility (coefficient of variation less than 15% for biomass growth, glucose consumption, lipid content, and pH) were achieved in the Duetz-microtiter plate system for Mucor circinelloides and Crypthecodinium cohnii. Mortierella alpina cultivation reproducibility might be improved with inoculation optimization. In conclusion, we have presented suitability of the Duetz-microtiter plate system for the reproducible, scalable, and cost-efficient high-throughput screening of oleaginous microorganisms.

FTIR spectroscopy as a unified method for simultaneous analysis of intra- and extracellular metabolites in high-throughput screening of microbial bioprocesses.

BACKGROUND: Analyses of substrate and metabolites are often bottleneck activities in high-throughput screening of microbial bioprocesses. We have assessed Fourier transform infrared spectroscopy (FTIR), in combination with high throughput micro-bioreactors and multivariate statistical analyses, for analysis of metabolites in high-throughput screening of microbial bioprocesses. In our previous study, we have demonstrated that high-throughput (HTS) FTIR can be used for estimating content and composition of intracellular metabolites, namely triglyceride accumulation in oleaginous filamentous fungi. As a continuation of that research, in the present study HTS FTIR was evaluated as a unified method for simultaneous quantification of intra- and extracellular metabolites and substrate consumption. As a proof of concept, a high-throughput microcultivation of oleaginous filamentous fungi was conducted in order to monitor production of citric acid (extracellular metabolite) and triglyceride lipids (intracellular metabolites), as well as consumption of glucose in the cultivation medium. RESULTS: HTS FTIR analyses of supernatant samples was compared with an attenuated total reflection (ATR) FTIR, which is an established method for bioprocess monitoring. Glucose and citric acid content of growth media was quantified by high performance liquid chromatography (HPLC). Partial least square regression (PLSR) between HPLC glucose and citric acid data and the corresponding FTIR spectral data was used to set up calibration models. PLSR results for HTS measurements were very similar to the results obtained with ATR methodology, with high coefficients of determination (0.91-0.98) and low error values (4.9-8.6%) for both glucose and citric acid estimates. CONCLUSIONS: The study has demonstrated that intra- and extracellular metabolites, as well as nutrients in the cultivation medium, can be monitored by a unified approach by HTS FTIR. The proof-of-concept study has validated that HTS FTIR, in combination with Duetz microtiter plate system and chemometrics, can be used for high throughput screening of microbial bioprocesses. It can be anticipated that the approach, demonstrated here on single-cell oil production by filamentous fungi, can find general application in screening studies of microbial bioprocesses, such as production of single-cell proteins, biopolymers, polysaccharides, carboxylic acids, and other type of metabolites.

Microtiter plate cultivation of oleaginous fungi and monitoring of lipogenesis by high-throughput FTIR spectroscopy.

BACKGROUND: Oleaginous fungi can accumulate lipids by utilizing a wide range of waste substrates. They are an important source for the industrial production of omega-6 polyunsaturated fatty acids (gamma-linolenic and arachidonic acid) and have been suggested as an alternative route for biodiesel production. Initial research steps for various applications include the screening of fungi in order to find efficient fungal producers with desired fatty acid composition. Traditional cultivation methods (shake flask) and lipid analysis (extraction-gas chromatography) are not applicable for large-scale screening due to their low throughput and time-consuming analysis. Here we present a microcultivation system combined with high-throughput Fourier transform infrared (FTIR) spectroscopy for efficient screening of oleaginous fungi. RESULTS: The microcultivation system enables highly reproducible fungal fermentations throughout 12 days of cultivation. Reproducibility was validated by FTIR and HPLC data. Analysis of FTIR spectral ester carbonyl peaks of fungal biomass offered a reliable high-throughput at-line method to monitor lipid accumulation. Partial least square regression between gas chromatography fatty acid data and corresponding FTIR spectral data was used to set up calibration models for the prediction of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, unsaturation index, total lipid content and main individual fatty acids. High coefficients of determination (R2 = 0.86-0.96) and satisfactory residual predictive deviation of cross-validation (RPDCV = 2.6-5.1) values demonstrated the goodness of these models. CONCLUSIONS: We have demonstrated in this study, that the presented microcultivation system combined with rapid, high-throughput FTIR spectroscopy is a suitable screening platform for oleaginous fungi. Sample preparation for FTIR measurements can be automated to further increase throughput of the system.

Production of docosahexaenoic acid from spruce sugars using Aurantiochytrium limacinum.

In this study lignocellulosic sugars from Norway spruce were used for production of docosahexaenoic acid (DHA) by the marine thraustochytrid Aurantiochytrium limacinum SR21. Enzymatically prepared spruce hydrolysate was combined with a complex nitrogen source and different amounts of salts. Shake flask batch cultivations revealed that addition of extra salts was not needed for optimal growth. Upscaling to fed-batch bioreactors yielded up to 55 g/L cell dry mass and a total fatty acid content of 44% (w/w) out of which 1/3 was DHA. Fourier transform infrared spectroscopy was successfully applied as a rapid method for monitoring lipid accumulation in A. limacinum SR21. Thus, this proof-of-principle study clearly demonstrates that crude spruce hydrolysates can be directly used as a novel and sustainable resource for production of DHA.

High-throughput screening of Mucoromycota fungi for production of low- and high-value lipids.

BACKGROUND: Mucoromycota fungi are important producers of low- and high-value lipids. Mortierella alpina is used for arachidonic acid production at industrial scale. In addition, oleaginous Mucoromycota fungi are promising candidates for biodiesel production. A critical step in the development of such biotechnological applications is the selection of suitable strains for lipid production. The aim of the present study was to use the Duetz-microtiter plate system combined with Fourier transform infrared (FTIR) spectroscopy for high-throughput screening of the potential of 100 Mucoromycota strains to produce low- and high-value lipids. RESULTS: With this reproducible, high-throughput method, we found several promising strains for high-value omega-6 polyunsaturated fatty acid (PUFA) and biodiesel production purposes. Gamma-linolenic acid content was the highest in Mucor fragilis UBOCC-A-109196 (24.5% of total fatty acids), and Cunninghamella echinulata VKM F-470 (24.0%). For the first time, we observed concomitant gamma-linolenic acid and alpha-linolenic acid (up to 13.0%) production in psychrophilic Mucor flavus strains. Arachidonic acid was present the highest amount in M. alpina ATCC 32222 (41.1% of total fatty acids). Low cultivation temperature (15 °C) activated the temperature sensitive ∆17 desaturase enzyme in Mortierella spp., resulting in eicosapentaenoic acid production with up to 11.0% of total fatty acids in M. humilis VKM F-1494. Cunninghamella blakesleeana CCM-705, Umbelopsis vinacea CCM F-539 and UBOCC-A-101347 showed very good growth (23-26 g/L) and lipid production (7.0-8.3 g/L) with high palmitic and oleic acid, and low PUFA content, which makes them attractive candidates for biodiesel production. Absidia glauca CCM 451 had the highest total lipid content (47.2% of biomass) of all tested strains. We also demonstrated the potential of FTIR spectroscopy for high-throughput screening of total lipid content of oleaginous fungi. CONCLUSIONS: The use of Duetz-microtiter plate system combined with FTIR spectroscopy and multivariate analysis, is a feasible approach for high-throughput screening of lipid production in Mucoromycota fungi. Several promising strains have been identified by this method for the production of high-value PUFA and biodiesel.

Comparative Assessment of Enzymatic Hydrolysis for Valorization of Different Protein-Rich Industrial Byproducts.

Hydrolyzed protein-rich byproducts from food production may find a variety of applications, for example, as rich ingredients of fermentation media. We have conducted a study of the enzymatic hydrolysis of three byproducts from Norwegian food industries: chicken byproducts, mixed pork and beef byproducts, and salmon viscera. The efficiency and optimization of the enzymatic hydrolysis were evaluated using endogenous enzymes alone and in combination with commercial proteases. Hydrolysis reactions were conducted with freshly thawed raw materials using short incubation times and including an initial temperature gradient from 4 to 60 °C to both harness the power of endogenous enzymes and minimize microbial contamination. Subsequently, hydrolysates were characterized by analyzing the total recovery of protein, the peptide molecular-weight distribution, and the composition of total and free amino acids. The action of endogenous enzymes played an important role in raw-material hydrolysis, particularly when hydrolyzing salmon viscera but less so when hydrolyzing chicken byproducts. For pork-beef and chicken byproducts, the addition of Alcalase or Papain improved protein recovery, reaching levels up to 90%. Next to showing efficient hydrolysis protocols, the present data also provide a comparison of the amino acid compositions of hydrolysates derived from these three different protein-rich byproducts. Growth studies showed that the obtained protein-rich hydrolysates from meat and fish industries are a promising alternative for expensive nitrogen sources that are commonly used for fermenting yeasts.

FTIR Spectroscopy for Evaluation and Monitoring of Lipid Extraction Efficiency for Oleaginous Fungi.

To assess whether Fourier Transform Infrared (FTIR) spectroscopy could be used to evaluate and monitor lipid extraction processes, the extraction methods of Folch, Bligh and Lewis were used. Biomass of the oleaginous fungi Mucor circinelloides and Mortierella alpina were employed as lipid-rich material for the lipid extraction. The presence of lipids was determined by recording infrared spectra of all components in the lipid extraction procedure, such as the biomass before and after extraction, the water and extract phases. Infrared spectra revealed the incomplete extraction after all three extraction methods applied to M.circinelloides and it was shown that mechanical disruption using bead beating and HCl treatment were necessary to complete the extraction in this species. FTIR spectroscopy was used to identify components, such as polyphosphates, that may have negatively affected the extraction process and resulted in differences in extraction efficiency between M.circinelloides and M.alpina. Residual lipids could not be detected in the infrared spectra of M.alpina biomass after extraction using the Folch and Lewis methods, indicating their complete lipid extraction in this species. Bligh extraction underestimated the fatty acid content of both M.circinelloides and M.alpina biomass and an increase in the initial solvent-to-sample ratio (from 3:1 to 20:1) was needed to achieve complete extraction and a lipid-free IR spectrum. In accordance with previous studies, the gravimetric lipid yield was shown to overestimate the potential of the SCO producers and FAME quantification in GC-FID was found to be the best-suited method for lipid quantification. We conclude that FTIR spectroscopy can serve as a tool for evaluating the lipid extraction efficiency, in addition to identifying components that may affect lipid extraction processes.