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.
- Klíčová slova
- Carotenoids, Infrared spectroscopy, Lipids, Partial least squares (PLS) regression, Process analytical technology, Raman spectroscopy, Real-time monitoring, Rhodotorula, Schizochytrium,
- MeSH
- biomasa MeSH
- fermentace MeSH
- glukosa metabolismus MeSH
- glycerol MeSH
- karotenoidy metabolismus MeSH
- Ramanova spektroskopie * metody MeSH
- triglyceridy MeSH
- uhlík * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glukosa MeSH
- glycerol MeSH
- karotenoidy MeSH
- triglyceridy MeSH
- uhlík * MeSH
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.
- Klíčová slova
- biodiesel, biopolymers, carotenoids, chitin, chitosan, fatty acids, fermentation, fungi, oleaginous microorganisms, pigments,
- MeSH
- analýza hlavních komponent MeSH
- biologické pigmenty analýza MeSH
- biomasa MeSH
- biotechnologie MeSH
- chromatografie plynová MeSH
- fosfor analýza metabolismus MeSH
- Fourierova analýza MeSH
- houby chemie růst a vývoj MeSH
- karotenoidy analýza MeSH
- lipidy analýza MeSH
- magnetická rezonanční spektroskopie MeSH
- Ramanova spektroskopie metody MeSH
- spektrofotometrie ultrafialová MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- vápník metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- biologické pigmenty MeSH
- fosfor MeSH
- karotenoidy MeSH
- lipidy MeSH
- vápník MeSH
Beta (β)-glucans are polysaccharides composed of D-glucose monomers. Nowadays, β-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 β-glucans. In the present study, we screened eleven Basidiomycetes for the coproduction of lipids and β-glucans. We examined the effect of four different C/N ratios and eight different osmolarity conditions on the coproduction of lipids and β-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 β-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 β-glucans. Increased C/N ratio led to increased biomass, lipid and β-glucans production for several yeast strains. Increased osmolarity had a negative effect on biomass and lipid production while the β-glucan production was positively affected.
- Klíčová slova
- carbon:nitrogen ratio, high-throughput screening, lipids, osmotic stress, red yeast, β-glucans,
- Publikační typ
- časopisecké články MeSH
