Chemometric modeling and two-dimensional fluorescence analysis of bioprocess with a new strain of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> to convert residual glycerol into 1,3-propanediol

The goal of this study was to show that the metabolism of Klebsiella pneumoniae under different aeration strategies could be monitored and predicted by the application of chemometric models and fluorescence spectroscopy. Multi-wavelength fluorescence was applied to the on-line monitoring of process parameters for K. pneumoniae cultivations. Differences observed in spectra collected under aerobiosis and anaerobiosis can be explained by the different metabolic states of the cells. To predict process variables such as biomass, glycerol, and 1,3-propanediol (1,3-PD), chemometric models were developed on the basis of the acquired fluorescence spectra, which were measured continuously. Although glycerol and 1,3-PD are not fluorescent compounds, the results showed that this technique could be successfully applied to the on-line monitoring of variables in order to understand the process and thus improve 1,3-PD production. The root mean square errors of predictions were 0.78 units, 10 g/L, and 2.6 g/L for optical density, glycerol, and 1,3-PD, respectively.     

In‐situ monitoring of Saccharomyces cerevisiae ITV01 bioethanol process using near‐infrared spectroscopy NIRS and chemometrics

The application feasibility of in‐situ or in‐line monitoring of S. cerevisiae ITV01 alcoholic fermentation process, employing Near‐Infrared Spectroscopy (NIRS) and Chemometrics, was investigated. During the process in a bioreactor, in the complex analytical matrix, biomass, glucose, ethanol and glycerol determinations were performed by a transflection fiber optic probe immersed in the culture broth and connected to a Near‐Infrared (NIR) process analyzer. The NIR spectra recorded between 800 and 2,200 nm were pretreated using Savitzky‐Golay smoothing and second derivative in order to perform a partial least squares regression (PLSR) and generate the calibration models. These calibration models were tested by external validation and then used to predict concentrations in batch alcoholic fermentations. The standard errors of calibration (SEC) for biomass, ethanol, glucose and glycerol were 0.212, 0.287, 0.532, and 0.296 g/L and standard errors of prediction (SEP) were 0.323, 0.369, 0.794, and 0.507 g/L, respectively. Calibration and validation criteria were defined and evaluated in order to generate robust and reliable models for an alcoholic fermentation process matrix. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:510–517, 2016     

Fermentation of crude glycerol from biodiesel production by <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis>

Clostridium pasteurianum can utilize glycerol as the sole carbon source for the production of butanol and 1,3-propanediol. Crude glycerol derived from biodiesel production has been shown to be toxic to the organism even in low concentrations. By examination of different pretreatments we found that storage combined with activated stone carbon addition facilitated the utilization of crude glycerol. A pH-controlled reactor with in situ removal of butanol by gas stripping was used to evaluate the performance. The fermentation pattern on pretreated crude glycerol was quite similar to that on technical grade glycerol. C. pasteurianum was able to utilize 111 g/l crude glycerol. The average consumption rate was 2.49 g/l/h and maximum consumption rate was 4.08 g/l/h. At the maximal glycerol consumption rate butanol was produced at 1.3 g/l/h. These rates are higher than those previously reported for fermentations on technical grade glycerol by the same strain. A process including pretreatment and subsequent fermentation of the crude glycerol could be usable for industrial production of butanol by C. pasteurianum.     

Use of different carbon sources in cultivation of recombinant Pichia pastoris for angiostatin production

To improve the growth of recombinant Pichia pastoris with a phenotype of Mut^S and expression of angiostatin, the effects of glycerol, sorbitol, acetate and lactic acid which were, respectively, added together with methanol in the expression phase, were studied in a 5-l fermentor. Methanol concentration was automatically controlled at 5 g/l by a methanol monitor and control system, while the feeding of the other carbon source was manually adjusted. The angiostatin production level was 108 mg/l when glycerol was added at an initial rate of 2.3 g/h and gradually increased to 9.9 g/h within an induction period of 96 h. The angiostatin concentration was 141 mg/l as sorbitol was used, while only 52 mg/l were obtained on acetate. The highest angiostatin production of 191 mg/l was achieved as lactic acid was used; whose feeding rate was gradually increased from 2.6 to 11.3 g/h. Lactic acid accumulated during the induction phase and reached 6.3 g/l at the end of fermentation. However, the accumulation of lactic acid did not interfere with angiostatin production, indicating that lactic acid to be a non-repressive carbon source. The average productivity and specific productivity of angiostatin obtained on lactic acid and methanol were, respectively, 2.96 and 0.044 mg/(g h), 1.7- and 2.5-fold of those obtained in the fermentation fed with glycerol and methanol.     

Role of Glycerol Addition on Xylose-to-Xylitol Bioconversion by Candida guilliermondii

The effect of glycerol on xylose-to-xylitol bioconversion by Candida guilliermondii was evaluated by its addition (0.7 and 6.5 g/l) to semidefined media (xylose as a substrate). The glycerol concentrations were chosen based on the amounts produced during previous studies on xylitol production by C. guilliermondii. Medium without glycerol addition (control) and medium containing glycerol (53 g/l) in substitution to xylose were also evaluated. According to the results, the addition of 0.7 g/l glycerol to the fermentation medium favored not only the yield (Y _P/S = 0.78 g/g) but also the xylitol productivity (Q _P = 1.13 g/l/h). During the xylose-to-xylitol bioconversion, the formation of byproducts (glycerol and ethanol) was observed for all conditions employed. In relation to the cellular growth, glycerol as the only carbon source for C. guilliermondii was better than xylose or xylose and glycerol mixtures, resulting in a maximum cellular concentration (5.34 g/l).     

Biotransformation of glycerol to <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid by <Emphasis Type="Italic">Acetobacter tropicalis</Emphasis>

Bacterial strains capable of converting glycerol to glyceric acid (GA) were screened among the genera Acetobacter and Gluconacetobacter. Most of the tested Acetobacter and Gluconacetobacter strains could produce 1.8 to 9.3 g/l GA from 10% (v/v) glycerol when intact cells were used as the enzyme source. Acetobacter tropicalis NBRC16470 was the best GA producer and was therefore further investigated. Based on the results of high-performance liquid chromatography analysis and specific rotation, the enantiomeric composition of the produced GA was d-glyceric acid (d-GA). The productivity of d-GA was enhanced with the addition of both 15% (v/v) glycerol and 20 g/l yeast extract. Under these optimized conditions, A. tropicalis NBRC16470 produced 22.7 g/l d-GA from 200 g/l glycerol during 4 days of incubation in a jar fermentor.     

The influence of increasing media methanol concentration on sophorolipid biosynthesis from glycerol-based feedstocks

Candida bombicola, a known producer of sophorolipids (SLs; glycolipid surfactants), was grown on glycerol and oleic acid with up to 1.5% (v/v) methanol in the fermentation growth media to assess the effects of methanol presence on SL synthesis and structural distribution. Increasing methanol concentrations had little effect on the growth of the organism resulting in average cell dry weights (CDW; after SL separation) of 20.8 ± 0.7 g/l between 0 and 1.5% methanol. However, increasing methanol concentrations decreased SL production by 56% (from 12.7 to 5.6 g/l at 1.5% methanol) which translated to SL yields on a cellular basis of between 0.60 g SL/g cells (in the absence of methanol) to 0.27 g SL/g cells (in the presence of 1.5% methanol). LC/MS revealed that increased methanol concentrations also resulted in larger concentrations (up to 20 mol%) of free acid SLs but had little effect on the ratios of diacetylated SL lactones synthesized with palmitic acid (4 mol%), linoleic acid (3 mol%), oleic acid (80 mol%), and stearic acid (13 mol%) as the hydrophobic moieties.     

Biomass and lipid production of heterotrophic microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> by using biodiesel-derived crude glycerol

Microalgal lipids may be a more sustainable biodiesel feedstock than crop oils. We have investigated the potential for using the crude glycerol as a carbon substrate. In batch mode, the biomass and lipid concentration of Chlorella protothecoides cultivated in a crude glycerol medium were, respectively, 23.5 and 14.6 g/l in a 6-day cultivation. In the fed-batch mode, the biomass and lipid concentration improved to 45.2 and 24.6 g/l after 8.2 days of cultivation, respectively. The maximum lipid productivity of 3 g/l day in the fed-batch mode was higher than that produced by batch cultivation. This work demonstrates the feasibility of crude biodiesel glycerol as an alternative carbon substrate to glucose for microalgal cultivation and a cost reduction of carbon substrate feed in microalgal lipid production may be expected.     

Real‐time monitoring of ethanol production during Pichia stipitis NRRL Y‐7124 alcoholic fermentation using transflection near infrared spectroscopy

The application of in situ near‐infrared spectroscopy monitoring of xylose metabolizing yeast such as Pichia stipitis for ethanol production with semisynthetic media, applying chemometrics, was investigated. During the process in a bioreactor, biomass, glucose, xylose, ethanol, acetic acid, and glycerol determinations were performed by a transflection probe immersed in the culture broth and connected to a near‐infrared process analyzer. Wavelength windows in near‐infrared spectra recorded between 800 and 2200 nm were pretreated using Savitzky–Golay smoothing, second derivative and multiplicative scattering correction in order to perform a partial least squares regression and generate the calibration models. These calibration models were tested by external validation (78 samples). Calibration and validation criteria were defined and evaluated in order to generate robust and reliable models for an alcoholic fermentation process matrix. Moreover, regressions coefficients (β) and variable influence in the projection plots were used to assess the results. A novelty is the use of β versus VIP dispersion plots to determine which vectors have more influence on the response in order to improve process comprehension and operability. Validated models were used in a real‐time monitoring during P. stipitis NRRL Y7124 semisynthetic media fermentations.     

Monitoring of Streptomyces peucetius cultivations using FTIR/ATR spectroscopy and quantitative models based on library type data

Cultivations of Streptomyces peucetius in two types of medium were monitored on-line using a Fourier transform infrared (FTIR) spectrometer combined with an attenuated total reflection probe. The quantitative measurements of the glucose, starch and acetate concentrations were implemented using partial least squares calibration models. These were regressed on spectral and concentration information obtained by adding together single constituent spectra of the main constituents in the medium according to a full factorial design. The accuracy achieved was considered to be satisfactory, with an average root mean square error of prediction of 1.5 g/l for glucose and 0.25 g/l for acetate. The methodology used is considered to be a rapid technique for generation of calibration data, and a step towards the use of library type data for calibration purposes in quantitative FTIR spectroscopy applications in bioprocesses.     

Effective and stable porcine interferon-α production by Pichia pastoris fed-batch cultivation with multi-variables clustering and analysis

Efficient porcine interferon-α (pIFN-α) expression in high density recombinant Pichia pastoris cultivation was achieved in a 5 l bench-scaled bioreactor. The results indicated that a high and stable oxygen uptake rate (OUR) during induction phase was closely related with pIFN-α production efficiency. The multi-variables clustering and analysis results showed that the achievement of a high and stable OUR relied on a higher glycerol consumption rate during fed-batch culture phase and a moderate methanol level (around 10 g/l) during induction phase. In the high and stable OUR environments (200–300 mmol/l/h), the highest pIFN-α antiviral activity could reach a level of 6.7 × 10⁶ IU/ml, which was more than 10–300-folds higher than those obtained at lower OUR (80–200 mmol/l/h) using the same bioreactor and those obtained in shaking flasks. Clustering and analysis of the specific growth and glycerol consumption rates data during culture phase could detect the ill fermentation state at early stage, potentially provided a simple and effective fault alarming/diagnosis method for the achievement of stable pIFN-α production.     

Metabolism of glucose and xylose as single and mixed feed in <Emphasis Type="Italic">Debaryomyces nepalensis</Emphasis> NCYC 3413: production of industrially important metabolites

Efficient conversion of hexose and pentose (glucose and xylose) by a single strain is a very important factor for the production of industrially important metabolites using lignocellulose as the substrate. The kinetics of growth and polyol production by Debaryomyces nepalensis NCYC 3413 was studied under single and mixed substrate conditions. In the presence of glucose, the strain produced ethanol (35.8 ± 2.3 g/l), glycerol (9.0 ± 0.2 g/l), and arabitol (6.3 ± 0.2 g/l). In the presence of xylose, the strain produced xylitol (38 ± 1.8 g/l) and glycerol (18 ± 1.0 g/l) as major metabolites. Diauxic growth was observed when the strain was grown with different combinations of glucose/xylose, and glucose was the preferred substrate. The presence of glucose enhanced the conversion of xylose to xylitol. By feeding a mixture of glucose at 100 g/l and xylose at 100 g/l, it was found that the strain produced a maximum of 72 ± 3 g/l of xylitol. A study of important enzymes involved in the synthesis of xylitol (xylose reductase (XR) and xylitol dehydrogenase (XDH)), glycerol (glycerol-3-phosphate dehydrogenase (G3PDH)) and ethanol (alcohol dehydrogenase (ADH)) in cells grown in the presence of glucose and xylose revealed high specific activity of G3PDH and ADH in cells grown in the presence of glucose, whereas high specific activity of XR, XDH, and G3PDH was observed in cells grown in the presence of xylose. To our knowledge, this is the first study to elaborate the glucose and xylose metabolic pathway in this yeast strain.     

Intra- and extra-cellular flux distributions of TCA and glyoxalate cycle and vancomycin production of <Emphasis Type="Italic">Amycolatopsis orientalis</Emphasis> grown in different glycerol concentration

The relationship between tricarboxylic acid (TCA) and glyoxalate cycle and the effect of their metabolites levels on the vancomycin production of Amycolatopsis orientalis were investigated in different concentration of glycerol (2.5–20 g/l). Intracellular glycerol levels increased with respect to increases in glycerol concentrations of the growth medium. Extracellular glycerol levels decreased slowly up to 24 h while uptake rates were increased during 36–48^th h for 10 and 15 g/l and during 36–60^th h at 20 g/l of glycerol. Intracellular citrate, α-ketoglutarate, fumarate levels increased up to 10 g/l glycerol concentration. However, intracellular succinate and malate levels were increased up to 15 g/l glycerol. Extracellular citrate, α-ketoglutarate, succinate and malate levels increased with respect to increases in glycerol concentration. The highest α-ketoglutarate dehydrogenase activity was determined at 15 g/l glycerol. Isocitrate lyase activity showed a positive correlation with the increases in glycerol concentration of the growth medium. Vancomycin production increased with the increases in glycerol concentration from 5 to 10 g/l. These results showed that A. orientalis grown in glycerol containing medium used glyoxalate shunt actively instead of TCA cycle which supports precursors of many amino acid which are effective on the antibiotic production.     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> growing on biodiesel-derived crude glycerol through a non-sterilized fermentation process

The aim of the present study was to investigate the production of 1,3-propanediol (PDO) under non-sterile fermentation conditions by employing the strain Clostridium butyricum VPI 1718. A series of batch cultures were performed by utilizing biodiesel-derived crude glycerol feedstocks of different origins as the sole carbon source, in various initial concentrations. The strain presented similarities in terms of PDO production when cultivated on crude glycerol of various origins, with final concentrations ranging between 11.1 and 11.5 g/L. Moreover, PDO fermentation was successfully concluded regardless of the initial crude glycerol concentration imposed (from 20 to 80 g/L), accompanied by sufficient PDO production yields (0.52–0.55 g per gram of glycerol consumed). During fed-batch operation under non-sterile culture conditions, 67.9 g/L of PDO were finally produced, with a yield of 0.55 g/g. Additionally, the sustainability of the bioprocess during a continuous operation was tested; indeed, the system was able to run at steady state for 16 days, during which PDO effluent level was 13.9 g/L. Furthermore, possible existence of a microbial community inside the chemostat was evaluated by operating a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis, and DGGE results revealed the presence of only one band corresponding to that of C. butyricum VPI 1718. Finally, non-sterile continuous cultures were carried out at different dilution rates (D), with inlet glycerol concentration at 80 g/L. Maximum PDO production was achieved at low D values (0.02 h⁻¹) corresponding to 30.1 g/L, while the elaboration of kinetic data from continuous cultures revealed the stability of the bioprocess proposed, with global PDO production yield corresponding to 0.52 g/g.     

Real-time update of calibration model for better monitoring of batch processes using spectroscopy

In order to reduce the large calibration matrix usually required for calibrating multiwavelength optical sensors, a simple algorithm based on the addition in process of new standards is proposed. A small calibration model, based on 14 standards, is periodically updated by spectra collected on-line during fermentation operation. Concentrations related to these spectra are reconciled into best-estimated values, by considering carbon and oxygen balances. Using this method, fructose, acetate, and gluconacetan were monitored during batch fermentations of Gluconacetobacter xylinus 12281 using mid-infrared spectroscopy. It is shown that this algorithm compensates for noncalibrated events such as production or consumption of by-products. The standard error of prediction (SEP) values were 0.99, 0.10, and 0.90 g/L for fructose, acetate, and gluconacetan, respectively. By contrast, without an updating of the calibration model, the SEP values were 2.46, 0.92, and 1.04 g/L for fructose, acetate, and gluconacetan, respectively. Using only 14 standards, it was therefore possible to approach the performance of an 88-standard-based calibration model having SEP values of 1.11, 0.37, and 0.79 g/L for fructose, acetate, and gluconacetan, respectively. Therefore, the proposed algorithm is a valuable approach to reduce the calibration time of multiwavelength optical sensors.     

Enhanced production of 2,3-butanediol from glycerol by forced pH fluctuations

The glycerol fermentation by Klebsiella pneumoniae occurs by receiving more than five liquid products—organic acids, diols, and ethanol. Aiming to direct the glycerol conversion towards predominant production of 2,3-butanediol (2,3-BD), the main influencing parameters (the aeration and the pH) were investigated during fed-batch processes. The regime of intensive aeration (2.2 vvm air supply) was evaluated as most favorable for 2,3-BD synthesis and ensured the decrease of all other metabolites. Thus, without pH control, 52.5 g/l 2,3-BD were produced, as the carbon conversion of glycerol into 2,3-BD reached 60.6%. Additional enhancement in 2,3-BD production (by significant increase of glycerol utilization) was achieved by the development of a new method of “forced pH fluctuations”. It was realized by consecutive raisings of pH using definite ΔpH value, at exact time intervals, allowing multiple variations. Thus, the optimal conditions for maximal glycerol consumption were defined, and 70 g/l 2,3-BD were produced, which is the highest amount obtained from glycerol as a sole carbon source until now. The forced pH fluctuations emphasized pH as a governing factor in microbial conversion processes.     

Monitoring biomass and glycerol in an Escherichia coli fermentation using near-infrared spectroscopy

Summary Near-infrared spectroscopy was used to determine biomass and glycerol concentrations in E.coli whole broth fermentation samples. For dry cell weight, a standard error of prediction (SEP) of 0.2 g/L and correlation coefficient (r) of 0.991 were obtained. The SEP and r for glycerol, carbon nutrient, were 0.3 g/L and 0.979. respectively. Off-line analysis was accomplished within 2 minutes of sampling and therefore provides the opportunity to monitor fermentations quickly enough to permit in-process development and troubleshooting.     

Cresols utilization by <Emphasis Type="Italic">Trametes versicolor</Emphasis> and substrate interactions in the mixture with phenol

The ability of the white rot fungus Trametes versicolor strain 1 to degrade and utilize methylated phenols (cresols) was established for the first time in a medium not containing any other carbon components. The data obtained demonstrated the better potential of the strain to assimilate p-cresol instead of o- or m- cresol. The 0.5 g/l p-cresol provided was degraded in full after 96 h. The effect of a dual substrate mixture (0.3 g/l phenol + 0.2 g/l p-cresol) on the growth behavior and degradation capacity of the investigated strain was examined. The cell-free supernatants were analyzed by HPLC. It was established that the presence of p-cresol had not prevented complete phenol degradation but had a significant delaying effect on the phenol degradation dynamics. Phenol hydroxylase, catechol 1.2-dioxygenase and cis,cis-muconate cyclase activities were obtained in conditions of single and mixed substrates cultivation. The influence of different phenolic substrates on phenol hydroxylase activity in Trametes versicolor 1 was established. The mathematical models describing the dynamics of single substrates’ utilization as well as the mutual influence of phenol and p-cresol in the mixture were developed on the bases of Haldane kinetics. The estimated interaction coefficients (I _ph/cr = 4.72, I _cr/ph = 7.46) demonstrated the significant inhibition of p-cresol on phenol biodegradation and comparatively low level of influence of phenol presence on the p-cresol degradation. Molecular 18S RNA gene taxonomy of the investigated strain was performed.     

Optimization of lipase production by <Emphasis Type="Italic">Staphylococcus warneri</Emphasis> EX17 using the polydimethylsiloxanes artificial oxygen carriers

In this research, the combined effects of polydimethylsiloxane (PDMS) and different conditions of oxygen volumetric mass transfer coefficient (k_La) on lipase production by Staphylococcus warneri EX17 were studied and optimized in bioreactor cultures. Raw glycerol from biodiesel synthesis was used as the sole carbon source. Full-factorial central composite design and the response surface methodology were employed for the experimental design and analysis of the results. The optimal polydimethylsiloxane concentration and mass coefficient transfer (k_La) were found to be 13.5% (v/v) and 181 h⁻¹, respectively. Under these conditions, the maximal cell production obtained was 10.0 g/l, and the volumetric lipase activities of approximately 490 U/l, after 6 h of cultivation. These results are in close agreement with the model predictions. Results obtained in this work reveal the positive effects of PDMS on oxygen volumetric mass transfer coefficient (k_La) in the Staphylococcus warneri EX17 cultivation and lipase production.