Succinic acid production from pulp and paper industry waste - A transcriptomic approach

The utilization of renewable resources for the production of bio-based products is essential in order to develop sustainable bioprocesses and biorefineries. Xylose-rich hydrolysates produced from hemicelluloses contained in lignocellulosic resources could be used for the production of succinic acid, one of the most important platform chemicals in the bio-economy era. Exploitation of spent sulphite liquor (SSL), a xylose rich by-product from pulp and paper industry has been previously investigated for succinic acid production by Actinobacillus succinogenes, one of the most efficient natural succinic acid producers. In this study, the metabolic potential of this strain was evaluated through the RNA expression of the metabolic pathways involved in succinic acid production utilizing glucose, xylose or SSL as carbon sources. A transcriptomic approach of the key enzymes of glucose and xylose catabolism, carboxylic acid production as well as oxidative phosphorylation led to an improved understanding on the energy consuming metabolic pathways. The transcriptomic analysis was carried out in batch cultures. A cDNA library was constructed at different phases of the fermentation where major metabolic changes in extracellular metabolites or biomass production were observed. Real time PCR was used to determine the expression levels of the genes of interest throughout the fermentation. The bottlenecks of the fermentative production of succinic acid by A. succinogenes were addressed with particular focus on the effect of glucose and xylose catabolism on pathways that involve ATP consumption and NADH oxidation. All subunits of ATP synthase were highly expressed in all substrates. In particular ATP synthase F0 (ATP SYN F0) was higher expressed when glucose was the only carbon source. Phosphoenol-pyruvate carboxykinase (PEPCK) expression was delayed when xylose was present in the medium. Despite the fact that extracellular lactic acid was not detected, low expression levels of lactic acid dehydrogenase (

Ανάπτυξη βιοδιεργασιών και μεταγραφομική ανάλυση για την παραγωγή ηλεκτρικού οξέος μέσω μικροβιακών ζυμώσεων με χρήση αποβλήτων από βιομηχανικές διεργασίες παραγωγής χαρτιού

The scope of this thesis is the evaluation of spent sulphite liquor (SSL) derived as by-product stream from the sulphite pulping process of Eucalyptus globulus wood for the production of succinic acid via microbial fermentations. The study was initiated with the analysis of the composition of SSL and the evaluation of the inhibitory effect of SSL components and metabolic products on succinic acid production and bacterial growth. The evaluation of acetic acid, furfural and methanol showed that concentrations of 12, 1 and 4 g/L, respectively, are inhibitory for Actinobacillus succinogenes growth, while concentrations of 12, 3 and 8 g/L, respectively, are inhibitory for Basfia succiniciproducens growth. Lignosulphonates also posed major inhibitory effect on succinic acid production at concentrations higher than 50 g/L. Based on the high lignosulphonate concentration of the thick SSL used in this study, it was concluded that pretreatment of SSL should be carried out before fermentation. The critical concentrations of succinic acid, lactic acid, formic acid and acetic acid that inhibit bacterial growth and succinic acid production were 55, 60, 18 and 38 g/L, respectively. Ultrafiltration and nanofiltration of SSL was employed in order to evaluate the sequential extraction of lignosulphonates and the production of bio-based succinic acid using the bacterial strains A. succinogenes and B. succiniciproducens. Furthermore, this pretreatment step produced a permeate liquid stream with low lignosulphonate content that led to efficient succinic acid production. Ultrafiltration with membranes of 10, 5 and 3 kDa molecular weight cut-off result in significant losses of lignosulphonates (26 - 50%) in the permeate stream, while nanofiltration using membrane with 500 Da molecular weight cut-off results in high retention yields of lignosulphonates (95.6%) in the retentate stream and 66% of total sugars in the permeate stream. Fed-batch bioreactor cultures using permeates from ultrafiltrated SSL resulted in similar succinic acid concentration (27.5 g/L) and productivity (0.4 g/L/h) by both strains. When permeates from nanofiltrated SSL were used, the strain B. succiniciproducens showed the highest succinic acid concentration (33.8 g/L), yield (0.58 g per g of consumed sugars) and productivity (0.48 g/L/h). Ultrafiltration of SSL resulted in higher succinic acid production per t of SSL used, whereas nanofiltration resulted in higher LS recovery per t of SSL used. The nanofiltration of 1 t of thick SSL could lead to the production of 306.3 kg of lignosulphonates and 52.7 kg of succinic acid when B. succiniciproducens is used or 51.8 kg of succinic acid when A. succinogenes is used. The ultrafiltration of 1 t of thick SSL using a 3 kDa membrane could result in the production of 237 kg of lignosulphonates and 71.8 kg of succinic acid when B. succiniciproducens is used.The metabolic potential of A. succinogenes was evaluated through RNA expression of the genes that encode the enzymes involved in succinic acid production when the bacterial strain was cultivated on glucose, xylose and SSL. Ultrafiltrated SSL was selected as the substrate to analyse RNA expression levels, which were compared with respective expression levels observed in glucose and xylose bioreactor cultures. Xylose and glucose were selected because they constitute 73% and 10% of the total sugars contained in SSL. A transcriptomic approach of the key enzymes of glucose and xylose catabolism, carboxylic acid production as well as oxidative phosphorylation led to an improved understanding on the energy consuming metabolic pathways. The transcriptomic analysis was carried out in batch cultures. A cDNA library was constructed at different phases of the fermentation where major metabolic changes in extracellular metabolites or biomass production were observed. RT-qPCR was used to determine the expression levels of the genes of interest throughout the fermentation. The bottlenecks of the fermentative production of succinic acid by A. succinogenes were addressed with particular focus on the effect of glucose and xylose catabolism on pathways that involve ATP consumption and NADH oxidation. All subunits of ATP synthase were highly expressed in SSL. In particular, ATP synthase F0 was higher expressed in SSL. Phosphoenol-pyruvate carboxykinase (PEPCK) expression was delayed when xylose was present in the medium. Despite the fact that extracellular lactic acid was not detected low expression levels of lactic acid dehydrogenase (LDH) were observed in all substrates.Finally, a breakthrough technology was applied in fed-batch B. succiniciproducens cultures that integrates succinic acid production via fermentation and in situ separation via electrochemical membrane extraction. The current drives the charged carboxylic acids across an anion exchange membrane by electromigration from the high-volume bioreactor into a low-volume extract that contains the succinic acid in higher concentrations. These membranes are permeable to many carboxylic acid anions (e.g. acetic acid, succinic acid), but impermeable to cells and solids, resulting in a combined extraction, clarification, acidification and concentration step in a single unit.Ο σκοπός της διατριβής αυτής έγκειται στην αξιολόγηση του παράπλευρου ρεύματος που παράγεται από βιομηχανίες χαρτοπολτού (SSL) για την παραγωγή ηλεκτρικού οξέος μέσω μικροβιακών ζυμώσεων. Η μελέτη επικεντρώθηκε αρχικά στην ποσοτικοποίηση των κυριοτέρων συστατικών. Ακολούθως μελετήθηκε η παρεμπόδιση συγκεκριμένων συστατικών του SSL στην παραγωγή ηλεκτρικού οξέος και στην μικροβιακή ανάπτυξη. Οι συγκεντρώσεις του οξικού οξέος, της φουρφουράλης και της μεθανόλης που παρεμποδίζουν την ανάπτυξη του Actinobacillus succinogenes είναι 12, 1 and 4 g/L αντίστοιχα, ενώ οι αντίστοιχες συγκεντρώσεις για τον Basfia succiniciproducens είναι 12, 3 and 8 g/L, αντίστοιχα. Τα λιγνοσουλφονικά άλατα αποτελούν παρεμποδιστικά συστατικά σε συγκεντρώσεις μεγαλύτερες από 50 g/L. Το γεγονός ότι το SSL που χρησιμοποιήθηκε σε αυτή τη μελέτη περιέχει υψηλή συγκέντρωση λιγνοσουλφονικών αλάτων οδήγησε στην απόφαση να προεπεξεργαστούμε το αρχικό SSL μέσω υπερδιήθησης ή νανοδιήθησης. Οι συγκεντρώσεις του ηλεκτρικού οξέος, του γαλακτικού οξέος, του μυρμηγκικού οξέος και του οξικού οξέος που παρεμποδίζουν την παραγωγή ηλεκτρικού οξέος είναι 55, 60, 18 και 38 g/L τόσο για τον A. succinogenes όσο και για τον B. succiniciproducens. Η επεξεργασία του SSL μέσω υπερδιήθησης ή νανοδιήθησης αξιολογήθηκε προκειμένου να εκτιμήσουμε τη δυνατότητα διαχωρισμού λιγνοσουλφονικών αλάτων και σακχάρων τα οποία χρησιμοποιήθηκαν ακολούθως για την παραγωγή ηλεκτρικού οξέος με χρήση των στελεχών A. succinogenes και B. succiniciproducens. Η χρήση υπερδιήθησης με μεμβράνες με μοριακό βάρος αποκοπής της τάξης των 10, 5 και 3 kDa είχε ως αποτέλεσμα την απώλεια σημαντικών ποσοτήτων λιγνοσουλφονικών αλάτων (26 - 50%) στο διήθημα. Η χρήση νανοδιήθησης με μεμβράνες με μοριακό βάρος αποκοπής της τάξης των 500 Da οδήγησε σε υψηλό βαθμό διαχωρισμού λιγνοσουλφονικών αλάτων (95.6%). Η πραγματοποίηση ζυμώσεων ημι-διαλείποντος έργου με χρήση διηθήματος από επεξεργασμένο SSL μέσω υπερδιήθησης οδήγησε στην παραγωγή 27.5 g/L ηλεκτρικού οξέος με παραγωγικότητα 0.4 g/L/h όταν χρησιμοποιήθηκαν και οι δύο μικροοργανισμοί. Η υψηλότερη συγκέντρωση ηλεκτρικού οξέος (33.8 g/L), παραγωγικότητα (0.48 g/L/h) και βαθμός μετατροπής σακχάρων σε ηλεκτρικό οξύ (0.58 g/g) επετεύχθησαν με το στέλεχος B. succiniciproducens όταν αναπτύχθηκε σε διήθημα που παρήχθη μέσω νανοδιήθησης του SSL. Η νανοδιήθηση 1 τόνου SSL δύναται να οδηγήσει στην παραγωγή 306.3 kg λιγνοσουλφονικών αλάτων και 52.7 kg ηλεκτρικού οξέος όταν χρησιμοποιηθεί ο μικροοργανισμός B. succiniciproducens. Η υπερδιήθηση 1 τόνου SSL με χρήση μεμβράνης με μοριακό βάρος αποκοπής της τάξης των 3 kDa δύναται να οδηγήσει στην παραγωγή 237 kg λιγνοσουλφονικών αλάτων και 71.8 kg ηλεκτρικού οξέος όταν χρησιμοποιηθεί ο μικροοργανισμός B. succiniproducens.Η δυναμική του μεταβολισμού του A. succinogenes αξιολογήθηκε μέσω της έφρασης του RNA των γονιδίων που είναι υπεύθυνα για τα ένζυμα που συμμετέχουν στην παραγωγή ηλεκτρικού οξέος, σε υποστρώματα που περιέχουν γλυκόζη, ξυλόζη και στο παραπροϊόν της βιομηχανίας χαρτοπολτού. Επιλέχθηκε η μέθοδος της υπερδιήθησης για την προεπεξεργασία του SSL που χρησιμοποιήθηκε σαν υπόστρωμα για την μελέτη της έκφρασης του RNA. Πραγματοποιήθηκε σύγκριση των αποτελεσμάτων από τη ζύμωση σε SSL με ζυμώσεις που πραγματοποιήθηκαν σε γλυκόζη ή ξυλόζη. Η ξυλόζη και η γλυκόζη επιλέχθηκαν διότι αποτελούν τα κύρια σάκχαρα που περιέχονται στο SSL με περιεκτικότητα 73% και 10%, αντίστοιχα. Η μεταγραφομική ανάλυση των σημαντικότερων ενζύμων που συμμετέχουν στον καταβολισμό της γλυκόζης και της ξυλόζης, στον κύκλο των τρικαρβοξυλικών οξέων και της οξειδωτικής φωσφορυλίωσης, οδήγησαν στην καλύτερη κατανόηση των μεταβολικών διαδικασιών. Η μεταγραφομική ανάλυση πραγματοποιήθηκε σε ζυμώσεις διαλείποντος έργου σε εργαστηριακό βιοαντιδραστήρα. Σε διαφορετικές φάσεις της ζύμωσης, όπου παρατηρήθηκαν αλλαγές στους εξωκυτταρικούς μεταβολίτες, κατασκευάστηκαν cDNA βιβλιοθήκες. Τα επίπεδα έκφρασης των γονιδίων «στόχων», σε διαφορετικές χρονικές στιγμές κατά τη διάρκεια της ζύμωσης, αναλύθηκαν με τη χρήση της RT-qPCR. Τα καθοριστικά σημεία του μεταβολισμού του A. succinogenes αναγνωρίστηκαν. Ιδιαίτερο ενδιαφέρον παρουσίασαν τα μεταβολικά μονοπάτια που συμμετέχουν στην κατανάλωση ATP και στην οξείδωση του NADH σε συνδυασμό με τον καταβολισμό της γλυκόζης και της ξυλόζης. Όλες οι υπομονάδες της ATP συνθάσης υπερεκφράστηκαν στο SSL. Συγκεκριμένα, η F0 περιοχή της ATP συνθάσης υπερεκφράστηκε στο SSL. H έκφραση της φωσφοενολοπυροσταφυλικής καρβοξυκινάσης (PEPCK) ήταν χαμηλότερη όταν το θρεπτικό μέσο περιείχε ξυλόζη. Παρά το γεγονός ότι δεν ανιχνεύθηκε εξωκυτταρικό γαλακτικό οξύ, ανιχνεύθηκε η έκφραση της γαλακτικής δεϋδρογονάσης (LDH) σε όλα τα υποστρώματα.Τέλος, μία καινοτόμος τεχνολογία εφαρμόσθηκε σε καλλιέργεις ημι-διαλείποντος έργου με τον B. succiniciproducens, η οποία συνδυάζει την παραγωγή ηλεκτρικού οξέος μέσω ζύμωσης με ταυτόχρονο διαχωρισμό του ηλεκτρικού οξέος μέσω μίας ηλεκτροχημικής κυψέλης στην οποία χρησιμοποιείται μία ανιονική μεμβράνη. Το ηλεκτρικό ρεύμα οδηγεί τα καρβοξυλικά οξέα από το υγρό της ζύμωσης, διαμέσου της ανιονικής μεμβράνης, σε ένα μικρότερου όγκου διάλυμα το οποίο περιέχει υψηλή συγκέντρωση ηλεκτρικού οξέος. Οι μεμβράνες αυτές είναι διαπερατές σε πολλά καρβοξυλικά οξέα (π.χ. οξικό οξύ, ηλεκτρικό οξύ), αλλά δεν είναι διαπερατές από μικροβιακά κύτταρα και στερεά συστατικά. Αυτό έχει ως αποτέλεσμα να επιτυγχάνεται διαχωρισμός του ηλεκτρικού οξέος από το υγρό της ζύμωσης σε συνδυασμένο με την ταυτόχρονη μετατροπή του άλατος των καρβοξυλικών οξέων στην όξινη μορφή γεγονός το οποίο οδηγεί στη μείωση των σταδίων καθαρισμού του ηλεκτρικού οξέος (π.χ. απομάκρυνση μικροβιακών κυττάρων και προσμίξεων που περιέχονται στο υγρό της ζύμωσης)

Succinic acid production by Actinobacillus succinogenes from batch fermentation of mixed sugars

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8–26.8 g/L and a total yield of 0.59–0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14–0.20 g/g and formate 0.08–0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO2 sparging could replace carbonate supply in the form of MgCO3 without affecting the succinate yield

Direct electrochemical extraction increases microbial succinic acid production from spent sulphite liquor

The fermentative production of succinic acid leads to increasing toxicity over time, and requires base addition to counteract acidification. Here we integrated a fed-batch Basfia succiniciproducens succinic acid fermentation with membrane electrolysis. This approach brings the broth in direct contact with an OH- and H-2 producing cathode, and enables in situ extraction of succinate towards the low volume H+ and O-2 producing anode compartment. In the latter, the succinate is acidified to succinic acid and precipitated. The key advantage of the cathodic process is the production of base maintaining fermentation pH with limited external base addition. The bacterial cells are recycled through the cathode compartment of the electrochemical cell, and exposed to H-2, creating biological reducing power. Fermentations were executed with glucose, xylose and ultrafiltered spent sulphite liquor (SSL), the side stream generated via acidic sulphite pulping of Eucalyptus globulus wood. The membrane was not permeable to cells, color and solids, which resulted in combined succinate extraction, clarification, acidification and concentration in a single unit operation. The succinic acid to by-product ratio increased, favoring the production of succinic acid over lactic, formic and acetic acids, with up to 15% higher total sugars to succinic acid conversion yield. The OH-production due to water reduction resulted in lower NaOH usage (up to 33% less) for maintaining the pH during fermentation. The maximum productivity also increased by 30% to a rate of 0.41 g L-1 h(-1) with the electrochemical system, with 1.65 kW h per kg succinic acid extracted in the case of SSL and 2.4 and 1.9 kW h per kg succinic acid extracted in the case of glucose and xylose, respectively. Considering the aforementioned advantages, this integrated system could evolve into a breakthrough technology in sustainable industrial succinic acid production from crude industrial side streams

Bioprocess development for the production of succinic acid from orange peel waste

A preliminary study has been conducted for the development of a bioprocess targeting the valorization of orange peel waste (OPW). Essential oils and pectin were recovered from OPW through distillation, acid hydrolysis and precipitation with ethanol. Optimal conditions for dilute-acid hydrolysis were investigated via estimation of the sugars released and fermentation. Hydrolysis conditions of 109 o C for 20 min and 116 o C for 10 min using 5 % of dry raw material produced the highest sugar yields at 0.76 and 0.77 (gtotal sugars gdry raw material-1) respectively. In order to test the efficiency of enzyme hydrolysis as a pretreatment method for OPW valorization, cellulase production from T. reesei was investigated, which was maximized following 5 days of cultivation. Furthermore, elemental analysis in hydrolyzates from dilute-acid hydrolysis and a combination of acid and enzyme hydrolysis was performed. The results indicate that during acid/enzyme hydrolysis, high concentrations of Mg2+ and Ca2+ ions are liberated in contrast to dilute-acid hydrolysis. A. succinogenes fermentations of glucose and fructose were performed and the succinic acid yields achieved were 0.66 (gsuccinic acid gglucose-1) and 0.33 (gsuccinic acid gfructose-1) respectively, while galactose was not fermented. Overall, OPW may serve as a promising raw material for simultaneous production of essential oils, pectin and succinic acid

Modelling succinic acid fermentation using a xylose based substrate

This study focuses on the development of unstructured models, including both substrate and product inhibition, that predict the cultivation of Actinobacillus succinogenes and Basfia succiniciproducens on a mixture of C5 and C6 sugars, similar to the sugar composition contained in spent sulphite liquor, the liquid waste stream from the sulphite pulping process. The main sugar monomer contained in the medium was xylose (72.6%) with galactose (12.2%), glucose (10.9%), mannose (4.2%) and arabinose (0.1%) making up the remaining sugar content. The growth inhibition caused by metabolic products (succinic, lactic, acetic, formic and mixed acids) and initial mixed sugar concentration was determined. The highest obtained succinic acid yield, final concentration and productivity in fermentations carried out in Duran bottles were 0.76 g/g, 26.0 g/L and 0.66 g/L/h for B. succiniciproducens and 0.69 g/g, 27.4 g/L and 0.60 g/L/h for A. succinogenes, respectively (the units in yield calculations are referred to grams of succinic acid produced per gram of total sugars consumed). The kinetic parameters for both strains were estimated from experimental results. The obtained R2 values for the fitted models were 0.96 for A. succinogenes and 0.94 for B. succiniciproducens. A sensitivity analysis on the obtained parameters showed that the maximum specific growth rates (μmax) and the growth associated substrate consumption parameters (γ) are the most influential model parameters for both microorganisms. The model was validated by fermentations conducted in lab-scale bioreactors showing good agreement between experimental data and model simulations

Actinobacillus succinogenes: Advances on succinic acid production and prospects for development of integrated biorefineries

Actinobacillus succinogenes is a wild-type bacterial strain, isolated from bovine rumen, known as one of the most efficient natural producers of succinic acid. Herein, the factors contributing to the fermentative production of succinic acid by A. succinogenes are reviewed with particular focus on raw materials, culture conditions, significance of carbon dioxide availability and downstream separation and purification. The metabolic potential of this strain is evaluated through discussion of the pathways involved in succinic acid production, genome analysis as well as the development of A. succinogenes mutants. The review also addresses the importance of by-product formation during fermentation that constitutes an important aspect regulating succinic acid production by A. succinogenes. The prospect of integrating succinic acid production in future biorefineries is assessed

Bioconversions of Biodiesel-Derived Glycerol into Sugar Alcohols by Newly Isolated Wild-Type <i>Yarrowia lipolytica</i> Strains

The utilization of crude glycerol, generated as a by-product from the biodiesel production process, for the production of high value-added products represents an opportunity to overcome the negative impact of low glycerol prices in the biodiesel industry. In this study, the biochemical behavior of Yarrowia lipolytica strains FMCC Y-74 and FMCC Y-75 was investigated using glycerol as a carbon source. Initially, the effect of pH value (3.0–7.0) was examined to produce polyols, intracellular lipids, and polysaccharides. At low pH values (initial pH 3.0–5.0), significant mannitol production was recorded. The highest mannitol production (19.64 g L−1) was obtained by Y. lipolytica FMCC Y-74 at pH = 3.0. At pH values ranging between 5.0 and 6.0, intracellular polysaccharides synthesis was favored, while polyols production was suppressed. Subsequently, the effect of crude glycerol and its concentration on polyols production was studied. Y. lipolytica FMCC Y-74 showed high tolerance to impurities of crude glycerol. Initial substrate concentrations influence polyols production and distribution with a metabolic shift toward erythritol production being observed when the initial glycerol concentration (Gly0) increased. The highest total polyols production (=56.64 g L−1) was obtained at Gly0 adjusted to ≈120 g L−1. The highest polyols conversion yield (0.59 g g−1) and productivity (4.36 g L−1 d−1) were reached at Gly0 = 80 g L−1. In fed-batch intermittent fermentation with glycerol concentration remaining ≤60 g L−1, the metabolism was shifted toward mannitol biosynthesis, which was the main polyol produced in significant quantities (=36.84 g L−1) with a corresponding conversion yield of 0.51 g g−1

Chemical Profiling, Bioactivity Evaluation and the Discovery of a Novel Biopigment Produced by <i>Penicillium purpurogenum</i> CBS 113139

Biobased pigments are environmentally friendly alternatives to synthetic variants with an increased market demand. Production of pigments via fermentation is a promising process, yet optimization of the production yield and rate is crucial. Herein, we evaluated the potential of Penicillium purpurogenum to produce biobased pigments. Optimum sugar concentration was 30 g/L and optimum C:N ratio was 36:1 resulting in the production of 4.1–4.5 AU (namely Pigment Complex A). Supplementation with ammonium nitrate resulted in the production of 4.1–4.9 AU (namely Pigment Complex B). Pigments showed excellent pH stability. The major biopigments in Pigment Complex A were N-threonyl-rubropunctamin or the acid form of PP-R (red pigment), N-GABA-PP-V (violet pigment), PP-O (orange pigment) and monascorubrin. In Pigment Complex B, a novel biopigment annotated as N-GLA-PP-V was identified. Its basic structure contains a polyketide azaphilone with the same carboxyl-monascorubramine base structure as PP-V (violet pigment) and γ-carboxyglutamic acid (GLA). The pigments were not cytotoxic up to 250 μg/mL