Constraint-based modelling of mixed microbial populations: Application to polyhydroxyalkanoates production

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

Production of Acetic Acid for CMA Deicer, HR-304, 1988

Although the overall objective for undertaking this project is to help decide on the best way to produce CMA, the tasks to be performed deal primarily with acetic acid itself. The objectives of our part of this project can be restated here: A. Evaluate the cost and composition of potential low-cost fermentation substrates that are available in large quantity at central locations in Iowa. B. Compare the nutritional and physiological properties of a variety of homoacetogenic bacteria relative to acetic acid production, based on information available in the literature. C. Using both of these pools of information, evaluate the possibilities for use of substrates for acetic acid production that are significantly cheaper than the previous sugar, starch hydrolysate or whole corn based studies; also, compare the different acetogens encountered with the most commonly discussed acetogen, Clostridium thermoaceticum; arrive at conclusions on 1-3 of the best agriculture-derived substrates that should be further examined, and on 1-3 of the best organisms to evaluate experimentally. D. Collect experimental data at the tube and fermentor scale on 1-2 of the possibilities in C above. E. Comment on our understanding of acetic acid production possibilities from our perspective as microbiologists, and provide all this above information to Paul Peterschmidt for him to consider for his portion of this report. F. In addition, we would like to point out the possible advantage of examining the use of an agricultural by-product, corn steep liquor, as a direct, non-fermented feedstock for a non-acetic acid deicer.

Utilisation of by-products from sunflower-based biodiesel production processes for the production of fermentation feedstock

By-products streams from a sunflower-based biodiesel plant were utilised for the production of fermentation media that can be used for the production of polyhydroxyalkanoates (PHA). Sunflower meal was utilised as substrate for the production of crude enzyme consortia through solid state fermentation (SSF) with the fungal strain Aspergillus oryzae. Fermented solids were subsequently mixed with unprocessed sunflower meal aiming at the production of a nutrient-rich fermentation feedstock. The highest free amino nitrogen (FAN) and inorganic phosphorus concentrations achieved were 1.5 g L-1 and 246 mg L-1, respectively, when an initial proteolytic activity of 6.4 U mL-1 was used. The FANconcentrationwas increased to 2.3 g L-1 when the initial proteolytic activity was increased to 16 U mL-1. Sunflower meal hydrolysates were mixed with crude glycerol to provide fermentationmedia that were evaluated for the production of poly(3-hydroxybutyrateco- 3-hydroxyvalerate) (P(3HB-co-3HV)) using Cupriavidus necator DSM545. The P(3HB-co-3HV) (9.9 g l-1) produced contained 3HB and 3HV units with 97 and 3 mol %, respectively. Integrating PHA production in existing 1st generation biodiesel production plants through valorisation of by-product streams could improve their sustainability.

Fermentation of Groundnut Shell Enzymatic Hydrolysate for Fuel Ethanol Production by Free and Sorghum Stalks Immobilized Cells of Pichia stipitis NCIM 3498

Groundnut shell (GS), after separation of pod, is readily available as a potential feedstock for production of fermentable sugars. The substrate was delignified with sodium sulfite. The delignified substrate released 670 mg/g of sugars after enzymatic hydrolysis (50 degrees C, 120 rpm, 50 hrs) using commercial cellulases (Dyadic Xylanase PLUS, Dyadic Inc. USA). The groundnut shell enzymatic hydrolysate (45.6 g/L reducing sugars) was fermented for ethanol production with free and sorghum stalks immobilized cells of Pichia stipitis NCIM 3498 under submerged cultivation conditions. Immobilization of yeast cells on sorghum stalks were confirmed by scanning electron microscopy (SEM). A maximum of ethanol production (17.83 g/L, yield 0.44 g/g and 20.45 g/L, yield 0.47 g/g) was observed with free and immobilized cells of P. stipitis respectively in batch fermentation conditions. Recycling of immobilized cells showed a stable ethanol production (20.45 g/L, yield 0.47 g/g) up to 5 batches followed by a gradual downfall in subsequent cycles.

Behavior and viability of spontaneous oxidative stress-resistant Lactococcus lactis mutants in experimental fermented milk processing

Previously, we isolated two strains of spontaneous oxidative (SpOx2 and SpOx3) stress mutants of Lactococcus lactis subsp cremoris. Herein, we compared these mutants to a parental wild- type strain (J60011) and a commercial starter in experimental fermented milk production. Total solid contents of milk and fermentation temperature both affected the acidification profile of the spontaneous oxidative stress- resistant L. lactis mutants during fermented milk production. Fermentation times to pH 4.7 ranged from 6.40 h (J60011) to 9.36 h (SpOx2); V(max) values were inversely proportional to fermentation time. Bacterial counts increased to above 8.50 log(10) cfu/mL. The counts of viable SpOx3 mutants were higher than those of the parental wild strain in all treatments. All fermented milk products showed post-fermentation acidification after 24 h of storage at 4 degrees C; they remained stable after one week of storage.

Increased CLA content in organic milk fermented by bifidobacteria or yoghurt cultures

This study investigates the kinetics of acidification, fatty acid (FA) profile and conjugated linoleic acid (CLA, C18:2 c9, t11) content in fermented milks prepared from organic and conventional milk. Fermented milks were manufactured with five mixed cultures: four different strains of Bifidobacterium animalis subsp. lactis (BL04, B94, BB12 and HN019) and Lactobacillus delbrueckii subsp. bulgaricus LB340, in co-culture with Streptococcus thermophilus TA040. The composition of milk was evaluated, and the kinetics of acidification was followed by continuous pH measurement using the Cinac system. The profile of FA, including CLA, was analyzed by gas chromatography. The chemical composition of conventional and organic milk was similar, with the exception of protein and Fe, the concentrations of which were higher in the organic milk. The rate of acidification was significantly influenced by the type of milk and the bacterial strain used. Co-cultures St-HN019 and St-BB12 showed higher maximal acidification rates in both milks. Final counts of S. thermophilus (9.0-10.1 log(10) colony forming units (CFU) . mL(-1), L)actobacillus bulgaricus (8.2-8.5 log(10) CFU . mL(-1)) and B. animalis subsp. lactis strains (8.3-9.3 log(10) CFU . mL(-1)) did not differ significantly in either milk. Unexpectedly, all fermented organic milks contained significantly higher amounts of CLA than the same milk before fermentation, whereas CLA amounts did not change during fermentation of conventional milk. Regardless of the type of milk, CLA was found to be significantly positively correlated with trans-vaccenic acid and negatively correlated with linoleic acid. Moreover, the CLA contents were significantly higher in fermented milks showing shorter fermentation times.

Effect of inulin as a prebiotic to improve growth and counts of a probiotic cocktail in fermented skim milk

Inulin was used as a prebiotic to improve quality of skim milk fermented by pure cultures of Lactobacillus acidophilus Lactobacillus rhamnosus Lactobacillus bulgaricus and Bifidobacterium lactis binary co-cultures with Streptococcus thermophilus or a cocktail containing all them Inulin supplementation to pure cultures lowered the generation time with particular concern to S thermophilus and L acidophilus The generation time of all micro-organisms decreased in the following order mono-cultures co-cultures cocktail It was demonstrated a synergism between S thermophilus and the other strains and a bifidogenic effect of inulin Enumerations of L rhamnosus in cocktail markedly decreased compared to co-cultures likely because of greater competition for the same substrates The results of this work highlight the industrial potential of the cocktail mainly in terms of fermentation acceleration (C) 2010 Elsevier Ltd All rights reserved

Use of lactulose as prebiotic and its influence on the growth, acidification profile and viable counts of different probiotics in fermented skim milk

Lactulose can be considered as a prebiotic, which is able to stimulate healthy intestinal microflora. In the present work, the use of this ingredient in fermented milk improved quality of skim milk fermented by Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus and Bifidobacterium lactis in co-culture with Streptococcus thermophilus. Compared to control fermentations without lactulose, the addition of such a prebiotic in skim milk increased the counts of all probiotics, with particular concern to B. lactis (bifidogenic effect), the acidification rate and the lactic acid acidity, and concurrently reduced the time to complete fermentation (t(pH4.5)) and the pH at the end of cold storage for 1 to 35 days. (c) 2010 Elsevier B.V. All rights reserved.

Effect of inulin as prebiotic and synbiotic interactions between probiotics to improve fermented milk firmness

Inulin behaved as a prebiotic to improve firmness of skim milk fermented by (a) pure cultures of Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), Lactobacillus bulgaricus (Lb) and Bifidobacterium lactis (Bl), (b) binary co-cultures of them with Streptococcus thermophilus (St), or (c) a cocktail containing all them. Inulin addition to co-cultures and cocktail enhanced products firmness, either after 1 day (D1) or 7 days (D7) of cold storage, likely due to the increase in microbial growth induced by metabolic interactions among lactic acid bacteria and partial inulin metabolization. Co-culture firmness did in fact range from 0.33 N without inulin (St-Lb) after D1 and 0.55 N with inulin (St-Lr) after D7. Also cocktail cultures exhibited high values of firmness, ranging, as an average, from 0.43 N (D1) to 0.46 N (D7), which suggests that they could have been potentiated by the reciprocal synergistic effects of microorganisms in complex mixture. (C) 2011 Elsevier Ltd. All rights reserved.

Liquid-liquid extraction by mixed micellar systems: A new approach for clavulanic acid recovery from fermented broth

This work is the first attempt to apply aqueous two-phase mixed micellar systems (ATPMS) of the nonionic surfactant Triton X-114 and the anionic one AOT to extract clavulanic acid (CA) from broth fermented by Streptomyces clavuligerus. Cloud points were determined in McIlvane buffer pH 6.5 with or without NaCl, and diagram phases/coexistence curves were constructed. CA partition was investigated following a 2(4)-full factorial design in which AOT (0.022, 0.033 and 0.044% w/w), Triton X-114 (1.0, 3.0 and 5.0% w/w) and NaCl (0, 2.85 and 5.70% w/w) concentrations and temperature (24,26 and 28 degrees C) were selected as independent variables, and CA partition coefficient (K(CA)) and yield in the top phase (eta(CA)) as responses. CA partitioned always to the top, micelle-poor phase. The regression analysis pointed out that NaCl concentration and interaction between temperature and Triton X-114 concentration had statistically significant effects on K(CA), while eta(CA) was mainly influenced by temperature, Triton X-114 concentration and their interaction. Different ATPMS compositions were then needed to maximize these responses, specifically 0.022% (w/w) AOT, 5% (w/w) Triton X-114 for K(CA) (2.08), and 0.044% (w/w) AOT, 1% (w/w) Triton X-114 for eta(CA) (98.7%), both at 24 degrees C without NaCl. Since at 0.022% (w/w) AOT, 1% (w/w) Triton X-114 and 28 degrees C without NaCl the system was able to ensure satisfactory intermediate results (K(CA) = 1.48; eta(CA) = 86.3%), these conditions were selected as the best ones. These preliminary results are of concern for possible industrial application, because CA partition to the dilute phase can simplify the subsequent purification protocol. (C) 2011 Elsevier B.V. All rights reserved.

The effect of inulin as a prebiotic on the production of probiotic fibre-enriched fermented milk

We investigated the effect of inulin as a prebiotic on the production of probiotic fibre-enriched fermented milk. The kinetics of acidification of inulin-supplemented milk (0, 0.01, 0.02 and 0.04 g/g), as well as probiotic survival, pH and firmness of fermented milk stored at 4 degrees C for 24 h and 7 days after preparation were examined. Probiotic fibre-enriched fermented milk quality was influenced both by the amount of inulin and by the co-culture composition. Depending on the co-culture, inulin addition to milk influenced acidification kinetic parameters, probiotic counts, pH and the firmness of the product.

Effect of different prebiotics on the fermentation kinetics, probiotic survival and fatty acids profiles in nonfat symbiotic fermented milk

The simultaneous effects of different binary co-cultures of Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus rhamnosus and Bifidobacterium lactis with Streptococcus thermophilus and of different prebiotics on the production of fermented milk were investigated in this paper. In particular, we determined and compared the kinetics of acidification of milk either as such or supplemented with 4% (w/w) maltodextrin, oligofructose and polydextrose, as well as the probiotic survival, chemical composition (pH, lactose, lactic acid and protein contents), fatty acids profile and conjugate linoleic acid (CIA) content of fermented milk after storage at 4 degrees C for 24 h. Fermented milk quality was strongly influenced both by the co-culture composition and the selected prebiotic. Depending on the co-culture, prebiotic addition to milk influenced to different extent kinetic acidification parameters. All probiotic counts were stimulated by oligofructose and polydextrose, and among these B. lactis always exhibited the highest counts in all supplemented milk samples. Polydextrose addition led to the highest post-acidification. Although the contents of the main fatty acids were only barely influenced. the highest amounts of conjugated linoleic acid (38% higher than in the control) were found in milk fermented by S. thermophilus-L. acidophilus co-culture and supplemented with maltodextrin. (C) 2008 Elsevier B.V. All rights reserved.

Continuous extraction of alpha-toxin from a fermented broth of Clostridium perfringens Type A in perforated rotating disc contactor using aqueous two-phase PEG-phosphate system

A 2(3-1) factorial experimental design was used to evaluate the performance of a perforated rotating disc contactor to extract alpha-toxin from the fermented broth of Clostridium perfringens Type A by aqueous two-phase system of polyethylene glycol-phosphate salts. The influence of three independent variables, specifically the dispersed phase flowrate, the continuous phase flowrate and the disc rotational speed, was investigated on the hold up, the mass transfer coefficient, the separation efficiency and the purification factor, taken as the response variables. The optimum dispersed phase flowrate was 3.0 mL/min for all these responses. Besides, maximum values of hold up (0.80), separation efficiency (0. 10) and purification factor (2.4) were obtained at this flowrate using the lowest disc rotational speed (35 rpm), while the optimum mass transfer coefficient (0. 165 h(-1)) was achieved at the highest agitation level (140 rpm). The results of this study demonstrated that the dispersed phase flowrate strongly influenced the performance of PRDC, in that both the mass transfer coefficient and hold up increased with this parameter. (c) 2007 Elsevier B. V. All rights reserved.

Liquid-liquid extraction of proteases from fermented broth by PEG/citrate aqueous two-phase system

This work deals with the use of an aqueous two-phase system (ATPS) of PEG/citrate to remove proteases from a Clostridium perfringens fermentation broth. To plan the experimental tests and evaluate the corresponding results, three successive experimental designs were employed, for which the PEG molar mass (M-PEG) and concentration (C-PEG), the citrate concentration (C-C) and the pH were selected as independent variables, while the purification factor (PF), the partition coefficient (K), the activity yield (Y) and the selectivity (S) were selected as responses. PF of proteases in the top phase was shown to increase with increasing MPEG and decreasing Cc, whereas a completely opposite trend was observed for K. On the other hand, Y was favored by simultaneous decreases in both these variables, while S decreased with increasing Cc. Therefore, selecting a simultaneous increase in PF and Y as the most desirable result, the best performance of the system was obtained using M-PEG = 10-000 g/mol C-PEG = 22% (w/w) and C-c = 8.0% (w/w) at pH 8.5. Under these conditions, the activity yield was very high (131 %) but the purification factor (4.2) and the selectivity (4.3) were lower than those ensured by more selective purification methods. According to these results, the ATPS seems to be an interesting alternative primary concentration/decontamination step for vaccine preparation from C. perfringens fermented broth. (C) 2007 Elsevier B.V. All rights reserved.

Performance of Petrifilm Aerobic Count plates on enumeration of lactic acid bacteria in fermented milks

This study aimed to compare Petrifilm Aerobic Count (AC) plates and the conventional pour plate methodology using the de Man-Rogosa-Sharpe (MRS) agar for the enumeration of lactic acid bacteria (LAB) in fermented milks (FMs), with different starter cultures added. FM samples (n = 66) were collected and plated on both methodologies, with incubation under anaerobic conditions at 35C for 48 h. The count results were compared by analysis of variance (P <= 0.05) and regression analysis. No differences between the mean counts obtained by both methodologies were observed, even when distinct FMs were compared. Considering all samples, a high correlation level was obtained between Petrifilm AC and MRS agar (r = 0.92), but these indexes were lower in FMs with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (r = 0.90) and Lactobacillus fortis (r = 0.81). Despite some slight interferences, Petrifilm AC has proven to be a convenient methodology on enumerating LAB in FM.