In vitro fermentation of lactulose-derived oligosaccharides by mixed fecal microbiota

Fermentation properties of oligosaccharides derived from lactulose (OsLu) and lactose (GOS) have been assessed in pH-controlled anaerobic batch cultures using lactulose and Vivinal-GOS as reference carbohydrates. Changes in gut bacterial populations and their metabolic activities were monitored over 24 h by fluorescent in situ hybridization (FISH) and by measurement of short-chain fatty acid (SCFA) production. Lactulose-derived oligosaccharides were selectively fermented by Bifidobacterium and lactic acid bacterial populations producing higher SCFA concentrations compared to GOS. The highest total SCFA production was from Vivinal-GOS > lactulose > OsLu > GOS. Longer incubation periods produced a selective fermentation of OsLu when they were used as a carbon source reaching the highest selective index scores. The new oligosaccharides may constitute a good alternative to lactulose, and they could belong to a new generation of prebiotics to be used as a functional ingredient for improving the composition of gut microflora.

In vitro fermentation of a red wine extract by human gut microbiota: changes in microbial groups and formation of phenolic metabolites

An in vitro batch culture fermentation experiment was conducted with fecal inocula from three healthy volunteers in the presence and absence of a red wine extract. Changes in main bacterial groups were determined by FISH during a 48 h fermentation period. The catabolism of main flavonoids (i.e., flavan-3-ols and anthocyanins) and the formation of a wide a range of phenolic microbial metabolites were determined by a targeted UPLC-PAD-ESI-TQ MS method. Statistical analysis revealed that catechol/pyrocatechol, as well as 4-hydroxy-5-(phenyl)-valeric, 3- and 4-hydroxyphenylacetic, phenylacetic, phenylpropionic, and benzoic acids, showed the greatest increases in concentration during fermentation, whereas 5-(3′-hydroxyphenyl)-γ-valerolactone, its open form 4-hydroxy-5-(3′-hydroxyphenyl)-valeric acid, and 3,4-dihydroxyphenylacetic acid represented the largest interindividual variations in the catabolism of red wine polyphenols. Despite these changes, microbial catabolism did not produce significant changes in the main bacterial groups detected, although a slight inhibition of the Clostridium histolyticum group was observed.

In vitro fermentation of commercial α-gluco-oligosaccharide by faecal microbiota from lean and obese human subjects

The fermentation selectivity of a commercial source of α-gluco-oligosaccharides (BioEcolians; Solabia) was investigated in vitro. Fermentation by faecal bacteria from four lean and four obese healthy adults was determined in anaerobic, pH-controlled faecal batch cultures. Inulin was used as a positive prebiotic control. Samples were obtained at 0, 10, 24 and 36 h for bacterial enumeration by fluorescent in situ hybridisation and SCFA analyses. Gas production during fermentation was investigated in non-pH-controlled batch cultures. α-Gluco-oligosaccharides significantly increased the Bifidobacterium sp. population compared with the control. Other bacterial groups enumerated were unaffected with the exception of an increase in the Bacteroides–Prevotella group and a decrease in Faecalibacterium prausnitzii on both α-gluco-oligosaccharides and inulin compared with baseline. An increase in acetate and propionate was seen on both substrates. The fermentation of α-gluco-oligosaccharides produced less total gas at a more gradual rate of production than inulin. Generally, substrates fermented with the obese microbiota produced similar results to the lean fermentation regarding bacteriology and metabolic activity. No significant difference at baseline (0 h) was detected between the lean and obese individuals in any of the faecal bacterial groups studied.

An assessment of the prebiotic potential of single and blended substrates in anaerobic in vitro batch culture fermentations using canine faecal samples as inocula

Previously, using an in vitro static batch culture system, it was found that rice bran (RB), inulin, fibersol, mannanoligosaccharides (MOS), larch arabinogalactan and citrus pectin elicited prebiotic effects (in terms of increased numbers of bifidobacteria and lactic acid bacteria) on the faecal microbiota of a dog. The aim of the present study was to confirm the prebiotic potential of each individual substrate using multiple faecal donors, as well as assessing the prebiotic potential of 15 substrate blends made from them. Anaerobic static and stirred, pH-controlled batch culture systems inoculated with faecal samples from healthy dogs were used for this purpose. Fluorescence in situ hybridization (FISH) analysis using seven oligonucleotide probes targeting selected bacterial groups and DAPI (total bacteria) was used to monitor bacterial populations during fermentation runs. High-performance liquid chromatography was used to measure butyrate produced as a result of bacterial fermentation of the substrates. RB and a MOS/RB blend (1:1, w/w) were shown to elicit prebiotic and butyrogenic effects on the canine microbiota in static batch culture fermentations. Further testing of these substrates in stirred, pH-controlled batch culture fermentation systems confirmed the prebiotic and butyrogenic effects of MOS/RB, with no enhancement of Clostridium clusters I and II and Escherichia coli populations.

Formulation of fermentation media from flour-rich waste streams for microbial lipid production by Lipomyces starkeyi

Flour-rich waste (FRW) and by-product streams generated by bakery, confectionery and wheat milling plants could be employed as the sole raw materials for generic fermentation media production, suitable for microbial oil synthesis. Wheat milling by-products were used in solid state fermentations (SSF) of Aspergillus awamori for the production of crude enzymes, mainly glucoamylase and protease. Enzyme-rich SSF solids were subsequently employed for hydrolysis of FRW streams into nutrient-rich fermentation media. Batch hydrolytic experiments using FRW concentrations up to 205 g/L resulted in higher than 90%(w/w) starch to glucose conversion yields and 40% (w/w) total Kjeldahl nitrogen to free amino nitro-gen conversion yields. Starch to glucose conversion yields of 98.2, 86.1 and 73.4% (w/w) were achieved when initial FRW concentrations of 235, 300 and 350 g/L were employed in fed-batch hydrolytic experiments, respectively. Crude hydrolysates were used as fermentation media in shake flask cultures with the oleaginous yeast Lipomyces starkeyi DSM 70296 reaching a total dry weight of 30.5 g/L with a microbial oil content of 40.4% (w/w), higher than that achieved in synthetic media. Fed-batch bioreactor cultures led to a total dry weight of 109.8 g/L with a microbial oil content of 57.8% (w/w) and productivity of 0.4 g/L/h.

Biorefinery development through utilization of biodiesel industry by-products as sole fermentation feedstock for 1,3-propanediol production

Rapeseed meal (RSM) hydrolysate was evaluated as substitute for commercial nutrient supplements in 1,3-propanediol (PDO) fermentation using the strain Clostridium butyricum VPI 1718. RSM was enzymatically converted into a generic fermentation feedstock, enriched in amino acids, peptides and various micro-nutrients, using crude enzyme consortia produced via solid state fermentation by a fungal strain of Aspergillus oryzae. Initial free amino nitrogen concentration influenced PDO production in batch cultures. RSM hydrolysates were compared with commercial nutrient supplements regarding PDO production in fed-batch cultures carried out in a bench-scale bioreactor. The utilization of RSM hydrolysates in repeated batch cultivation resulted in a PDO concentration of 65.5 g/L with an overall productivity of 1.15 g/L/h that was almost 2 times higher than the productivity achieved when yeast extract was used as nutrient supplement.

Effect of 3,4,4 '-trichlorocarbanilide on growth of lactic acid bacteria contaminants in alcoholic fermentation

3,4,4'-trichlorocarbanilide (TCC) was rested as a new method of bacterial growth control for S. cerevisiae alcoholic fermentations of diluted high test molasses (HTM). Minimal inhibitory concentration (MIC) was tested to determine the necessary concentration of TCC to control bacterial growth. The fed-batch alcoholic fermentation process was used with cell recycle similar to industrial conditions and Lactobacillus fermentum CCT 1407 was mixed in the first inoculum to grow with the yeast. Yeast extract was added into the must to stimulate bacterial growth. The best results of TCC's MIC to bacterial growth of Lactobacillus fermentum and Leuconostoc mesenteroides (< 0.125-1.0 mu g/ml) and Saccharomyces cerevisiae (16 mu g/ml) occurred when it was combined with sodium dodecylsulphate (SDS) in a 1: 4 TCC/SDS ratio (wt/wt) in distilled water solution. 1.8 g/l TCC entrapped in calcium alginate added to the must with yeast extract inhibited the growth of Lactobacillus fermentum CCT 1407 maintaining a controlled acidity, higher yeast viability and up to 20.8% of improvement in the average of alcoholic efficiency. Addition of 0.075 g/l TCC entrapped in calcium alginate and 1.67 mg/l SDS in the wort with yeast extract (0-5.0 g/l), inhibited and controlled the extensive bacterial contamination for 19 cycles of fermentation. (C) 1998 Published by Elsevier B.V. Ltd.

The dead core model applied to beads with immobilized cells in a fed-batch cephalosporin C production bioprocess

Viable cells immobilized in inert supports are currently studied for a wide range of bioprocesses. The intrinsic advantages of such systems over suspended cultures incite new research, including studies on fundamental aspects as well as on the industrial viability of these non-conventional processes. In aerobic culture of filamentous fungi, scale-up is hindered by oxygen mass transfer limitation through the support material and bioprocess kinetics must be studied together with mass transfer limitation. In this work, experimental and simulated data of cephalosporin C production were compared. Concentrations in the bulk fermentation medium and cellular mass profiles inside the bioparticles are focused. Immobilized cells were used in a tower bioreactor, operated in fed-batch mode. To describe the radial variation of oxygen concentration within the pellet, a dead core model was used. Despite the extremely low sugar concentrations, bioreaction rates in the pellets were limited by the dissolved oxygen concentration. Cell growth occurs only in the outer layers, a result also confirmed by scanning electron microscopy. (C) 2001 Elsevier B.V. Ltd. All rights reserved.

Chlorine dioxide against bacteria and yeasts from the alcoholic fermentation

The ethanol production in Brazil is carried out by fed-batch or continuous process with cell recycle, in such way that bacterial contaminants are also recycled and may be troublesome due to the substrate competition. Addition of sulphuric acid when inoculum cells are washed can control the bacterial growth or alternatively biocides are used. This work aimed to verify the effect of chlorine dioxide, a well-known biocide for bacterial decontamination of water and equipments, against contaminant bacteria ( Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides) from alcoholic fermentation, through the method of minimum inhibitory concentration ( MIC), as well as its effect on the industrial yeast inoculum. Lower MIC was found for B. subtilis ( 10 ppm) and Leuconostoc mesenteroides ( 50 ppm) than for Lactobacillus fermentum ( 75 ppm) and Lactobacillus plantarum ( 125 ppm). Additionally, these concentrations of chlorine dioxide had similar effects on bacteria as 3 ppm of Kamoran (R) ( recommended dosage for fermentation tanks), exception for B. subtilis, which could not be controlled at this Kamoran (R) dosage. The growth of industrial yeasts was affected when the concentration of chlorine dioxide was higher than 50 ppm, but the effect was slightly dependent on the type of yeast strain. Smooth yeast colonies ( dispersed cells) seemed to be more sensitive than wrinkled yeast colonies ( clustered cells/pseudohyphal growth), both isolated from an alcohol-producing unit during the 2006/2007 sugar cane harvest. The main advantage in the usage of chlorine dioxide that it can replace antibiotics, avoiding the selection of resistant populations of microorganisms.

Bioconversion of Cellulose to Hydrogen by dark fermentation

Hydrogen is known as a clean energy resource. The biological production of hydrogen has been attracting attention as an environmentally friendly processs that does not consume fossil fuels. Cellulosic plant and waste materials are potential resources for fermentative hydrogen production. Cellulose is a linear biopolymer of glucose molecules, connected by β-1,4-glycosidic bonds. Enzymatic hydrolysis of cellulose requires the presence of cellulase. The present study aimed to investigate the efficiency of acid pretreatment on ruminal fluid in order to enrich H2 producing bacteria consortia to enhance biohydrogen rate and substrate removal efficiency. In this study, fermentative hydrogen producers were enriched on cellulose (2g/L) in a modificated Del Nery medium (DNM) at 37ºC and initial pH 7.0 using rumen fluid (10% v/v) as inoculum. To increase the hydrogen production it was added cellulose (10mL) to the medium. The gas products (mainly H2 and CO2) was analyzed by gas chromatography (Shimadzu GC 2010) using a thermal conductivity detector. The volatile fatty acids and ethanol were also detected by GC using a flame ionization detector. Cellulose degradation was quantified by using the phenolsulfuric acid method. Analysis showed that the biogas produced from the anaerobic fermentation contained only hydrogen and carbon dioxide, without detectable methane after acid pretreatment test. On DNM the hydrogen production started with 4 h (5,3 x 105 mmol H2/L) of incubation, and the maximum H2 concentration was observed with 34 h (7,1 x 106 mmol H2/L) of incubation. During the process, it was observed a predominance of acetic acid and butyric acid as well as a low production of acetone, ethanol and nbutanol in all experimental phases. Butyrate accounted for more than 77% of total. As a result of the accumulation of volatile fatty acids (VFAs), the pH value in anaerobic digestion system was reduced to 4,0. On microscopy analyses there were observed rods with endospores. The batch anaerobic fermentation assays performed on anaerobic mixed inoculum from rumen fluid demonstrated the feasibility of H2 generation utilizing cellulose as substrate. Based on the results, it can be concluded that the acid treatment was efficient to inhibit the methanogenic archaea cells present in rumen fluid. The rumen fluid cells present a potential route in converting renewable biomass such as cellulose into hydrogen energy.

Kinetic and thermodynamic investigation of Arthrospira (Spirulina) platensis fed-batch cultivation in a tubular photobioreactor using urea as nitrogen source

BACKGROUND: Fed-batch culture allows the cultivation of Arthrospira platensis using urea as nitrogen source. Tubular photobioreactors substantially increase cell growth, but the successful use of this cheap nitrogen source requires a knowledge of the kinetic and thermodynamic parameters of the process. This work aims at identifying the effect of two independent variables, temperature (T) and urea daily molar flow-rate (U), on cell growth, biomass composition and thermodynamic parameters involved in this photosynthetic cultivation. RESULTS: The optimal values obtained were T = 32 degrees C and U = 1.16 mmol L-1 d-1, under which the maximum cell concentration was 4186 +/- 39 mg L-1, cell productivity 541 +/- 5 mg L-1 d-1 and yield of biomass on nitrogen 14.3 +/- 0.1 mg mg-1. Applying an Arrhenius-type approach, the thermodynamic parameters of growth (?H* = 98.2 kJ mol-1; ?S* = - 0.020 kJ mol-1 K-1; ?G* = 104.1 kJ mol-1) and its thermal inactivation (Delta H-D(0) =168.9 kJ mol-1; Delta S-D(0) = 0.459 kJ mol-1 K-1; Delta G(D)(0) =31.98 kJ mol-1) were estimated. CONCLUSIONS: To maximize cell growth T and U were simultaneously optimized. Biomass lipid content was not influenced by the experimental conditions, while protein content was dependent on both independent variables. Using urea as nitrogen source prevented the inhibitory effect already observed with ammonium salts. Copyright (c) 2012 Society of Chemical Industry

Arthrospira (Spirulina) platensis cultivation in tubular photobioreactor: Use of no-cost CO2 from ethanol fermentation

The present study aimed at evaluating the production of Arthrospira platensis in tubular photobioreactor using CO2 from ethanol fermentation. The results of these cultivations were compared to those obtained using CO2 from cylinder at different protocols of simultaneous ammonium sulfate and sodium nitrate feeding. Maximum cell concentration (X-m), cell productivity (P-x), nitrogen-to-cell conversion factor (Y-X/N), and biomass composition (total lipids and proteins) were selected as responses and evaluated by analysis of variance. The source of CO2 did not exert any significant statistical influence on these responses, which means that the flue gas from ethanol fermentation could successfully be used as a carbon source as well as to control the medium pH, thus contributing to reduce the greenhouse effect. The results taken as a whole demonstrated that the best combination of responses mean values (X-m = 4.543 g L-1; P-x = 0.460 g L-1 d(-1); Y-X/N = 15.6 g g(-1); total lipids = 8.39%; total proteins = 18.7%) was obtained using as nitrogen source a mixture of 25% NaNO3 and 75% (NH4)(2)SO4, both expressed as nitrogen. (C) 2011 Elsevier Ltd. All rights reserved.

Culture medium of diluted skimmed milk for the production of nisin in batch cultivations

Nisin is a promising alternative to chemical preservatives for use as a natural biopreservative in foods. This bacteriocin has also potential biomedical applications. Lactic acid bacteria are commonly cultivated in expensive standard complex media. We have evaluated the cell growth and nisin production of Lactococcus lactis in a low-cost natural medium consisting of diluted skimmed milk in a 2-L bioreactor. The assays were performed at 30 degrees C for 56 h, at varying agitation speeds and airflow rates: (1) 200 rpm (no airflow, and airflow at 0.5, 1.0 and 2.0 L/min); (2) 100 rpm (no airflow, and airflow at 0.5 L/min). Nisin activity was evaluated using agar diffusion assays. The highest nisin concentration, 49.88 mg/L (3.3 log AU/mL or 1,995.29 AU/mL), was obtained at 16 h of culture, 200 rpm and no airflow (k(L)a = 5.29 x 10(-3)). These results show that a cultivation medium composed of diluted skimmed milk supports cell growth to facilitate nisin biosynthesis.

Produzione di bioidrogeno in dark fermentation da scarti dell'industria agroalimentale mediante l'impiego di batteri ipertermofili

La presente tesi di dottorato ha come argomento la produzione d’idrogeno per via fermentativa sfruttando il metabolismo anaerobico di particolari batteri estremofili del genere Thermotoga. In questo lavoro, svolto in seno al progetto Bio-Hydro, sfruttando reattori batch da 116 mL, è stato selezionato il ceppo migliore di Thermotoga fra i quatto ceppi testati: T. neapolitana. Una volta individuato il candidato batterico migliore è stato individuato il valore ottimale di pH (8.5 a t.amb) per la produzione d’idrogeno. Un intenso lavoro è stato svolto sul medium di coltura permettendone la minimizzazione e rendendolo così economicamente sostenibile per il suo utilizzo nel reattore da 19L; in questo caso il glucosio è stato completamente sostituito con due sottoprodotti agroindustriali individuati in precedenza, il melasso di barbabietola e il siero di latte. Sono stati poi eliminati i gravosi micronutrienti e le vitamine. È stata sfruttata la capacità di T. neapolitana di produrre biofilm e sono stati testati 4 diversi supporti in vetro sinterizzato e ceramici, tali test hanno permesso di individuare Biomax come supporto migliore. Sono stati svolti studi sul metabolismo di T. neapolitana volti ad individuare le concentrazioni inibenti di ogni substrato testato, l’inibizione da prodotto (idrogeno) e l’inibizione da ossigeno. Tutte queste prove hanno dato le conoscenze di base per la conduzione di esperienze su reattore da 19L. L’innovativo reattore di tipo SPCSTR è stato interamente studiato, progettato e costruito presso il DICMA. dell’Università di Bologna. La conduzione di esperienze batch su SPCSTR ha dato la possibilità di verificare il funzionamento del nuovo tipo d’impianto. Presso il Wageningen UR (NL), è stata svolta la selezione del miglior ceppo di Caldicellulosisruptor fra 3 testati e del miglior supporto per la produzione d’idrogeno; è stato poi costruito testato e condotto in continuo l’innovativo reattore CMTB.

The influence of diet on the effectiveness of garlic oil and cinnamaldehyde to manipulate in vitro ruminal fermentation and methane production.

The objective of this study was to evaluate the effects of increasing doses [0 (control: CON), 20, 60, 180 and 540 mg/L incubation medium] of garlic oil (GO) and cinnamaldehyde (CIN) on in vitro ruminal fermentation of two diets. Batch cultures of mixed ruminal microorganisms were inoculated with ruminal fluid from four sheep fed a medium-concentrate diet (MC; 50 : 50 alfalfa hay : concentrate) or four sheep fed a high-concentrate diet (HC; 15 : 85 barley straw : concentrate). Diets MC and HC were representative of those fed to dairy and fattening ruminants, respectively. Samples of each diet were used as incubation substrates for the corresponding inoculum, and the incubation was repeated on 4 different days (four replicates per experimental treatment). There were GO × diet-type and CIN × diet-type interactions (P < 0.001–0.05) for many of the parameters determined, indicating different effects of both oils depending on the diet type. In general, effects of GO were more pronounced for MC compared with HC diet. Supplementation of GO did not affect (P > 0.05) total volatile fatty acid (VFA) production at any dose. For MC diet, GO at 60, 180 and 540 mg/L decreased (P < 0.05) molar proportion of acetate (608, 569 and 547 mmol/mol total VFA, respectively), and increased (P < 0.05) propionate proportion (233, 256 and 268 mmol/mol total VFA, respectively), compared with CON values (629 and 215 mmol/mol total VFA for acetate and propionate, respectively). A minimum dose of 180 mg of GO/L was required to produce similar modifications in acetate and propionate proportions with HC diet, but no effects (P > 0.05) on butyrate proportion were detected. Methane/VFA ratio was reduced (P < 0.05) by GO at 60, 180 and 540 mg/L for MC diet (0.23, 0.16 and 0.10 mol/mol, respectively), and by GO at 20, 60, 180 and 540 mg/L for HC diet (0.19, 0.19, 0.16 and 0.08 mol/mol, respectively), compared with CON (0.26 and 0.21 mol/mol for MC and HC diets, respectively). No effects (P = 0.16–0.85) of GO on final pH and concentrations of NH3-N and lactate were detected. For both diet types, the highest CIN dose decreased (P < 0.05) production of total VFA, gas and methane, which would indicate an inhibition of fermentation. Compared with CON, CIN at 180 mg/L increased (P < 0.05) acetate proportion for the MC (629 and 644 mmol/mol total VFA for CON and CIN, respectively) and HC (525 and 540 mmol/mol total VFA, respectively) diets, without affecting the proportions of any other VFA or total VFA production. Whereas for MC diet CIN at 60 and 180 mg/L decreased (P < 0.05) NH3-N concentrations compared with CON, only a trend (P < 0.10) was observed for CIN at 180 mg/L with the HC diet. Supplementation of CIN up to 180 mg/L did not affect (P = 0.18–0.99) lactate concentrations and production of gas and methane for any diet. The results show that effectiveness of GO and CIN to modify ruminal fermentation may depend on diet type, which would have practical implications if they are confirmed in vivo.