Biocompatible polyhydroxybutyrate (PHB) production by marine Vibrio azureus BTKB33 under submerged fermentation

Polyhydroxybutyrate (PHB) is known to have applications as medical implants and drug delivery carriers and is consequently in high demand. In the present study the possibilities of harnessing potential PHB-producing vibrios from marine sediments as a new source of PHB was investigated since marine environments are underexplored. Screening of polyhydroxyalkanoate (PHA)-producing vibrios from marine sediments was performed using a fluorescent plate assay followed by spectrophotometric analysis of liquid cultures. Out of 828 isolates, Vibrio sp. BTKB33 showed maximum PHA production of 0.21 g/L and PHA content of 193.33 mg/g of CDW. The strain was identified as Vibrio azureus based on phenotypic characterization and partial 16S rDNA sequence analysis. The strain also produced several industrial enzymes: amylase, caseinase, lipase, gelatinase, and DNase. The FTIR analysis of extracted PHA and its comparison with standard PHB indicated that the accumulated PHA is PHB. Bioprocess development studies for enhancing PHA production were carried out under submerged fermentation conditions. Optimal submerged fermentation conditions for enhanced intracellular accumulation of PHA production were found to be 35 °C, pH −7, 1.5 % NaCl concentration, agitation at 120 rpm, 12 h of inoculum age, 2.5 % initial inoculum concentration, and 36 h incubation along with supplementation of magnesium sulphate, glucose, and ammonium chloride. The PHA production after optimization was found to be increased to 0.48 g/L and PHA content to426.88 mg/g of CDW, indicating a 2.28-fold increase in production. Results indicated that V. azureus BTKB33 has potential for industrial production of PHB.

Optimization of Lignin Peroxidase, Manganese Peroxidase, and Lac Production from Ganoderma lucidum Under Solid State Fermentation of Pineapple Leaf

This study was undertaken to isolate ligninase-producing white-rot fungi for use in the extraction of fibre from pineapple leaf agriwaste. Fifteen fungal strains were isolated from dead tree trunks and leaf litter. Ligninolytic enzymes (lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac)), were produced by solid-state fermentation (SSF) using pineapple leaves as the substrate. Of the isolated strains, the one showing maximum production of ligninolytic enzymes was identified to be Ganoderma lucidum by 18S ribotyping. Single parameter optimization and response surface methodology of different process variables were carried out for enzyme production. Incubation period, agitation, and Tween-80 were identified to be the most significant variables through Plackett-Burman design. These variables were further optimized by Box-Behnken design. The overall maximum yield of ligninolytic enzymes was achieved by experimental analysis under these optimal conditions. Quantitative lignin analysis of pineapple leaves by Klason lignin method showed significant degradation of lignin by Ganoderma lucidum under SSF

Digestion, rumen fermentation and circulating concentrations of insulin, growth hormone and IGF-1 in steers fed diets based on different proportions of maize silage and grass silage

Replacing grass silage with maize silage results in a fundamental change in the ratio of structural to non-structural carbohydrates with commensurate changes in rumen fermentation patterns and nutrient utilisation. This study investigated the effects of feeding four forage mixtures, namely grass silage (G); 67 g/100 g grass silage133 g/100 g maize silage (GGM); 67 g/100 g maize silage133/100 g grass silage (MMG); maize silage (M) to four ruminally and duodenally canulated Holstein Friesian steers. All diets were formulated to be isonitrogenous (22.4 g N/kg DM) using a concentrate mixture. Dietary dry matter (DM) and organic matter (OM) digestibility increased with ascending maize silage inclusion (P,0.1) whereas starch and neutral detergent fibre digestibility declined (P,0.05). Ratio of non-glucogenic to glucogenic precursors in the rumen fluid increased with maize silage inclusion (P,0.01) with a commensurate reduction in rumen pH (P,0.05). Mean circulating concentrations of insulin were greatest and similar in diets MMG and GGM, lower in diet M and lowest in diet G (P,0.01). There were no effects of diet on the mean circulating concentration of growth hormone (GH), or the frequency, amplitude and duration of GH pulses, or the mean circulating concentrations of IGF-1. Increasing levels of DM, OM and starch intakes with the substitution of grass silage with maize silage affected overall digestion, nutrient partitioning and subsequent circulating concentrations of insulin.

A further study of the recovery and purification of surfactin from fermentation broth by membrane filtration

Surfactin is a bacterial lipopeptide produced by Bacillus subtilis and is a powerful surfactant, having also antiviral, antibacterial and antitumor properties. The recovery and purification of surfactin from complex fermentation broths is a major obstacle to its commercialization; therefore, a two-step membrane filtration process was developed using a lab scale tangential flow filtration (TFF) unit with 10 kDa MWCO regenerated cellulose (RC) and polyethersulfone (PES)membranes at three different transmembrane pressure (TMP) of 1.5 bar, 2.0 bar and 2.5 bar. Two modes of filtrations were studied, with and without cleaning of membranes prior to UF-2. In a first step of ultrafiltration (UF-1), surfactin was retained effectively by membranes at above its critical micelle concentration (CMC); subsequently in UF-2, the retentate micelles were disrupted by addition of 50% (v/v) methanol solution to allow recovery of surfactin in the permeate. Main protein contaminants were effectively retained by the membrane in UF-2. Flux of permeates, rejection coefficient (R) of surfactin and proteinwere measured during the filtrations. Overall the three different TMPs applied have no significant effect in the filtrations and PES is the more suitable membrane to selectively separate surfactin from fermentation broth, achieving high recovery and level of purity. In addition this two-step UF process is scalable for larger volume of samples without affecting the original functionality of surfactin, although membranes permeability can be affected due to exposure to methanolic solution used in UF-2.

Comparison of methane produced by straw fed sheep in open-circuit respiration with methane predicted by fermentation characteristics measured by an in vitro gas procedure

Reducing carbon conversion of ruminally degraded feed into methane increases feed efficiency and reduces emission of this potent greenhouse gas into the environment. Accurate, yet simple, predictions of methane production of ruminants on any feeding regime are important in the nutrition of ruminants, and in modeling methane produced by them. The current work investigated feed intake, digestibility and methane production by open-circuit respiration measurements in sheep fed 15 untreated, sodium hydroxide (NaOH) treated and anhydrous ammonia (NH3) treated wheat, barley and oat straws. In vitro fermentation characteristics of straws were obtained from incubations using the Hohenheim gas production system that measured gas production, true substrate degradability, short-chain fatty acid production and efficiency of microbial production from the ratio of truly degraded substrate to gas volume. In the 15 straws, organic matter (OM) intake and in vivo OM digestibility ranged from 563 to 1201 g and from 0.464 to 0.643, respectively. Total daily methane production ranged from 13.0 to 34.4 l, whereas methane produced/kg OM matter apparently digested in vivo varied from 35.0 to 61.8 l. The OM intake was positively related to total methane production (R2 = 0.81, P<0.0001), and in vivo OM digestibility was also positively associated with methane production (R2 = 0.67, P<0.001), but negatively associated with methane production/kg digestible OM intake (R2 = 0.61, P<0.001). In the in vitro incubations of the 15 straws, the ratio of acetate to propionate ranged from 2.3 to 2.8 (P<0.05) and efficiencies of microbial production ranged from 0.21 to 0.37 (P<0.05) at half asymptotic gas production. Total daily methane production, calculated from in vitro fermentation characteristics (i.e., true degradability, SCFA ratio and efficiency of microbial production) and OM intake, compared well with methane measured in the open-circuit respiration chamber (y = 2.5 + 0.86x, R2 = 0.89, P<0.0001, Sy.x = 2.3). Methane production from forage fed ruminants can be predicted accurately by simple in vitro incubations combining true substrate degradability and gas volume measurements, if feed intake is known.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - II. Effects on rate of acidification, fibre degradation during ensiling and rumen fermentation

A study was carried out to determine the influence of fibrolytic enzymes derived from mesophilic or thermophilic fungal sources, added at ensiling, on time-course fermentation characteristics and in vitro rumen degradation of maize silage. The mesophilic enzyme was a commercial product derived from Trichodenna reesei (L), whereas the thermophilic enzyme was a crude extract produced from Thermoascus aurantiacus (Ta) in this laboratory. The fungus was cultured using maize cobs as a carbon source. The resulting fermentation extract was deionised to remove sugars and characterised for its protein concentration, main and side enzymic activities, optimal pH, protein molecular mass and isoelectric point. In an additional study, both enzymes were added to maize forage (333.5 g DM/kg, 70.0, 469.8, 227.1 and 307.5 g/kg DM of CP, NDF, ADF and starch, respectively) at two levels each, normalized according to xylanase activity, and ensiled in 0.5 kg capacity laboratory minisilos. Duplicate silos were opened at 2, 4, 8, 15, and 60 days after ensiling, and analysed for chemical characteristics. Silages from 60 days were bulked and in vitro gas production (GP) and organic matter degradability (OMD) profiles evaluated using the Reading Pressure Technique (RPT), in a completely randomised design. The crude enzyme extract contained mainly xylanase and endoglucanase activities, with very low levels of exoglucanase, which probably limited hydrolysis of filter paper. The extract contained three major protein bands of between 29 and 55 kDa, with mainly acidic isoelectric points. Ensiling maize with enzymes lowered (P < 0.05) the final silage pH, with this effect being observed throughout the ensiling process. All enzyme treatments reduced (P < 0.05) ADF contents. Treatments including Ta produced more gas (P < 0.05) than the controls after 24 h incubation in vitro, whereas end point gas production at 96 h was not affected. Addition of Ta increased (P < 0.01) OMD after 12 h (410 and 416 g/kg versus 373 g/kg), whereas both L and Ta increased (P < 0.05) OMD after 24 h. Addition of enzymes from mesophilic or thermophilic sources to maize forage at ensiling increased the rate of acidification of the silages and improved in vitro degradation kinetics, suggesting an improvement in the nutritive quality. (C) 2003 Elsevier B.V All rights reserved.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - III. Comparison of enzymes derived from psychrophilic, mesophilic or thermophilic sources

A completely randomised study was completed to examine the influence of fibrolytic enzymes derived from psychrophilic, (F), mesophilic, (L) or thermophilic (Ta) sources, applied at ensiling, on the chemical characteristics and in vitro rumen fermentation of maize silage, assessed using the Reading Pressure Technique (RPT). Treatments, all in triplicate, consisted of untreated maize forage or treated with preparations F, L, Ta or a mixture (1: 1, v/v) of F and L (FL), at two levels each, and ensiled for 210 days in plastic mini-silos. Addition of enzymes L decreased (P < 0.05) silage pH relative to the control, whereas enzyme Ta tended (P < 0.10) to reduce it. Preparations F, L and Ta tended to reduce (P < 0.10) the fibre contents of the silages, with effects being attributable to a decrease in the cellulose fraction. Starch contents were reduced (P < 0.05) in the treatments including enzyme F. End-point (96 h) gas production (GP) values did not differ among treatments, suggesting that enzymes did not change the total amount of fermentable substrate. However, consistent with the decrease in starch contents, adding enzyme F reduced (P < 0.05) GP at most incubation times. Addition of enzymes increased (P < 0.05) the initial (6 h) organic matter degradation (OMD) levels in all but one treatment (F), with increases of 14, 19, and 26% for preparations L, Ta, and FL, respectively, averaged across levels. Furthermore, the addition of enzymes increased (P < 0.05) the soluble OM losses, however, these increases did not fully account for the initial increase in OMD. The latter suggests that enzymes increased solubility and also altered silage structure, making it more amenable to degradation by ruminal microorganisms. As a result of the increase in OMD, without a concomitant increase in GP, the fermentation efficiency was greatly increased (P < 0.05) in enzyme treatments. Addition of enzymes to maize at ensiling, particularly those from the mesophilic and thermophilic sources used here, have the potential to increase the initial rate of silage OMD. (C) 2003 Elsevier B.V. All rights reserved.

Chemical composition and fermentation characteristics of grain and different parts of the stover from maize land races harvested at different growing periods in two zones of central Mexico

The objective of this work was to determine the rumen fermentation characteristics of maize land races used as forage in central Mexico. In vitro gas production (ml per 200 mg dry matter (DM)) incubations were carried out, and cumulative gas volumes were fitted to the Krishnamoorthy et al. (1991) model. The trial used a split-plot design with cultivation practices associated with maize colour (COL) as the main plot with three levels: white, yellow and black maize; growing periods (PER) were the split plots where PER1, PER2 and PER3 represented the first, second and third periods, respectively and two contrasting zones (Z1 = valley and Z2 = mountain) were used as blocking factors. The principal effects observed were associated with the maturity of the plants and potential gas production increased (P < 0.05) in stems (PER 1 = 51.8, PER2 = 56.3, PER3 = 58.4 ml per 200 mg DM) and in whole plant (PER 1 = 60.9, PER2 = 60.8, PER3= 70.9 ml per 200 mg DM). An inverse effect was observed with fermentation rates in leaves (P < 0.01) with 0.061, 0.053 and 0.0509 (per h) and in whole plant (P < 0.05) with 0.068, 0.057, 0.050 (per h) in PER1, PER2 and PER3 respectively. The digestibility of the neutral-detergent fibre (NDF) decreased with maturity especially in leaves (P < 0.05) with values of 0.71, 0.67 and 0.66 g/kg; in rachis (P < 0.01) 0.75, 0.72, and 0.65 in PER1, PER2 and PER3 respectively. The NDF content in leaves in leaves (668, 705 and 713 g/kg DM for PER1, PER2 and PER3, respectively), stems (580, 594 and 644 g/kg DM) and, husk (663, 774 and, 808 g/kg DM) increased (P < 0.05) with increasing plant maturity, rachis were significantly different between periods (P < 0.01). The structure with-the best nutritive characteristics was the husk, because it had the lowest fibre contents, especially in acid-detergent lignin, with values of 22.6, 28.6 and 37.6 g/kg DM in PER1, PER2 and PER3, respectively.

Digestion, rumen fermentation and circulating concentrations of insulin, growth hormone and IGF-1 in steers given maize silages harvested at three stages of maturity

Advancing maturity of forage maize is associated with increases in the proportion of dry matter (DM) and starch and decreases in the proportions of structural carbohydrates in the ensiled crop. Three maize silages (286 (low, L), 329 (medium, M) and 379 (high, H) g DM per kg fresh weight) plus a concentrate formulated to give isonitrogenous intakes were offered to Holstein-Friesian steers fitted with a cannula in the dorsal sac of the rumen and a 'T' piece cannula in the proximal duodenum in an experiment with a cross-over design that allowed four collection periods. Nutrient flow to the duodenum was estimated using chromium-EDTA. Steers consumed approximately 0(.)6 kg DM per day less of diet L compared with the other two diets (P=0(.)026), resulting in less DM being digested (P=0(.)005) but digestibility did not differ between diets. Similar results were obtained for organic matter. There were no differences between diets in the intake or digestibility of neutral-detergent fibre. Intake, duodenal flow and faecal output of starch were greater for steers offered diets M and H compared with those given diet L (P < 0(.)05). In all diets rumen digestion contributed to over 90% of total digestion of starch, although rumen digestibility declined significantly with advancing maize maturity (P=0(.)002). Molar proportions of acetic acid were higher in diet H (P < 0(.)05) whilst proportions of propionic acid and n-butyric acid were higher in diets M and L. There were no significant differences between diets in mean rumen pH or ammonia concentrations. Mean circulating concentrations of insulin were higher (P=0(.)009) in cattle given diets L and M compared with diet H. There were no differences between diets in the mean circulating concentration of growth hormone, or the frequency, amplitude and duration of growth hormone pulses, or the mean circulating concentrations of IGF-1. Changes in forage composition that accompany advancing maize maturity affect overall silage digestion and circulating concentrations of insulin.

Effects of essential oils on ruminal microorganisms and their protein metabolism

A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet fed to dairy cattle in order to determine their influence on protein metabolism by ruminal microorganisms. EO inhibited (P < 0.05) the rate of deamination of amino acids. Pure-culture studies indicated that the species most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi.

Chemical composition and in vitro fermentation of tannin-rich tree fruits

Dry and mature tree fruits are a potential source of protein for goats in the semi-arid areas of southern Africa, but their chemical composition and feeding value is largely unknown. This study presents the chemical composition and in vitro fermentation of indehiscent whole fruits and separated seed and hull fractions from Acacia nilotica, Acacia erubescens, Acacia sieberiana, Acacia erioloba, Piliostigma thonningii and Dichrostachys cinerea trees. Results indicate that the N contents of whole fruits ranged between 13.5 g/kg DM (A. nilotica) and 27.1 g/kg DM (A. erubescens). Seeds had a higher N content than hulls for all tree species. A. nilotica, D. cinerea and P thonningii fruits had high levels of extractable phenolics (758, 458 and 299 g/kg DM, respectively). Soluble phenolics (SPh) and ytterbium precipitable phenolics (YbPh) levels were negatively correlated to in vitro gas production but positively correlated to in vitro organic matter degradability (iOMD). Partition factors for whole fruits at 48 h ranged between 3.6 mg/ml for A. erioloba and 7.8 mg/ml for A. nilotica. Seeds of A. erioloba, A. erubescens and P thonningii were consistently fermented more efficiently throughout the incubation period compared to their whole fruits or hulls. Estimating in vitro degradability of phenolic-rich substrates through filtration procedures can give erroneous results due to the loss of soluble phenolics, which are not necessarily degradable. The feeding value of fruits from D. cinerea and A. nilotica tree species may be reduced due to the presence of high levels of phenolics. (C) 2007 Elsevier B.V. All rights reserved.

The effectiveness of adapted rumen fluid versus PEG to ferment tannin-containing substrates in vitro

This study investigated the potential of the goat's ruminal adaptation to reduce the negative effect of tannins on in vitro fermentation. Rumen fluid was obtained from goats fed a mixture of tannin-containing tree fruits (adapted rumen fluid) or tannin-free commercial protein supplements (unadapted rumen fluid) for 85 days. Dry, mature fruits of Acacia nilotica, Acacia erubescens, Acacia erioloba, Dichrostachys cinerea and Piliostigma thonningii were used as substrates for the in vitro fermentation. The effectiveness of adapted rumen fluid to ferment tannin-containing substrates was compared to the extent of fermentation when tannins were inactivated with polyethylene glycol (PEG), a known tannin-binding agent. Adapted rumen fluid (P < 0.05) increased gas production from all five substrates between 15.8% and 73.7%. In A. nilotica, D. cinerea and P thonningii, this increase was less than that obtained through PEG treatment. When PEG was added to adapted rumen fluid a further improvement in extent of fermentation was observed in four out of the five fruit samples. The largest PEG effect when incubated with adapted rumen fluid was observed in A. nilotica (43.1%) and D. cinerea (42.9%) fruits. It is concluded that some tannin-rich feedstuffs may still benefit from treatment even when these are offered to adapted animals. (c) 2006 Elsevier B.V. All rights reserved.

Quantification of ruminal Clostridium proteoclasticum by real-time PCR using a molecular beacon approach

Aims: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18 : 2) via conjugated 18 : 2 metabolites (mainly cis-9,trans-11-18 : 2, conjugated linoleic acid) to vaccenic acid (trans-11-18 : 1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18 : 0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. Materials and Results: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2-9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. Conclusion: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. Signifance and Impact of the Study: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.