M.A.M. Rodrigues

Modification of wheat straw lignin by solid state fermentation with white-rot fungi

The potential of crude enzyme extracts, obtained from solid state cultivation of four white-rot fungi (Trametes versicolor, Bjerkandera adusta, Ganoderma applanatum and Phlebia rufa), was exploited to modify wheat straw cell wall. At different fermentation times, manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), laccase, carboxymethylcellulase (CMCase), avicelase, xylanase and feruloyl esterase activities were screened and the content of lignin as well as hydroxycinnamic acids in fermented straw were determined. All fungi secreted feruloyl esterase while LiP was only detected in crude extracts from B. adusta. Since no significant differences (P>0.05) were observed in remaining lignin content of fermented straw, LiP activity was not a limiting factor of enzymatic lignin removal process. The levels of esterified hydroxycinnamic acids degradation were considerably higher than previous reports with lignocellulosic biomass. The data show that P. rufa, may be considered for more specific studies as higher ferulic and p-coumaric acids degradation was observed for earlier incubation times.

Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi

Wheat straw was submitted to a pre-treatment by the basidiomycetous fungi Euc-1 and Irpex lacteus, aiming to improve the accessibility of cellulose towards enzymatic hydrolysis via previous selective bio-delignification. This allowed the increase of substrate saccharification nearly four and three times while applying the basidiomycetes Euc-1 and I. lacteus, respectively. The cellulose/lignin ratio increased from 2.7 in the untreated wheat straw to 5.9 and 4.6 after the bio-treatment by the basidiomycetes Euc-1 and I. lacteus, respectively, thus evidencing the highly selective lignin biodegradation. The enzymatic profile of both fungi upon bio-treatment of wheat straw have been assessed including laccase, manganese-dependent peroxidase, lignin peroxidase, carboxymethylcellulase, xylanase, avicelase and feruloyl esterase activities. The difference in efficiency and selectivity of delignification within the two fungi treatments was interpreted in terms of specific lignolytic enzyme profiles and moderate xylanase and cellulolytic activities.

Influence of ligninolytic enzymes on straw saccharification during fungal pretreatment.

Solid state and submerged fermentations in the presence of white-rot basidiomycetes (Bjerkandera adusta, Fomes fomentarius, Ganoderma resinaceum, Irpex lacteus, Phanerochaete chrysosporium, Trametes versicolor and basidiomycete Euc-1) and the litter-decomposing basidiomycete Lepista nuda were evaluated as a pretreatment to increase enzymatic saccharification of wheat straw. Enzymatic hydrolysis of holocellulose after solid state pretreatment showed a significant (P<0.05) increase of saccharification process for T. versicolor, Euc-1, G. resinaceum and I. lacteus, being T. versicolor (strain Tv2) the best one with a sugar yield increase of 91% compared with untreated straw. In submerged medium the pretreatment with I. lacteus, Euc-1 and P. chrysosporium enhanced saccharification but at a lesser extent. Covariance analysis was used to evaluate the relationships between ligninolytic enzymes (lignin peroxidase, manganese-dependent peroxidase and laccase) and saccharification increase. Results showed that only the presence of lignin peroxidase during pretreatment can lead to a significant (P<0.05) increase in the saccharification yield.

Understanding the equine cecum-colon ecosystem: current knowledge and future perspectives

Having evolved as a grazing animal, a horses digestive physiology is characterized by rapid gastric transit, a rapid but intense enzymatic digestion along the small intestine, and a long and intense microbial fermentation in the large intestine. The process of understanding and describing feed degradation mechanisms in the equine digestive system in general, and in the hindgut ecosystem in particular, is essential. Regardless of its importance for the nutritional status of the host, the significance of the cecum-colon ecosystem has not yet been fully understood, and few reports have focused deeply on the contribution of the hindgut microbial population to the nitrogen and energy requirements of the horse. Compared to ruminal activity, very little is known about hindgut ecosystem activity in the horse. Information concerning the metabolism of this microbial population and its requirements is lacking. The use of internal bacterial markers for quantifying microbial outflow in ruminants is widely reported. These techniques can be applied to cecum-colon microbial quantification, contributing to a better characterization of this ecosystem. It is likely wrong to believe that the optimization strategy in the hindgut is similar to what happens in the rumen - that is, to maximize microbial growth and, therefore, fermentation. If we consider the type of substrate that, in normal conditions, arrives in the hindgut, we can expect it to be nitrogen limiting, providing limited nitrogen-based substrates for microbial fermentation. In this review paper, we intend to gather existing information on the equine ecosystem and to provide future perspectives of research.

Estimation of feed intake by cattle using controlled-release capsules containing n-alkanes or chromium sesquioxide

An experiment was carried out to evaluate the utilization of the n-alkane technique and the chromium sesquioxide (Cr 2 O 3 )/acid insoluble ash (AIA) procedure to estimate feed intake of meadow hay by two breeds of cattle. The animals were housed in individual pens, and offered known amounts of meadow hay as the sole diet component. One group of 4 Barrosa cows (average weight, 457±24 kg) and another group of 4 Holstein–Friesian cows (average weight, 635±148 kg) were dosed with intra-ruminal controlled-release capsules (CRC) that contained alkane markers or Cr 2 O 3 in separate capsules. During intake estimation, meadow hay and faeces were sampled twice daily. There was no significant effect of grab sampling time on the meadow hay intake estimation, due to the fact that no differences were observed in the faecal marker concentrations. The mean real intakes, measured as the difference between the dry weight of feed offered each day and the dry weight of daily feed refusal, were 5·64 and 7·76 kg DM/day for Barrosa and Holstein–Friesian cows, respectively. For Barrosa cows the mean estimated intakes when using the CRC release rates documented by the manufacturer (MRR) were 5·66, 6·59 and 6·90 kg DM/day, using the Cr 2 O 3 /AIA procedure, C 31 [ratio ]C 32 and C 33 [ratio ]C 32 n-alkane pairs, respectively. For Holstein–Friesian cows the same markers gave values of 7·72, 8·51 and 8·95 kg DM/day, respectively. Mean daily intake estimation was improved when the release rate calculated as the reduction rate in CRCs payload performed in a additional experiment (CRR), was used. The differences from the real intake values, obtained using C 31 [ratio ]C 32 and C 33 [ratio ]C 32 alkane pairs, decreased from 950 and 1260 g/day to 140 and 420 g/day, respectively, for Barrosa cows and from 750 and 1190 g/day to 290 and 90 g/day for Holstein–Friesian cows. The intake estimation using the Cr 2 O 3 /AIA procedure was not affected by correcting the CRC release rate, because the release rates of both MRR and the CRR were similar. The results indicate that controlled-release capsules provide a uniform marker release in cattle, but also suggest that to obtain accurate estimates of intake, it is better to measure release rates within the context of particular experiments.

The potential of white‐rot fungi to degrade phorbol esters of Jatropha curcas L. seed cake

The potential of solid‐state cultivation, with three white‐rot fungi (Bjerkandera adusta, Ganoderma resinaceum and Phlebia rufa), to decrease phorbol esters concentration of Jatropha curcas L. was evaluated in this study. Incubation was conducted in 250 mL Erlenmeyer flasks without agitation at 28°C for 30 days. Phorbol esters were analyzed by reverse‐phase HPLC after an extraction procedure using dichloromethane. All fungi studied were able to decrease the concentration of phorbol esters, mainly B. adusta and P. rufa which significantly reduced (p<0.05) phorbol esters contents to non‐toxic levels. These results suggest that white‐rot fungi could be potentially used as a possible approach for the biological treatment of the oilseed cake.

Assessment of very long-chain fatty acids as complementary or alternative natural fecal markers to n-alkanes for estimating diet composition of goats feeding on mixed diets

This study aimed to assess the use of very long-chain fatty acids (VLCFA) as an alternative or a complement to n-alkane markers for estimation of diet composition of goats fed 7 different diets, composed of different proportions of herbaceous (Lolium perenne and Trifolium repens) and heathland woody species (Erica umbellata, Erica cinerea, Calluna vulgaris, Erica arborea, and Ulex gallii), in a metabolism study. Diet composition was estimated from VLCFA (i.e., C(22) to C(34)) and alkane (i.e., C(25) to C(33)) concentrations in diet and feces, by least squares procedures. For all plant species VLCFA concentrations were greater than their alkane concentrations, especially for the herbaceous species and U. gallii. In general, fecal recovery of both markers was incomplete and increased in a curvilinear (P < 0.001) fashion with carbon-chain length. The plants comprising the diets had a significant effect (P < 0.001) on fecal recovery of VLCFA and alkanes. Diet composition estimates based on VLCFA alone were less accurate (P = 0.013) than those obtained using alkanes alone. Combination of VLCFA and alkane data resulted in the most accurate (P < 0.05) estimates, indicating an increase on the discriminatory power among plant species. Use of uncorrected fecal marker concentrations provided the poorest estimates of diet composition, and use of individual recovery data and mean fecal recovery data of the dietary treatment yielded the most accurate ones. Results obtained in this study show that VLCFA have potential to be used as diet composition markers along with alkanes.

The influence of casein and urea as nitrogen sources on in vitro equine caecal fermentation.

To access the fermentative response of equine caecal microbial population to nitrogen availability, an in vitro study was conducted using caecal contents provided with adequate energy sources and nitrogen as limiting nutrient. Two nitrogen (N) sources were provided, protein (casein) and non-protein (urea). Caecal fluid, taken from three cannulated horses receiving a hay-concentrate diet, was mixed with a N-free buffer-mineral solution. The influence of four N levels (3.7, 6.3, 12.5 or 25 mg of N in casein or urea) was studied using the gas production technique. Total volatile fatty acids (VFA), NH3-N and gas production were measured after a 24-h incubation period. Microbial biomass was estimated using adenine and guanine bases as internal markers, and ATP production was estimated stoichiometrically. Microbial growth efficiency (YATP) and gas efficiency (Egas) were estimated. Fermentation with casein as the sole N source was generally characterized by lower total VFA, NH3-N, total gas production and higher acetate : propionate (A : P) ratio and YATP than with urea. Results herein presented indicate that, under these in vitro conditions, caecal microbial population does in fact use urea N, but less efficiently than casein in terms of microbial growth.

Evaluation of long-chain alcohols as diet composition markers in goats grazing heathland areas.

An experiment was conducted to assess the potential of long-chain alcohols (LCOH), in alternative or combined with alkanes and long-chain fatty acids (LCFA), as faecal markers to estimate the diet composition of goats grazing heathland vegetation with associated improved pastures. A total of seven diets were offered across the grazing season. The diets were composed of mixtures of herb species (Lolium perenne and Trifolium repens) and woody species (Erica spp., Calluna vulgaris and Ulex gallii) in an attempt to simulate diet selection of goats on these complex vegetation areas. The diet composition was estimated using LCOH markers alone or combined with alkanes, LCFA and alkanes+LCFA, by least square optimization procedures. The data showed large differences between plant species in their LCOH profile. Generally, plant species showed higher LCOH concentrations than those of alkanes and lower than LCFA markers. Faecal recovery of LCOH was incomplete and increased in a linear manner (P < 0.001) with the carbon-chain length, and was influenced by diet composition and its digestibility. The diet composition estimates based on LCOH alone were more accurate (P < 0.05) than those using alkanes or LCFA alone. Results showed that the combination of LCOH with alkanes, LCFA and alkanes+LCFA resulted in more accurate (P < 0.05) estimates of diet composition, indicating that LCOH provided different discriminatory information to that of alkanes and LCFA, helping in the discrimination of the plant species used in this experiment. Results indicate that correction of faecal LCOH concentrations to incomplete faecal recovery is necessary to obtain more accurate estimates of diet composition. Nevertheless, it seemed that the use of a less accurate recovery correction on LCOH markers had a lower impact on the accuracy of estimates than that of alkanes and LCFA.

European cowpea landraces for a more sustainable agriculture system and novel foods

BACKGROUND Genetic diversity is fundamental to breeding programs and consequently has an important role in obtaining new varieties. To properly use the genetic diversity present in germplasm collections, a good knowledge of the agro-morphological traits of each accession is needed. The aim of this study was to explore the production capacity of 24 cowpea landraces from southern Europe, through phenotypic characterization and evaluation in three different locations in Greece and Portugal. RESULTS Most qualitative parameters tested showed a high stability among the three locations. A wide difference was observed among the three locations with respect to number of days to flowering, ranging from 55 to 99 days. Quantitative traits showed a higher genotype × environment than genetic variance component. In general, an inverse relationship between σ2ge /σ2g ratio (where σ2ge is genotype × genotype interaction and σ2g is genotype impact) and heritability value was observed. Principal component analysis was able to group accessions based on their origin. The first two principal components explained 97.52% of variation, being the number of seeds per plant, plant height and seed protein content, the traits which contributed most to variability. CONCLUSION The results show that sufficient variation exists in different traits within landraces in the studied cowpea germplasm to pursue a breeding program. However, the quantitative traits showed a higher genotype × environment component.