Microbial and chemical changes during the spontaneous ensilage of grape pomace

Pilot scale fermentations with grape pomace from two different wineries were investigated during the 24 weeks of the ensiling period, along with laboratory scale experiments in which the environmental temperatures were held constant at 20, 25, 30 and 35 °C. During this period, yeast and lactic acid bacteria (LAB) counts were made, after which the identity of both groups of organisms was studied, as were the major microbial metabolites present. Major microbial and chemical alterations occurred during the first 3 weeks of ensilage, leaving a more stable product differing significantly from the initial substrate. The results obtained indicated that after initial growth, yeast and LAB populations undergo progressive inactivation at environmental temperatures above 20 °C, although LAB seem to adjust better to this specific, post-fermentation environment. Homofermentative species of Lactobacillus were the dominant LAB. The initial yeast flora of non- Saccharomyces species was replaced by a typical wine yeast flora, i.e. predominantly Saccharomyces cerevisiae. At the chemical level, major alterations were due to an alcoholic fermentation and a malolactic conversion within the first 3 weeks.     

The response of sheep to big bale grass silage ensiled with,or supplemented separately with,molassed sugar beet feed

Second cut big bale grass silage was prepared with grass ensiled alone or with added pelleted, molassed sugar beet feed (MSBF) at 32 or 64 g MSBF kg⁻¹ fresh weight of grass. Addition of MSBF at ensilage increased silage dry matter (DM), water soluble carbohydrate and lactic acid concentrations and decreased ammonia concentration. When fed to wether lambs, MSBF addition, whether at ensilage or given separately, variably improved DM intake, live-weight gain and feed conversion efficiency. Ruminal concentrations of ammonia were reduced and acetate molar proportion increased in diets supplemented with MSBF separately compared with the silage only control diet. Animal performance was generally improved by MSBF supplementation with responses to silages made with added MSBF comparable to those from silage supplemented with MSBF at feeding. There was little difference in animal performance between silages made with 32 or 64 g kg⁻¹ MSBF added at ensiling.     

Preservation of potential fermentables in sweet sorghum by ensiling

Pressed and wilted samples of sweet sorghum [Sorghum bicolor (L.) Moench var. Rio] were ensiled for periods up to 155 days. A kinetic study of the biochemical changes which occurred during ensiling showed that in wilted sorghum ensilage invert sugars and mannitol levels collectively were maintained at 65% of the original ferment able sugar content of the sorghum. The acidic environment produced by ensiling also served as a pretreatment that resulted in enhanced yields of reducing sugar when the sorghum was contacted with cellulolytic enzymes. The quantity of sugar obtained from enzymatic hydrolysis more than compensated for carbohydrate used by organisms during the ensiling process. Both Saccharomyces uvarum and Clostridium acetobutylicum were able to ferment a medium constituted from pressed sorghum juice and the solution resulting from enzymatic hydrolysis of sweet sorghum ensilage.     

Bacterial community dynamics during the ensilage of wilted grass

Aims:  Grass silage is the product formed by a natural lactic acid bacterial fermentation when grass is stored under anaerobic conditions, and represents an important ruminant feedstuff on farms during winter. Of the two commonly employed methods of ensiling forage, baled silage composition frequently differs from that of comparable precision-chop silage reflecting a different ensiling environment. The aim of this study was to investigate the dynamics of the silage fermentation in wilted grass and between ensiling systems.
Methods and Results:  Fermentation dynamics were examined using traditional methods of silage analyses, including microbial enumeration and analysis of fermentation products, and culture-independent terminal restriction fragment length polymorphism (T-RFLP). A successful fermentation was achieved in both systems, with the fermentation (increase in lactic acid bacteria and lactic acid concentration, decrease in pH) proceeding rapidly once the herbage was ensiled.
Conclusions:  Under controlled conditions, little difference in silage quality and microbial composition were observed between ensiling systems and this was further reflected in the T-RFLP community analysis.
Significance and Impact of the Study:  T-RFLP proved a potentially useful tool to study the ensilage process and could provide valid support to traditional methods, or a viable alternative to these methods, for investigating the dynamics of the bacterial community over the course of the fermentation.     

Bioconversion of corn straw by coupling ensiling and solid-state fermentation

A two-stage process that combined solid-state fermentation (SSF) and ensiling was used for bioconversion of corn straw, in order to increase nutritional value and palatability for animal feed. SSF of corn straw increased the level of protein from 6.7% to 14.7% and decreased the cellulose by 38.0% and hemicellulose by 21.2%. Cellulase and xylanase were produced during SSF. After SSF, the fermented substrate was directly ensiled by inoculating with lactic acid bacteria (LAB). In situ produced enzymes and bacterial inoculation resulted in a rapid drop in pH, a high level of lactic acid production, partial degradation of cell wall components and generation of reducing sugars (RSs). Efficiency of ensiling at 25 degrees C, 30 degrees C, 35 degrees C, 40 degrees C was evaluated. Temperature influenced the effect of ensiling; the higher the temperature, the shorter the ensiling period. The combined fermentation upgraded the nutritional value, enhanced the efficiency of ensiling and reduced bioprocessing costs.     

Fate of Escherichia coli O26 in corn silage experimentally contaminated at ensiling, at silo opening, or after aerobic exposure, and protective effect of various bacterial inoculants

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for human illness. Ruminants are recognized as a major reservoir of STEC, and animal feeds, such as silages, have been pointed out as a possible vehicle for the spread of STEC. The present study aimed to monitor the fate of pathogenic E. coli O26 strains in corn material experimentally inoculated (10⁵ CFU/g) during ensiling, just after silo opening, and after several days of aerobic exposure. The addition of 3 bacterial inoculants, Propionibacterium sp., Lactobacillus buchneri, and Leuconostoc mesenteroides (10⁶ CFU/g), was evaluated for their abilities to control these pathogens. The results showed that E. coli O26 could not survive in corn silage 5 days postensiling, and the 3 inoculants tested did not modify the fate of pathogen survival during ensiling. In the case of direct contamination at silo opening, E. coli O26 could be totally eradicated from corn silage previously inoculated with Leuconostoc mesenteroides. The combination of proper ensiling techniques and the utilization of selected bacterial inoculants appears to represent a good strategy to guarantee nutritional qualities of cattle feed while at the same time limiting the entry of pathogenic E. coli into the epidemiological cycle to improve the microbial safety of the food chain.     

Identification and characterization of yeasts in sugarcane silages

Aims: To enumerate the micro‐organisms and to identify the yeast species present during the ensilage of different sugarcane (Saccharum spp.) cultivars. Method: Samples of sugarcane silage were collected at 10, 20, 30 and 40 days from the start of fermentation. Population levels of lactic acid bacteria (LAB), mesophilic facultative anaerobic (MFA) bacteria, filamentous fungi and yeasts were determined. Nine species of yeasts were classified according to traditional methods and confirmed using molecular techniques. Conclusions: LAB dominated the ensiling process of sugarcane, although yeasts were present at relatively high population levels throughout the whole fermentation period. The detected species of yeasts varied according to sugarcane cultivar and time of fermentation. Torulaspora delbrueckii was the predominant yeast, followed by Pichia anomala and Saccharomyces cerevisiae. Significance and Impact of the Study: Knowledge of the population of micro‐organisms in general, and of yeasts in particular, present during the fermentation of sugarcane is of fundamental importance in the development of more effective ensiling processes.     

The effect of an inoculant and enzymes on fermentation and nutritive value of sorghum straw silages

The objectives of this study were to determine the effect of inoculant, enzymes and inoculant-enzymes mixture on fermentation quality, nutritive value, and microbial changes of sorghum straw silage. Sorghum straws were collected and treated with distilled water (control), inoculant, enzymes and inoculant+enzymes prior to ensiling. Three bag silos for each silage (denoted C, I, E and I+E, respectively) were opened after 3, 7, 11, 15, 30 and 60 days for chemical and microbial analyses. For all the silages, there was a rapid decline in pH during the first 3 days of ensiling. Relative to silage C, all the treatment (I, E and I+E) had higher (P<0.05) lactic acid concentration at all ensiling periods. Population of LAB during all ensiling time was numerically greater for treated than control silages. Separate addition of two additives, especially for enzymes, can effectively (P<0.05) decrease aNDF and ADF concentration. Treatments with enzymes (E, I+E) can also improve significantly silage IVDMD and IVNDFD concentration. These results indicated that the addition of additives can improve the sorghum straw silage fermentation quality at different extent.     

The effect of temperature and Lactobacillus amylovorus and Lact. plantarum, applied at ensiling, on wheat silage

The effects of applying Lactobacillus plantarum and Lact. amylovorus at ensiling on wheat silage stored at 25 and41 °C was studied under laboratory conditions. The inoculants were applied at 10⁶ cfu g⁻¹.Silages with no additives served as controls. Three jars per treatment were sampled on days 2, 8 and 60 after ensiling, for chemical and microbiological analyses. After the ensiling period, the silages were subjected to an aerobic stability test. The control and Lact. plantarum inoculated wheat fermented faster at 25 than at 41 °C, whereas silages inoculated with Lact. amylovorus fermented faster at 41 °C. This was apparent from the rate of pH decrease and from the contents of residual sugars and lactic acid in the final silages. The numbers of lactobacilli in the control and Lact. plantarum silages at 41 °C after 2 and 8 days of ensiling were lower than in the corresponding silages at 25 °C. For the Lact. amylovorus silage the opposite held true. The control silages at both temperatures and the Lact. plantarum silage at 41 °C were the most stable silages under aerobic exposure.     

Molasses and microbial inoculants improve fermentability and silage quality of cotton waste-based spent mushroom substrate

A small-silo study was conducted to develop an effective ensiling storage method for the use of cotton waste-based spent mushroom substrate (SMS) as an animal feed. The SMS was ensiled with 5% molasses (DM basis), 0.5% (v/w) lactic acid bacteria (LAB, Lactobacillus plantarum) inoculant or 0.5% (v/w) yeast (Saccharomyces cerevisiae) inoculant. The treatments included 100% SMS (control), 95% SMS+5% molasses (T1), 95% SMS+5% molasses+0.5% LAB (T2) and 95% SMS+5% molasses+5% LAB+0.5% yeast (T3). The treatments were ensiled for 10. Change in chemical compositions was little (P>0.05) according to the ensiling process and treatments. Compared with those before ensiling, 100% SMS (control) after ensiling showed unstable fermentative properties with high pH (5.2) and little lactic acid production. Compared with the ensiled control, treatments (T1, T2 and T3) resulted in decreased pH, 18-20 times higher concentrations of lactic acid, and greater populations of total bacteria (P<0.07), LAB and yeast (P<0.07). The addition of 5% molasses, 0.5% LAB and 0.5% yeast (T3) to the SMS resulted in the lowest pH (4.25) and the greatest microbial populations. Treatment T3 was selected for a large scale silo study which was ensiled for 10, 20 and 30 d. As in the small-silo study, the T3 treatment showed favorable fermentative and microbial parameters, compared with the control, by decreasing pH and increasing lactic acid concentrations, LAB and yeast populations. The minimum ensiling period was 20 d, when pH was reasonably low and LAB and yeast populations were greatest. In conclusion, molasses and microbial inoculation improved silage quality of SMS.     

Fermentation dynamics and diversity of bacterial community in four typical woody forages

Woody forage is a new feeding resource used worldwide, and silage is the main long-term storage, mediated by micro-organisms present during their processing. The objectives of our work were to evaluate the fermentation dynamics and to characterize the bacterial community of our typical woody forages. We selected four typical woody forages: paper mulberry (Broussonetia papyrifera), mulberry (Morus alba L.), moringa tree (Moringa oleifera), and Anthocephalus chinensis (Neolamarckia cadamba). The materials were ensiled and sampled after ensiling 1, 3, 5, 7, 15, 30, and 60 days. Our results indicate that woody forages have good forage properties with relatively high crude protein content and low neutral detergent fiber and acid detergent fiber content. However, the water soluble carbohydrate content in paper mulberry was considerably low (18.67 g kg−1), which makes ensiling difficult. The lactic acid and acetic acid contents in each of the four materials were reduced after 3 days of ensiling and increased again after 30 days of ensiling, with the exception of Anthocephalus chinensis. Anthocephalus chinensis and moringa tree were well-preserved after 7 and 60 days of ensiling, respectively, with low pH and ammonia nitrogen content. Cyanobacteria was predominant in moringa tree and Anthocephalus chinensis before ensiling, and Lactobacillus became dominant after 15 days of ensiling. Enterobacter dominated the paper mulberry and mulberry during fermentation process and accelerated their poor silage quality. Therefore, the conformity of bacterial community succession with ensiling parameters guaranteed the final quality of woody forage silages, and this might aid in controlling the manufacturing process.     

Nutritive value of ensiled broiler litter for cattle

Broiler litter, consisting of bedding material (chopped wheat straw or rice hulls), excreta, wasted feed and feathers was ensiled at 40 or 50% moisture for 42 days. Cheddar cheese whey was added to adjust the moisture level in some of the silage. The dry matter digestibility in vitro (IVDMD), after 21 days of ensiling was greater for silage containing the wheat straw base than for that with the rice hull base. Whey improved the IVDMD of the wheat straw base silage but not that of the rice hull base silage. Moisture levels did not influence the IVDMD. In a second trial, broiler litter consisting of chopped wheat straw bedding, excreta, wasted feed and feathers was ensiled for 28 days at approximately 45% dry matter. The litter was ensiled as: (1) litter alone; (2) litter plus Irish potato cannery waste (IPW); (3) litter plus ground maize, and (4) litter plus ground grain sorghum. The materials added supplied 33% of the dry matter of the silages. The pH of the silages 1 and 4 weeks after ensiling was (1) 6.00, 5.83; (2) 5.00, 4.56; (3) 4.96, 4.80; and (4) 4.92, 4.78. Total faecal and urine collection trials were conducted using 12 Holstein steers having an average body weight of 200 kg. Dry matter digestibility was greater (P < 0.01) for silages 2, 3 and 4. Digestible energy and protein were 61.2, 70.6; 65.3, 74.6; 65.2, 71.2; and 68.2, 76.4% for silages 1 through 4.     

Identification of sulcatol, a potential pheromone of the ambrosia beetle Gnathotrichus materiarius (Col., Scolytidae)

Abstract: We report the identification of a potential pheromone for Gnathotrichus materiarius (Fitch) (Col., Scolytidae). The population sex ratio is close to 1 : 1, and males initiate attacks on host trees. Headspace and hindgut samples from single males showed the presence of the putative pheromone 6-methyl-5-hepten-2-ol, sulcatol. Unmated males released sulcatol for at least 12 days, and ceased producing the pheromone after 20 days. The peak sulcatol release occurred after 2 days. Males cease production of sulcatol 24 h after being paired with females. Single females were unable to initiate galleries, and no sulcatol was detected from their headspace and hindgut samples. The chiral ratio of the pheromone, observed from headspace samples, was 31% (S)-(+)- and 69% (R)-(−)-sulcatol.     

Saccharification of Miscanthus x giganteus, incorporation of lignocellulosic by-product in cementitious matrix

Given the non competition of miscanthus with food and animal feed, this lignocellulosic species has attracted attention as a possible biofuel resource. However, sustainability of ethanol production from lignocelluloses biomass would imply reduction in the consumption of chemicals and/or energetic means, but also valorization of the lignocellulosic by-product remaining from enzymatic saccharification. Introduction of these by-products into a cementitious matrix could be used in manufacturing a lightweight composite. Miscanthus biomass was submitted to chemical pretreatments followed by saccharification using an enzymatic cocktail. Residues from saccharification were then mixed with a cementitious matrix. Given their mechanical properties and a good adherence between cement and by-product, the hardened materials could be used. However, the delay in the beginning of setting time is too long, which prevents the direct use of by-product into cementitious matrix. Preliminary experiments using a setting accelerator in the cementitious matrix permitted significant reduction in the setting time delay.     

Steam reforming of biodiesel by-product to make renewable hydrogen

The aim of this paper was to investigate the viability of steam reforming the combined glycerol and water by-product streams of a biodiesel plant. A platinum alumina catalyst was used to optimise the operating conditions for glycerol steam reforming and mass spectroscopy was chosen to measure reformer gas yield. The problem is that glycerol steam reforming is relatively untested even with pure glycerol and the by-product quality may be too poor. The strategy was therefore to optimise the process using pure glycerol and compare the performance with by-product glycerol. To test catalyst degradation caused by carbon deposition, a Solid Oxide fuel cell (SOFC) was used as a separate reformer and electrical performance was measured to indicate carbon deposition. This is the first time a SOFC has been run on glycerol. The results showed that thermodynamic theory can be used to predict reformer performance. At high temperatures high gas yield can be reached (almost 100%) and selectivities of 70% (dry basis) obtained. The optimum conditions for glycerol reforming were 860 °C temperature (maximum tested), 0.12 mols/min glycerol flow per kg of catalyst and 2.5 steam/carbon ratio. Reforming catalysts lasted for several days of continuous operation with minimal degradation, 0.4% of feed deposited. By-product glycerol performed slightly worse with a lower yield and more carbon deposition, 2% of feed. The results show that glycerol steam reforming is a viable alternative use for glycerol and potentially a better option than purification.     

Differentiation Among Bean Leafroll Virus Susceptible and Resistant Lentil and Faba Bean Genotypes on the Basis of Virus Movement and Multiplication

Systemic movement of Bean leafroll virus (BLRV) in susceptible and resistant lentil and faba bean genotypes was studied using plants grown in a plastic house. All the plants studied were inoculated with BLRV by viruliferous pea aphids (Acyrthosiphon pisum). Five plants/genotype of lentil and faba bean were harvested, respectively, at 3, 6, 9, 12 and 18 days and 1, 2, 3, 4 and 5 weeks after inoculation. Each plant was split into growing point, stem, stem base and root, and each was tested using tissue blot immunoassays (TBIA). Virus concentration in each section was estimated using a 0–3 score and a relative TBIA value was estimated accordingly for each genotype. In susceptible lentil genotypes (ILL 8063 and ILL 2581), BLRV was present in low concentrations in the growing point 3 days after inoculation and in high concentrations in all parts of the plant after 6 days. By contrast, the virus was not detected in the highly resistant genotype (ILL 74) until 18 days after inoculation. In the faba bean genotypes studied, BLRV was detected in high concentrations in all parts of the highly susceptible genotype (Fiord) 1 week after inoculation but only after 3 weeks in resistant genotypes (e.g. BPL 5274), but was not detected in the highly resistant genotypes (BPL 5278 and BPL 5279) 5 weeks after inoculation. The replication and systemic movement of BLRV was thus slower in resistant genotypes than in susceptible genotypes. Moreover, the use of TBIA scores clearly and easily differentiated resistant and susceptible genotypes. Our results suggest that BLRV movement and multiplication can be useful criteria when differentiating resistant from susceptible genotypes. In addition, undertaking the preliminary screening in a plastic house requires less space than direct planting in the field.     

Assessing the impact of various ensilage factors on the fermentation of grass silage using conventional culture and bacterial community analysis techniques

Aims: Grass silage is an important ruminant feedstuff on farms during winter. The ensilage of grass involves a natural lactic acid bacterial fermentation under anaerobic conditions, and numerous factors can influence the outcome of preservation. The aim of this study was to investigate the effect of dry matter concentration, ensiling system, compaction and air infiltration on silage bacterial community composition. Methods and Results: The impact of these factors was examined using conventional methods of microbial analysis and culture‐independent Terminal Restriction Fragment Length Polymorphism (T‐RFLP). Silage fermentation was restricted in herbage with a high dry matter concentration, and this was reflected in a shift in the bacterial population present. In contrast, ensiling system had little effect on bacterial community composition. Air infiltration, in the absence of compaction, altered silage bacterial community composition and silage pH. Conclusions: Dry matter concentration and the absence of compaction were the main factors affecting silage microbial community composition, and this was reflected in both the conventional culture‐based and T‐RFLP data. Significance and Impact of the Study: T‐RFLP proved a useful tool to study the factors affecting ensilage. Apart from monitoring the presence or absence of members of the population, shifts in the relative presence of members could be monitored.     

Mesophilic anaerobic digestion in a fluidised-bed reactor of wastewater from the production of protein isolates from chickpea flour

A study of the anaerobic digestion of wastewater derived from the production of protein isolates from chickpea flour was carried out in a laboratory-scale, mesophilic (35 °C) fluidised-bed reactor with saponite as bacterial support. Soluble chemical oxygen demand (SCOD) removal efficiencies in the range of 96.8–85.2% were achieved in the reactor at organic loading rates (OLR) of between 0.58 and 2.10 g chemical oxygen demand (COD)/l per day, hydraulic retention times (HRT) of between 14.9 and 4.5 days and average feed COD concentration of 9.1 g/l. Eighty-five percent of feed COD could be removed up to OLR of 2.1 g COD/l per day. The yield coefficient of methane production was 0.34 l of methane (at STP) per gram COD removed and was virtually independent of the OLR applied. Because the buffering capacity of the experimental system was maintained at favourable levels with excess total alkalinity present at all loadings, the rate of methanogenesis was not affected by loading. Experimental data indicated that a total alkalinity in the range of 1090–2130 mg/l as CaCO₃ was sufficient to prevent the pH from decreasing to below 7.2 for OLR of up to 2.7 g COD/l per day. The volatile fatty acid (VFA) levels and the VFA/alkalinity ratio were lower than the suggested limits for digester failure (0.3–0.4) for OLR and HRT up to 2.7 g COD/l per day and 3.5 days, respectively. For a HRT of 2.8 days (OLR of 3.00 g COD/l per day) the start of acidification was observed in the reactor.     

Effect of milk deprivation during the lactation period on performance and digestive enzyme activities of the piglets following weaning

Post-weaning performance of piglets from systems where lactation is disrupted (e.g. from multisuckling systems) is superior to conventionally reared piglets. The objective of this study was to establish whether restricted growth prior to weaning caused by disruption of suckling was an important factor in post-weaning performance and also whether there were related changes in gastro-intestinal development. Ten litters of eight piglets were used in a split-plot design. Half of each litter (limited suckling, LS) had suckling disrupted by separation from their dam for 7 h/day from day 14 to 28 after farrowing. The remainder of each litter was allowed to suck normally (normal suckling, NS). The same amount of creep feed was offered to LS piglets as consumed by NS littermates on the previous day. There were no differences in weight between LS and NS piglets at 14 days of age, but restricting access to the sow reduced weaning weight at 28 days of age (7.96 v. 9.00 kg; LS v. NS; P < 0.01; s.e.d. 0.23). Feed intakes were greater for LS than NS piglets over the first 28 days post weaning, particularly in the 1st week after weaning when feed efficiency was also improved (0.91 v. 0.62 kg gain per kg feed; P < 0.01; s.e.d. 0.08). As a result, LS piglets grew more rapidly in the first 28 days post weaning, particularly in the first 7 days after weaning. Subsequent performance to 8 weeks was similar for both groups. Digestive organ weights were not different at 2 and 9 days after weaning; nor were small intestine specific enzyme activities significantly different ( P>0.05). Pancreatic trypsin activity was, however, greater ( P < 0.01) for LS pigs on both days 2 and 9 post weaning. In conclusion the restriction of growth as a result of limited suckling itself is an important factor in determining post-weaning performance and may be related to development of pancreatic trypsin activity.     

Effect of inoculation of herbage prior to ensiling with Streptomyces achromogenes ISP 5028 on chemical composition of silage

Enzymes derived from bacteria and fungi have been used as additives in silage production. Increasing attention has been given to the use of cell-wall degrading enzymes such as cellulases, hemicellulases and peroxidases. However, little information has been published on the potential role of actinomycetes in silage production. The experiment reported in this paper examined the changes in chemical composition and predicted nutritive value of herbage inoculated with Streptomyces achromogenes ISP 5028 during 60 days of ensiling. Changes in chemical composition and nutritive value of herbage were examined after 1, 2, 3, 5, 10, 30 and 60 days ensiling. Colonisation of inoculated silage by S. achromogenes ISP 5028 occurred rapidly and after 30 days of ensiling the difference in viable spores was 1000-fold between the control and treated silages (P<0.001). Inoculation reduced the concentration of NDF (P<0.01) and crude hemicellulose (P<0.001) and accelerated the rate of loss of NDF (P<0.05) but not crude hemicellulose (P>0.05) from the cell wall. The changes in the composition of the cell wall facilitated an increase in the concentration of WSC (P<0.001), thus allowing greater concentrations of lactic (P<0.001) and acetic (P<0.01) acids to be formed. Elevated concentrations of various isomeric forms of butyric and valeric acid were also observed in the treated silage compared to the control. Inoculation of herbage with S. achromogenes ISP 5028 prior to ensiling altered the chemical and nutritive composition of silage, however it is not known whether the risks associated with other actinomycetes (respiratory disorders and toxin release) apply to the test organism in these experiments.