A.A. AbuGhazaleh

The effect of replacing corn with glycerol on ruminal bacteria in continuous culture fermenters

The effects of substituting corn with glycerol on DNA concentration of selected ruminal bacteria were investigated using continuous fermenters. Four continuous culture fermenters were used in a 4 × 4 Latin Square design with four 10 days consecutive periods. Treatment diets (60:40 forage to concentrate) were fed at 45 g/day dry matter (DM) in three equal portions. Glycerol (0.995 g/g glycerol) was used to replace corn in a grain mix at proportions of 0% (T0; control), 15% (T15), 30% (T30) and 45% (T45). On day 10 of each period, samples were collected from each fermenter 3 h after the morning feeding and analysed for volatile fatty acid and bacterial DNA concentration. Glycerol substitution was related to significantly higher butyrate, valerate and isovalerate concentrations. Compared with the T0 diet, acetate concentration was significantly lower with the T30 and T45 diets whilst propionate concentration was higher only with the T45 diet. The DNA concentrations for Butyrivibrio fibrisolvens and Selenomonas ruminantium decreased with the T30 and T45 diets compared with the T0 diet. No differences in the DNA concentrations for Ruminococcus albus and Succinivibrio dextrinosolvens amongst diets were observed. The findings show that substituting 15% of the dietary corn with glycerol had no substantive effects on fermentation processing or ruminal bacteria. Higher substitution levels, however, may adversely affect ruminal bacteria and negatively impact acetate production.

Short communication: The effect of substituting fish oil in dairy cow diets with docosahexaenoic acid-micro algae on milk composition and fatty acids profile

The effects of substituting fish oil (FO) with docosahexaenoic acid (DHA)-micro algae on milk chemical and fatty acid composition were examined in this study. Twenty-four Holstein cows in mid lactation grazing on an alfalfa-grass based pasture were divided into 4 treatment groups (6 cows/treatment) and supplemented with 7 kg/d grain mix plus 350 g of soybean oil and one of the following: 1) 150 g of FO, 2) 100 g of FO plus 50 g of algae, 3) 50 g of FO plus 100 g of algae, or 4) 150 g of algae. Cows were fed treatment diets for 3 wk, and milk samples were collected from each cow during the last 3 d of the study. Milk production (17.96, 17.56, 17.55, and 19.26 kg/d for treatment diets 1 to 4, respectively), milk fat percentages (3.17, 3.49, 3.74, and 3.43%), and milk protein percentages (3.35, 3.50, 3.71, and 3.42%) were similar between treatment diets. Concentrations (g/100 g of fatty acids) of milk cis-9 trans-11 (c9t11) conjugated linoleic acid (CLA; 3.41, 3.69, 4.47, and 4.21 for treatment diets 1 to 4, respectively) and vaccenic acid (11.80, 12.83, 13.87, and 13.53) were similar between treatment diets. Results of this study suggest that DHA-micro algae can partially or fully substitute FO in a cows diet without any adverse effects on milk production, milk composition, or milk c9t11 CLA content. The DHA-micro algae may be used as a viable alternative for FO in cows diet to modify rumen biohydrogenation to increase milk c9t11 CLA content.

The effect of lipid supplements on ruminal bacteria in continuous culture fermenters varies with the fatty acid composition

A single flow continuous culture fermenter system was used in this study to investigate the influence of dietary lipid supplements varying in their fatty acid content on the DNA concentration of selected rumen bacteria. Four continuous culture fermenters were used in a 4×4 Latin square design with four periods of 10 d each. Treatment diets were fed at 45 g/d (DM basis) in three equal portions during the day. The diets were: 1) control (CON), 2) control with animal fat source (SAT), 3) control with soybean oil (SBO), and 4) control with fish oil (FO). Lipid supplements were added at 3% of diet DM. The concentrations of total volatile fatty acids and acetate were not affected (P>0.05) by lipid supplements. Concentrations of propionate, iso-butyrate, valerate and iso-valerate were highest (P<0.05) with the FO diet compared with the other treatment diets. The concentration of til C18:l (vaccenic acid, VA) in effluents increased (P<0.05) with SBO and FO diets and was highest with the SBO diet. The concentrations of C18:0 in effluents were lowest (P<0.05) for the FO diet compared with the other treatment diets. Concentrations of DNA for Anaerovibrio lipolytica, and Butyrivibrio proteoclasticus in fermenters were similar (P>0.05) for all diets. The DNA concentrations of Butyrivibrio fibrisolvens and Ruminococcus albus in fermenters were lowest (P<0.05) with the FO diet but were similar (P>0.05) among the other treatment diets. Selenomonas ruminantium DNA concentration in fermenters was highest (P<0.05) with the FO diet. In conclusion, SBO had no effect on bacterial DNA concentrations tested in this study and the VA accumulation in the rumen observed on the FO diet may be due in part to FO influence on B. fibrisolvens, R. albus, and S. ruminantium.

Characterization of the FAD2 Gene Family in Soybean Reveals the Limitations of Gel-Based TILLING in Genes with High Copy Number

Soybean seed oil typically contains 18–20% oleic acid. Increasing the content of oleic acid is beneficial for health and biodiesel production. Mutations in FAD2-1 genes have been reported to increase seed oleic acid content. A subset of 1,037 mutant families from a mutagenized soybean cultivar (cv.) Forrest population was screened using reverse genetics (TILLING) to identify mutations within FAD2 genes. Although no fad2 mutants were identified using gel-based TILLING, four fad2-1A and one fad2-1B mutants were identified to have high seed oleic acid content using forward genetic screening and subsequent target sequencing. TILLING has been successfully used as a non-transgenic reverse genetic approach to identify mutations in genes controlling important agronomic traits. However, this technique presents limitations in traits such as oil composition due to gene copy number and similarities within the soybean genome. In soybean, FAD2 are present as two copies, FAD2-1 and FAD2-2. Two FAD2-1 members: FAD2-1A and FAD2-1B; and three FAD2-2 members: FAD2-2A, FAD2-2B, and FAD2-2C have been reported. Syntenic, phylogenetic, and in silico analysis revealed two additional members constituting the FAD2 gene family: GmFAD2-2D and GmFAD2-2E, located on chromosomes 09 and 15, respectively. They are presumed to have diverged from other FAD2-2 members localized on chromosomes 19 (GmFAD2-2A and GmFAD2-2B) and 03 (GmFAD2-2C). This work discusses alternative solutions to the limitations of gel-based TILLING in functional genomics due to high copy number and multiple paralogs of the FAD2 gene family in soybean.

Effects of incremental amounts of fish oil on trans fatty acids and Butyrivibrio bacteria in continuous culture fermenters

Previous studies have shown that adding fish oil (FO) to ruminant animal diets increased vaccenic acid (VA; t11 C18:1) accumulation in the rumen. Therefore, the objective of this study was to evaluate the effect of dietary FO amounts on selected strains of rumen bacteria involved in biohydrogenation. A single-flow continuous culture system consisting of four fermenters was used in a 4 × 4 Latin square design with four 9 days consecutive periods. Treatment diets were as follows: (i) control diet (53:47 forage to concentrate; CON), (ii) control plus FO at 0.5% (DM basis; FOL), (iii) control plus FO at 2% (DM basis; FOM) and (iv) control plus FO at 3.5% (DM basis; FOH). Fermenters were fed treatment diets three times daily at 120 g/day. Samples were collected from each fermenter on day 9 of each period at 1.5, 3 and 6 h post-morning feeding and then composited into one sample per fermenter. Increasing dietary FO amounts resulted in a linear decrease in acetate and isobutyrate concentrations and a linear decrease in acetate-to-propionate ratio. Propionate, butyrate, valerate and isovalerate concentrations were not affected by FO supplementation. Concentrations of C18:0 in fermenters linearly decreased, while concentrations of t10 C18:1 and VA linearly increased as dietary FO amounts increased. The concentrations of c9t11 and t10c12 conjugated linoleic acid were not affected by FO supplementation. The DNA abundance for Butyrivibrio fibrisolvens, Butyrivibrio vaccenic acid subgroup, Butyrivibrio stearic acid subgroup and Butyrivibrio proteoclasticus linearly decreased as dietary FO amounts increased. In conclusion, FO effects on trans fatty acid accumulation in the rumen may be explained in part by FO influence on Butyrivibrio group.

The Effect of Forage Level and Oil Supplement on Butyrivibrio fibrisolvens and Anaerovibrio lipolytica in Continuous Culture Fermenters.

The objective of this study was to evaluate the effects of forage level and oil supplement on selected strains of rumen bacteria believed to be involved in biohydrogenation (BH). A continuous culture system consisting of four fermenters was used in a 4×4 Latin square design with a factorial arrangement of treatments, with four 10 d consecutive periods. Treatment diets were: i) high forage diet (70:30 forage to concentrate (dry matter basis); HFC), ii) high forage plus oil supplement (HFO), iii) low forage diet (30:70 forage to concentrate; LFC), and iv) low forage plus oil supplement (LFO). The oil supplement was a blend of fish oil and soybean oil added at 1 and 2 g/100 g dry matter, respectively. Treatment diets were fed for 10 days and samples were collected from each fermenter on the last day of each period 3 h post morning feeding. The concentrations of vaccenic acid (t11C18:1; VA) and c9t11 conjugated linoleic acid (CLA) were greater with the high forage diet while the concentrations of t10 C18:1 and t10c12 CLA were greater with the low forage diet and addition of oil supplement increased their concentrations at both forage levels. The DNA abundance of Anaerovibrio lipolytica, and Butyrivibrio fibrisolvens vaccenic acid subgroup (Butyrivibrio VA) were lower with the low forage diets but not affected by oil supplement. The DNA abundance of Butyrivibrio fibrisolvens stearic acid producer subgroup (Butyrivibrio SA) was not affected by forage level or oil supplement. In conclusion, oil supplement had no effects on the tested rumen bacteria and forage level affected Anaerovibrio lipolytica and Butyrivibrio VA.

Short communication: Effect of blackberry and pomegranate oils on vaccenic acid formation in a single-flow continuous culture fermentation system

A single-flow continuous culture fermenter system was used to study the effect of blackberry and pomegranate oils on vaccenic acid (trans-11 C18:1; VA) formation. Four continuous culture fermenters were used in a 4 × 4 Latin square design with 4 periods of 10d each. Diets were (1) control (CON), (2) control plus soybean oil (SBO), (3) control plus blackberry oil (BBO), and (4) control plus pomegranate oil (PMO). Oil supplements were added at 30 g/kg of diet dry matter. Effluents were collected from each fermenter during the last 3d of each period and analyzed for nutrient and fatty acid composition. The concentration of VA in effluents increased with oil supplements and was greatest with the BBO diet. The concentration of stearic acid (C18:0) increased with the addition of soybean oil but decreased with the addition of pomegranate oil compared with the CON diet. The concentration of cis-9,trans-11 conjugated linoleic acid increased with oil supplements and was greatest with the PMO diet. In conclusion, all 3 oil sources were effective in increasing the production of VA. The effect of PMO and BBO on VA may have resulted in part from inhibition of the final step in the biohydrogenation of VA to stearic acid.

Lactic Acid Production from Apple Skin Waste by Immobilized Cells of Lactobacillus reuteri

Apple skins generally are not used in food products and often are discarded as waste. Apple skin contains several useful nutrients particularly sugars and more complex carbohydrates. This study was undertaken to determine the feasibility of apple skin waste as a media source for the production of lactic acid (LA) using Lactobacillus reuteri. One to nine ratios (10%) of apple skin to water was boiled for 5 min and its filtrate was then added with each of following supplements, respectively; ammonium phosphate, manganese sulfate, peptone, yeast extract, and potassium phosphate monobasic and dibasic. The efficacy of apple skin filtrates with and without supplements was determined by measuring pH, glucose and lactic acid after 2, 4 and 8 h fermentation at 37°C. Moreover, apple skin filtrate was also compared with MRS broth and G-M17 broth for the efficacy of glucose utilization and LA production. At the end of 8 h incubation, pH of apple skin filtrates with peptone and yeast extract reduced from 7 to ∼4. The LA concentrations produced from the filtrates with either peptone or yeast extract supplement were the greatest ranging from 0.9 g/L to 1.9 g/L. The efficacy of LA production using apple skin filtrate was about the same as G-M17 broth. Higher amount of LA was yielded with media inoculated with immobilized (encapsulated) cells than free cell. Results also indicated that immobilized L. reuteri MM2-3 produced 40% higher amount of LA than free cell. The results obtained from this study indicate that apple skins as a carbohydrate source and immobilization of lactic acid bacteria (LAB) could be a promising method for the production of LA. Further study is necessary to identify ways to increase glucose availability and promote LAB growth and LA production.

Evaluating the effects of essential oils on methane production and fermentation under in vitro conditions

Abstract The effects of adding essential oils (EO) on rumen fermentation and methane production were examined. The aim of experiment one was to screen the effects of four different EO (clove oil (CLO), white thyme oil (WTO), citronella oil (CTO) and anise oil (ANO)) at 500 mg/L of culture fluid on methane production under in vitro conditions. Rumen contents collected from a cannulated Holstein dairy cow was used in a 24-hour batch culture experiment. Treatments were a control (CON) or CON plus EO at 500 mg/L. Results showed that all EOs, except CTO, decreased (p ≤ .05) methane production. The aim of experiment two was to test the effects of three different dose levels of CLO, WTO, and ANO on methane production and fermentation in 24-h batch culture experiments. Treatments were CON or CON plus EO supplemented at 125, 250, and 500 mg/L. Relative to CON, methane production decreased (p ≤ .05) with the three EO at the 500 mg/L dose. At the 250 mg/L dose, ANO and CLO decreased (p ≤ .05) methane production and at the 125 mg/L dose, only CLO decreased methane production. Relative to CON, total VFA concentration declined (p ≤.05) in cultures incubated with WTO and with ANO at 500 mg/L dose. Relative to CON, the addition of CLO, WTO and ANO at 500 mg/L decreased (p ≤ .05) dry matter (DM) digestibility. In conclusion, our results showed that EO effects on methane production depend on EO source and dose level. Although the addition of ANO and WTO at the high doses resulted in lower methane production, they had negatively impacted on rumen microbial fermentation. Clove oil on the other hand reduced methane production without negatively impacting rumen fermentation.

Rapid and Simple Method for the Encapsulation of Lactobacillus reuteri in the Production of Lactic Acid

Lactic acid (LA) is the most widely used multifunctional organic acid and approximately 50% of it is produced by biotechnological process. Immobilizing microbial cells not only improves cell retention but also protects cells from harsh environmental conditions during LA production. The objectives of this research were therefore to develop a simple method to encapsulate (immobilize) Lactobacillus reuteri for the continuous production of LA and to evaluate LA production in three different types of media using free and immobilized L. reuteri.Five strains of L. reuteri grown in lactobacillusMRS broth at 37°C for 24 h were washed in peptone water and suspended in 2% sodium alginate solution. Encapsulation of cells was performed by dripping the mixture of sodium alginate and culture into ice-cold (2°C) 0.4 M calcium chloride solution using a separator funnel. The beads were then subjected to each of 500 ml MRS, modified MRS and whey-based broth and then incubated at 37°C for 12 h. Samples were withdrawn at 2-hour intervals during incubation period and analyzed for LA as represented in pH. Results show that the developed method is a rapid and simple microbial encapsulation procedure for the continuous production of LA. The efficacy of LA production as measured in pH was not significantly different in all tested media. At the end of fermentation process, pH of whey medium containing conventional (free) and encapsulated cells reached to 4.20 and 3.85, respectively. This indicates that higher amount of acid is yielded with encapsulated cells than free cells. In addition, immobilized cell strain MM2-3 produced the highest pH (3.5) while free cell strain SD2112 produced the lowest pH (4.05). Hence, results from this study suggest that we were able to develop a simple and rapid method for the encapsulation of L. reuteri.