Granular biomass capable of partial nitritation and anammox

A novel and efficient way of removing nitrogen from wastewater poor in biodegradable organic carbon, is the combination of partial nitritation and anoxic ammonium oxidation (anammox), as in the one-stage oxygen-limited autotrophic nitrification/denitrification (OLAND) process. Since anoxic ammonium-oxidizing bacteria grow very slowly, maximum biomass retention in the reactor is required. In this study, a lab-scale sequencing batch reactor (SBR) was used to develop granular, rapidly settling biomass. With SBR cycles of one hour and a minimum biomass settling velocity of 0.7 m/h, OLAND granules were formed in 1.5 months and the nitrogen removal rate increased from 50 to 450 mg N L(-1) d(-1) in 2 months. The granules had a mean diameter of 1.8 mm and their aerobic and anoxic ammonium-oxidizing activities were well equilibrated to perform the OLAND reaction. Fluorescent in-situ hybridization (FISH) demonstrated the presence of both beta-proteobacterial aerobic ammonium oxidizers and planctomycetes (among which anoxic ammonium oxidizers) in the granules. The presented results show the applicability of rapidly settling granular biomass for one-stage partial nitritation and anammox.     

Effect of pH and level of concentrate in the diet on the production of biohydrogenation intermediates in a dual-flow continuous culture

Milk fat depression in cows fed high-grain diets has been related to an increase in the concentration of trans-10 C_18:1 and trans-10,cis-12 conjugated linoleic acid (CLA) in milk. These fatty acids (FA) are produced as a result of the alteration in rumen biohydrogenation of dietary unsaturated FA. Because a reduction in ruminal pH is usually observed when high-concentrate diets are fed, the main cause that determines the alteration in the biohydrogenation pathways is not clear. The effect of pH (6.4 vs. 5.6) and dietary forage to concentrate ratios (F:C; 70:30 F:C vs. 30:70 F:C) on rumen microbial fermentation, effluent FA profile, and DNA concentration of bacteria involved in lipolysis and biohydrogenation processes were investigated in a continuous culture trial. The dual-flow continuous culture consisted of 2 periods of 8 d (5 d for adaptation and 3 d for sampling), with a 2 × 2 factorial arrangement of treatments. Samples from solid and liquid mixed effluents were taken for determination of total N, ammonia-N, and volatile fatty acid concentrations, and the remainder of the sample was lyophilized. Dry samples were analyzed for dry matter, ash, neutral and acid detergent fiber, FA, and purine contents. The pH 5.6 reduced organic matter and fiber digestibility, ammonia-N concentration and flow, and crude protein degradation, and increased nonammonia and dietary N flows. The pH 5.6 decreased the flow of C_18:0, trans-11 C_18:1 and cis-9, trans-11 CLA, and increased the flow of trans-10 C_18:1, C_18:2n-6, C_18:3n-3, trans-11,cis-15 C_18:2 and trans-10,cis-12 CLA in the 1 h after feeding effluent. The pH 5.6 reduced Anaerovibrio lipolytica (32.7 vs. 72.1 pg/10 ng of total DNA) and Butyrivibrio fibrisolvens vaccenic acid subgroup (588 vs. 1,394 pg/10 ng of total DNA) DNA concentrations. The high-concentrate diet increased organic matter and fiber digestibility, nonammonia and bacterial N flows, and reduced ammonia-N concentration and flow. The high-concentrate diet reduced trans-11 C_18:1 and trans-10 C_18:1, and increased C_18:2n-6, C_18:3n-3 and trans-10,cis-12 CLA proportions in the 1 h after feeding effluent. The increase observed in trans-10,cis-12 CLA proportion in the 1 h after feeding effluent due to the high-concentrate diet was smaller that that observed at pH 5.6. Results indicate that the pH is the main cause of the accumulation of trans-10 C_18:1 and trans-10, cis-12 CLA in the effluent, but the trans-10,cis-12 CLA proportion can be also affected by high levels of concentrate in the diet.     

Floc-based sequential partial nitritation and anammox at full scale with contrasting N2O emissions

New Activated Sludge (NAS^®) is a hybrid, floc-based nitrogen removal process without carbon addition, based on the control of sludge retention times (SRT) and dissolved oxygen (DO) levels. The aim of this study was to examine the performance of a retrofitted four-stage NAS^® plant, including on-line measurements of greenhouse gas emissions (N₂O and CH₄). The plant treated anaerobically digested industrial wastewater, containing 264 mg N L⁻¹, 1154 mg chemical oxygen demand (COD) L⁻¹ and an inorganic carbon alkalinity of 34 meq L⁻¹. The batch-fed partial nitritation step received an overall nitrogen loading rate of 0.18-0.22 kg N m⁻³ d⁻¹, thereby oxidized nitrogen to nitrite (45-47%) and some nitrate (13-15%), but also to N₂O (5.1-6.6%). This was achieved at a SRT of 1.7 d and DO around 1.0 mg O₂ L⁻¹. Subsequently, anammox, denitrification and nitrification compartments were followed by a final settler, at an overall SRT of 46 d. None of the latter three reactors emitted N₂O. In the anammox step, 0.26 kg N m⁻³ d⁻¹ was removed, with an estimated contribution of 71% by the genus Kuenenia, which constituted 3.1% of the biomass. Overall, a nitrogen removal efficiency of 95% was obtained, yielding a dischargeable effluent. Retrofitting floc-based nitrification/denitrification with carbon addition to NAS^® allowed to save 40% of the operational wastewater treatment costs. Yet, a decrease of the N₂O emissions by about 50% is necessary in order to obtain a CO₂ neutral footprint. The impact of emitted CH₄ was 20 times lower.     

Effects of chemically or technologically treated linseed products and docosahexaenoic acid addition to linseed oil on biohydrogenation of C18:3n-3 in vitro

Rumen biohydrogenation kinetics of C18:3n-3 from several chemically or technologically treated linseed products and docosahexaenoic acid (DHA; C22:6n-3) addition to linseed oil were evaluated in vitro. Linseed products evaluated were linseed oil, crushed linseed, formaldehyde treated crushed linseed, sodium hydroxide/formaldehyde treated crushed linseed, extruded whole linseed (2 processing variants), extruded crushed linseed (2 processing variants), micronized crushed linseed, commercially available extruded linseed, lipid encapsulated linseed oil, and DHA addition to linseed oil. Each product was incubated with rumen liquid using equal amounts of supplemented C18:3n-3 and fermentable substrate (freeze-dried total mixed ration) for 0, 0.5, 1, 2, 4, 6, 12, and 24 h using a batch culture technique. Disappearance of C18:3n-3 was measured to estimate the fractional biohydrogenation rate and lag time according to an exponential model and to calculate effective biohydrogenation of C18:3n-3, assuming a fractional passage rate of 0.060/h. Treatments showed no differences in rumen fermentation parameters, including gas production rate and volatile fatty acid concentration. Technological pretreatment (crushing) followed by chemical treatment applied as formaldehyde of linseed resulted in effective protection of C18:3n-3 against biohydrogenation. Additional chemical pretreatment (sodium hydroxide) before applying formaldehyde treatment did not further improve the effectiveness of protection. Extrusion of whole linseed compared with extrusion of crushed linseed was effective in reducing C18:3n-3 biohydrogenation, whereas the processing variants were not different in C18:3n-3 biohydrogenation. Crushed linseed, micronized crushed linseed, lipid encapsulated linseed oil, and DHA addition to linseed oil did not reduce C18:3n-3 biohydrogenation. Compared with the other treatments, docosahexaenoic acid addition to linseed oil resulted in a comparable trans11,cis15-C18:2 biohydrogenation but a lesser trans10+11-C18:1 biohydrogenation. This suggests that addition of DHA in combination with linseed oil was effective only in inhibiting the last step of biohydrogenation from trans10+11-C18:1 to C18:0.     

Effect of in vitro docosahexaenoic acid supplementation to marine algae-adapted and unadapted rumen inoculum on the biohydrogenation of unsaturated fatty acids in freeze-dried grass

The objective of this study was to examine the ruminal biohydrogenation of linoleic (18:2n-6) and linolenic (18:3n-3) acid during in vitro incubations with rumen inoculum from dairy cattle adapted or not to marine algae and with or without additional in vitro docosahexaenoic acid (DHA, 22:6n-3) supplementation. Treatments were incubated in 100-mL flasks containing 400 mg of freeze-dried grass, 5 mL of strained ruminal fluid, and 20 mL of phosphate buffer. Ruminal fluid was collected just before the morning feeding from 3 cows receiving a control diet (49% ryegrass silage, 39% corn silage, 1% straw, and 11% concentrate, fresh-weight basis) supplemented with marine algae for 21 d (adapted rumen fluid, aRF) or from the same cows receiving the control diet only for 14 d after marine algae supplementation was stopped (unadapted rumen fluid, uRF). In half of the incubation flasks, pure DHA (5 mg) was added as an oil-ethanol solution (100 mL). Incubations were carried out during 0, 0.5, 1, 2, 4, 6, and 24 h. After 24 h, in vitro addition of DHA resulted in greater amounts (mg/incubation) of 18:3n-3 (0.23, 0.43, 0.26, and 0.34 for aRF, aRF+DHA, uRF, and uRF+DHA), 18:2n-6 (0.14, 0.22, 0.15, and 0.20 for aRF, aRF+DHA, uRF, and uRF+DHA) and trans-11, cis-15-18:2 (0.27, 2.40, 0.06, and 2.21 for aRF, aRF+DHA, uRF, and uRF+DHA), whereas no effect of inoculum source was observed. Trans-11-18:1 accumulated after 24 h when aRF was incubated irrespective of in vitro DHA supplementation, whereas in incubations with uRF, accumulation of trans-11-18:1 only occurred when DHA was added (6.40, 4.35, 1.06, and 3.91 for aRF, aRF+DHA, uRF, and uRF+DHA). The increased amounts of trans-11-18:1 were due to the strong inhibition of the reduction to 18:0 because no 18:0 was formed when trans-11-18:1 accumulated after 24 h. The results of the current experiment shows hydrogenation of trans-11, cis-15-18:2 occurred in the absence of in vitro DHA only, whereas substantial hydrogenation of trans-11-18:1 to 18:0 only took place in incubations without DHA and with unadapted rumen inoculum, confirming the higher sensitivity of the latter process to DHA.     

Functional studies of synthetic gramicidin hybrid ion channels in CHO cells

The function of a gramicidin hybrid ion channel in living Chinese hamster ovary (CHO) cells was investigated by the patch clamp method. The synthetic ion channel 1 consists of two cyclohexyl ether amino acids that link two mini-gramicidin strands. With 1 at a concentration of 1.0 microM, an increase in the whole-cell membrane conductance was observed after 1.37 min. The conductance showed larger currents when Cs(+) was used as charge carrier than when Na(+) and K(+) were used. In single-channel recordings with Cs(+) as charge carrier, the substance showed comparable single-channel amplitudes in the membrane of living cells and artificial black lipid bilayers. In addition to functioning as a cation channel, compound 1 appeared to be a water channel. Exposure of the CHO cells to an extracellular hypoosmotic solution did not substantially change the cell volume. Extracellular hypoosmotic conditions in the presence of 1 increased the cell size to 146.5 % that of the control. Thus, the synthetic hybrid channel 1 can function as a cation channel with some Cs(+) specificity, and as a water channel in CHO cells.     

Effects of feeding different linseed sources on omasal fatty acid flows and fatty acid profiles of plasma and milk fat in lactating dairy cows

The aim of this experiment was to study the effects of feeding different linseed sources on omasal fatty acid (FA) flows, and plasma and milk FA profiles in dairy cows. Four ruminally cannulated lactating Holstein-Friesian cows were assigned to 4 dietary treatments in a 4×4 Latin square design. Dietary treatments consisted of supplementing crushed linseed (CL), extruded whole linseed (EL), formaldehyde-treated linseed oil (FL) and linseed oil in combination with marine algae rich in docosahexaenoic acid (DL). Each period in the Latin square design lasted 21 d, with the first 16 d for adaptation. Omasal flow was estimated by the omasal sampling technique using Cr-EDTA, Yb-acetate, and acid detergent lignin as digesta flow markers. The average DM intake was 20.6 ± 2.5 kg/d, C18:3n-3 intake was 341 ± 51 g/d, and milk yield was 32.0 ± 4.6 kg/d. Milk fat yield was lower for the DL treatment (0.96 kg/d) compared with the other linseed treatments (CL, 1.36 kg/d; EL, 1.49 kg/d; FL, 1.54 kg/d). Omasal flow of C18:3n-3 was higher and C18:3n-3 biohydrogenation was lower for the EL treatment (33.8 g/d; 90.9%) compared with the CL (21.8 g/d; 94.0%), FL (15.5 g/d; 95.4%), and DL (4.6 g/d; 98.5%) treatments, whereas whole-tract digestibility of crude fat was lower for the EL treatment (64.8%) compared with the CL (71.3%), FL (78.5%), and DL (80.4%) treatments. The proportion of C18:3n-3 (g/100 g of FA) was higher for the FL treatment compared with the other treatments in plasma triacylglycerols (FL, 3.60; CL, 1.22; EL, 1.35; DL, 1.12) and milk fat (FL, 3.19; CL, 0.87; EL, 0.83; DL, 0.46). Omasal flow and proportion of C18:0 in plasma and milk fat were lower, whereas omasal flow and proportions of biohydrogenation intermediates in plasma and milk fat were higher for the DL treatment compared with the other linseed treatments. The results demonstrate that feeding EL did not result in a higher C18:3n-3 proportion in plasma and milk fat despite the higher omasal C18:3n-3 flow. This was related to the decreased total-tract digestibility of crude fat. Feeding FL resulted in a higher C18:3n-3 proportion in plasma and milk fat, although the omasal C18:3n-3 flow was similar or lower than for the CL and EL treatment, respectively. Feeding DL inhibited biohydrogenation of trans-11,cis-15-C18:2 to C18:0, as indicated by the increased omasal flows and proportions of biohydrogenation intermediates in plasma and milk fat.     

Prediction of ruminal volatile fatty acid proportions of lactating dairy cows based on milk odd- and branched-chain fatty acid profiles: new models, better predictions

The volatile fatty acids (VFA) produced in the rumen and the proportions in which they are produced are important determinants of a ruminant's metabolism, but their monitoring requires rumen-fistulated animals, which is not feasible under practical conditions or in experimental setups at herd level. An alternative approach was suggested earlier, consisting of predicting the VFA proportions from measured odd- and branched-chain fatty acid concentrations in the milk with a linear model. Here, we have improved this strategy through the development and application of 2 new model structures: the quadratic model, containing quadratic terms and interactions, and the rational model, consisting of a ratio of linear expressions. Both were found to improve prediction accuracy significantly compared with the linear model. Although the quadratic model achieved the best prediction accuracy, the rational model has the interesting property that it takes the dependence of the 3 predicted VFA into account and guarantees that the 3 proportions add up to 1. Adding a study effect to correct for a possible study bias in the multi-study data improved prediction substantially for all 3 methods. Our results demonstrate the potential of using milk odd- and branched-chain fatty acid concentrations to predict rumen VFA proportions.     

Susceptibility of dairy cows to subacute ruminal acidosis is reflected in milk fatty acid proportions,with C18:1 trans-10 as primary and C15:0 and C18:1 trans-11 as secondary indicators

The current study was carried out to assess 2 hypotheses: (1) cows differ in susceptibility to a subacute ruminal acidosis (SARA) challenge, and (2) the milk fatty acid (FA) pattern can be used to differentiate susceptible from nonsusceptible cows. For this, 2 consecutive experiments were performed. During experiment 1, the milk FA pattern was determined on 125 cows fed an increasing amount of concentrate during the first 4 wk in milk (WIM). The coefficient of variation of several SARA indicative milk FA (i.e., C15:0, C18:1 trans-10, C18:2 cis-9,trans-11, and C18:1 trans-10 to C18:1 trans-11 ratio) increased, indicating that cows reacted differently upon the concentrate build-up. A first grouping was based on the milk fat C18:1 trans-10 proportion in the third WIM. Fifteen cows with the highest proportion of the latter FA (HT10) and their counterparts with low C18:1 trans-10 and equal parity distribution (LT10) were compared, which revealed that milk fat content and milk fat to protein ratio were lower for the HT10 group. From each of the HT10 and LT10 groups, 5 animals were selected for experiment 2. The subselection of the HT10 group, referred to as HT10s, showed a high proportion of C18:1 trans-10 at 3 WIM (>0.31 g/100 g of FA), a high level of C15:0 (on average ≥1.18 g/100 g of FA over the 4 WIM), and a sharp decrease of C18:1 trans-11 (Δ ≥ 0.25 g/100 g of FA during the 4 WIM). Their counterparts (LT10s) had a low milk fat C18:1 trans-10 proportion at 3 WIM (<0.23 g/100 g of FA), an average C15:0 proportion of 0.99 g/100 g of FA or lower, and a rather stable C18:1 trans-11 proportion. The HT10s group was hypothesized to be more susceptible to a SARA challenge, achieved by increasing amounts of rapidly fermentable carbohydrates in experiment 2. The HT10s cows had a lower nadir, mean, and maximum reticulo-ruminal pH; longer period of reticulo-ruminal pH below 6.0; and higher daily reticulo-ruminal pH variation compared with LT10s cows. Throughout experiment 2, HT10s and LT10s cows differed in levels of SARA indicative milk FA. Five animals, including one LT10s and 4 HT10s cows, experienced SARA, defined as reticulo-ruminal pH <6.0 for more than 360 min/d. These results indicate that it is possible to distinguish cows with different susceptibility to a SARA challenge within a herd by monitoring the milk FA composition when cows receive the same diet.