PSVI-20 Impact of Saccharomyces cerevisiae fermentation products on in vitro rumen microbial fermentation of new crop corn silage

Dairy cows fed silage towards the front edge of a bunker have shown decreased performance often referred to as the new crop corn silage slump. The objective of this study was to evaluate the effect of Saccharomyces cerevisiae fermentation product, NutriTek® (NutriTek), on in vitro volatile fatty acid (VFA) production using new crop corn silage collected at various distances from the front edge of the bunker as substrate. Silage was sampled at 1.8, 3.6 and 7.2 m from the front edge of the bunker by taking the entire face of the silage and mixing. NutriTek was added to each serum bottle relative to the recommended feeding rate of 19 g/d along with 0.3 g of silage as substrate. Each serum bottle was inoculated with 40 mL of buffered rumen fluid from cows on a diet consisting of 70% forage and 30% concentrate and was incubated for 24 h. Ten replicates were run per treatment. After incubation, VFA production was measured. Data were analyzed using the GLM model of JMP. Means were compared using Tukey test and significance was defined as P < 0.05. There was a linear trend of decreasing silage pH as sample depth increased; 4.01 at 1.8 m, 3.71 at 3.6 m, and 3.56 at 7.2 m. Results showed that VFA production was reduced for 1.8 m sample compared to the 3.6 m and 7.2 m samples (Table 1). NutriTek was able to improve acetate, propionate, and total VFA production over Control at all corn silages tested (Table 2). In addition, NutriTek showed the ability to elevate VFA production of the 1.8 m sample to Control samples at 3.6 and 7.2 m. In conclusion, NutriTek could be a management tool to address the performance slump of dairy cows when transitioning to a new crop corn silage.

The Use of Lactic Acid Bacteria as Ruminant Probiotic Candidates Based on In Vitro Rumen Fermentation Characteristics

This research was conducted to select, to identify LAB isolates and to investigate the effects of the LAB as probiotics candidate in the rumen fermentation. Nine isolates exhibited the potency as candidate probiotics for cattle. The experiment was arranged in randomized block design with ten treatments and three different times of in vitro as a block. The substrate consisted of 70% forage and 30% concentrate proportion. The substrate was incubated at 39oC using serum bottle of 100 ml capacity for fermentation. Approximately 0.75 g of substrates was put inside the serum bottle glass and filled with 73 ml of buffered rumen fluid and 2 ml of LAB inoculant. Gas production was measured every 2, 4, 6, 8, 10, 12, 24, 48 and 72 h of the incubation period. Gas production kinetic was estimated by the Ørskov’s equation. The LAB with the highest gas production, as probiotics candidate, were identified using partial 16S rDNA sequence. The results of this research indicated that nine LAB produced high gas production in the range of 193-198 ml compare to that of control (173 ml). The addition of LAB in rumen fermentation resulted in digestibility 65-75%, organic matter digestibility 51-73%, and 6.67-6.68 pH. Based on the molecular identification, 8 isolates are Lactobacillus plantarum and 1 of uncultured bacteria. The LAB strain 32 L. plantarum showed the best for a ruminant probiotic candidate based on the in vitro rumen fermentation characteristic.

Process development for co-digestion of toxic effluents : development of screening procedures

The primary objective of this project was to establish a screening protocol which could be used to access high strength/toxic effluent for toxicity and degradability prior to being disposed in wastewater treatment works. The serum bottle method (materials and method section) is simple, makes use of small glass vials (125 mℓ-volume were used in this research) which do not require any stirring nor feeding device or other engineered tool: a serum bottle is sealed immediately after all components are poured inside and thereafter conducted in a batch mode and occasionally shaken to ensure adequate homogenisation of the components. The only variables which are regularly measured are the volume of biogas produced and gas composition. The two assays, originally developed by Owen et al. (1979) to address the toxicity and the biodegradability have been combined in a single test called AAT, Anaerobic Activity Test, which enables one to assess simultaneously the inhibitory effect on the methanogenic biomass and the biodegradability of the test material as well as the ability of the biomass to adapt to the test material and therefore to overcome the initial inhibition. The screening protocol is illustrated in Annexure A. The protocol consists of a sequence of assays which employ the serum bottle methodology. A first step of the procedure is aimed at rapidly estimating whether the effluent is potentially toxic to the methanogenic biomass and in what concentration. The second step is a more extensive screening, aimed at precisely characterising the toxicity of the effluent, the extent of biodegradation that can be achieved, as well as at establishing whether a potential for adaptation of the biomass exists upon exposure. If the sample passes the screening stage, the same serum bottle method will be used to conduct a series of batch co-digestion experiments aimed at evaluating a convenient volumetric ratio between the test material and the readily biodegradable substrate. Finally, a laboratory-scale codigestion trial could simulate the full-scale process, thus enabling the selection of appropriate operating conditions for the start-up of the full-scale implementation. This the protocol has been used to assess the amenability to be anaerobically (co)digested of four industrial effluents, i.e. size and distillery effluents which are classified as high strength and scour and synthetic dye effluents classified as toxic. From the biodegradability and toxicity assays the following conclusions were drawn. The size and distillery effluent were found to be ii degradable at 32 g COD/ℓ and 16 g COD /ℓ concentrations respectively. Concentrations higher than these stipulated above were found inhibitory. Scour effluent was found to be recalcitrant at all concentration tested and synthetic dye was 100 % degradable at 0.12 g COD/ℓ and lower and highly inhibitory at concentration higher than 1.1 g COD/ℓ. Co-digestion experiment using serum bottle AAT method were undertaken between effluents i.e. size + distillery, size + scour, distillery + synthetic dye in an attempt to verify whether the digestion performance benefits from simultaneous presence of the two substrates. The volumetric ratios between the effluents were 1:1, 1:2, 2:1. The presence of two mixtures in the case of size and distillery had better methane production compared to individual substrate i.e. size or distillery separate. The mixture with volumetric flow rate ratio of 2:1 (size: distillery) was preferable in terms of process performance as it had highest COD removal compared to the other mixtures /ratios and individual substrates. The mixture of size and scour (2:1) had highest degradation percentage compared to other ratios but not high enough to qualify as degradable (less than 50 %). The mixture of distillery and synthetic dye had the same pattern with ratio of 2:1 giving the best COD conversion. The pattern than can be drawn from the degradability of mixtures is: the degradability of mixtures increase with the increasing amount of the most biodegradable compound/effluent in the mixture. Serum bottle results provided the detailed information regarding the safe operating parameters which should be used during the starting point for the larger scale investigation i.e. lab-scale investigations. The lab scale investigations were conducted primarily to validate screening and monitor how the digestion progresses and also to provide data for future project i.e. pilot plant investigation. Other effluents i.e. scour and synthetic dye and their co-digestion mixture were excluded from the lab-scale investigations since they were found to be non- biodegradable i.e. their COD conversion was less the 50 % in the screening protocol. Due to time constrains and other technical difficulties in the laboratory, the co-digestion of size and distillery mixture trials we not conducted on the laboratory scale. Laboratory-scale digestion trials showed that the best organic loading rate for distillery effluent in terms of reactor performance and stability was 1.0g COD/ℓ with efficiency of about 45 %, and for size was 2.0g COD/ℓ with an efficiency of 40 %. The efficiencies obtained in both effluents trials could be greatly improved by acclimation; however these results showed that the digestion of these effluents on the bigger scale is possible.

The effect of moisture content and alkalinity on the anaerobic biodegradation of pit latrine sludge

This study investigated the effect of additional moisture and/or alkalinity on the rate of anaerobic digestion in samples of material obtained from pit latrines. In modified serum bottle tests it was shown that poor gas production rates were observed from all experiments with material collected at the lower part of one pit. Using material from the top layer of a second pit it was shown that experimental bottles produced significant amounts of gas for all treatments. Analysis of data indicated that treatment with additional alkalinity had no discernible effect on anaerobic gas production rates, but that there was some correlation between moisture content and gas production rate. These results did not support the hypothesis that low pH buffering capacity was a limiting factor in the rate of digestion of pit latrine sludge, but confirmed that low moisture content could reduce the rate of stabilisation. This implies that increasing the moisture content in a pit latrine has the potential to increase biological stabilisation rates in the pit when the material is not already well-stabilised.

The effect of moisture content and alkalinity on the anaerobic biodegradation of VIP contents

This study investigated the effect of additional moisture and/or alkalinity on the rate of anaerobic digestion in samples of material obtained from pit latrines. In modified serum bottle tests it was shown that poor gas production rates were observed from all experiments with material collected at the lower part of one pit. Using material from the top layer of a second pit it was shown that experimental bottles produced significant amounts of gas with both the addition of water and the addition of alkalinity. The results supports the motivating hypotheses that moisture content and pH buffering capacity obtained in VIP material are low and may be limiting factors in the rate of stabilisation that may occur in these pits. This implies that increasing the moisture content and alkalinity in the pit has the potential to increase stabilisation rate in the pit when the material is not already well-stabilised.

Anaerobic degradation of 2,4,6-trinitrotoluene in granular activated carbon fluidized bed and batch reactors

In this study, an anaerobic fluidized bed reactor (AFBR) was used to treat a synthetically produced pink water waste stream containing trinitrotoluene (TNT). The synthesized waste consisted of 95 mg/l-TNT, the main contaminant in pink water, which was to be co-metabolized with 560-mg/l ethanol. Granular activated carbon was used as the attachment medium for biological growth. TNT was reduced to a variety of compounds, mainly 2,4,6-triaminotoluene (2,4,6-TAT), 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT), 2,6-diamino-4-nitrotoluene (2,6-DA-4-NT), 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT), and 4-amino-2, 6-dinitrotoluene (4-A-2,6-DNT). These conversions resulted through the oxidation of ethanol to carbon dioxide under anoxic conditions, or reduction to methane under methanogenic conditions. The anaerobic reactor was charged with 1.0 kg of 16×20 U.S. Mesh Granular Activated Carbon (GAC) and was pre-loaded with 200g of TNT prior to the addition of the mixed seed culture. During the first three weeks of operation, ethanol was completely degraded and no methane was produced. Effluent inorganic carbon revealed stoichiometric conversion of the feed ethanol to dissolved inorganic carbon with accumulation of carbon dioxide in the headspace of the reactor. GAC extraction showed incremental reduction of the nitro groups to amino groups, with 2,4,6-TAT as the final product. After three weeks, the oxygen from the nitro groups was depleted and methane production commenced. The reproducibility of this phenomenon was confirmed by repeating the experiment in the same manner using an identical AFBR. Furthermore, serum bottle tests were conducted using TNT loading ratios of 0.2, 0.4, 0.8, 1.0 g-TNT/g-GAC as well as experiments in the absence of GAC. Similar behavior to that of the columns was observed, with degradation rates varying according to the particular condition. GAC greatly enhanced the degradation rates and the higher TNT loading resulted in slower degradation rates of ethanol.

Hydrolysis of (ligno)cellulosic materials under sulfidogenic and methanogenic conditions

A Biochemical methane potential (BMP) test and Serum Bottle Reactor (SBR) test were used to compare hydrolysis (mineralization) of lignocellulosic materials under sulfidogenic and methanogenic conditions. Lignocellulosic carbon mineralization under sulfidogenic conditions was found to be more than 2 times higher than under methanogenic conditions. The percentages of lignocellulosic carbon mineralized under methanogenic condition were 18.0% and 10.71% while under sulfidogenic conditions 36.69% and 27.44% for office paper and newspaper, respectively. Although a poor linear relationship between the percentage of carbon mineralization and percentage lignin content was observed, but in general a decrease in mineralization of lignocellulosic carbon was observed with the increase in lignin content. A method based on selective inhibition of microorganism activity, by 3% toluene, was used to measure the initial rate of lignocellulosic material mineralization and the accumulation of mineralized products (i. e. sugars). Sugars linearly accumulated over time and the accumulation rates of glucose and xylose were calculated. The accumulation rates of glucose under methanogenic condition were 1.302, μM/g-dry wt hr and 0.004, μM/g-dry wt hr while under sulfidogenic condition they were 2.624, μM/g-dry wt hr and 2.279 μM/g-dry wt hr for offce and newspaper, respectively.

Anaerobic Toxicity of Fines in Chemi-Thermomechanical Pulp Wastewaters: A Batch Assay-Reactor Study Comparison

Toxicity of chemi-thermomechanical pulping (CTMP) wastewaters was tested in batch assays and in anaerobic sludge blanket reactors. In batch serum bottle tests particulate constituents were responsible for 80-90% of the inhibition of acetoclastic activity, and the soluble (fines-free) fraction accounted for 10-20%. Performance of two 2-stage anaerobic sludge blanket reactor systems was compared, one receiving fines-free (NF) and the other unaltered (F) CTMP waste for 140 days. Both reactors became acclimatized to CTMP and developed a tolerance to resin acid concentrations as high as 300-1500 mg·1−1 in the sludge bed. The fines-free reactor was superior throughout, but the differences in performance did not reflect the batch test results. At the organic loading rate of 17-22 g soluble COD.g VSS−1·d−1 NF treated 100% CTMP waste with 42% and F with 38% removal efficiency. Fines accumulation in the sludge bed of F reactor caused a 70% increase in bed volume, resulted in deterioration of sludge settleability, and led to an increased likelihood of biomass loss.

Modified Anaerobic Serum Bottle Testing Procedures for Industrial Wastewaters L

Serum bottle bioassay techniques are batch procedures which provide a cost efficient alternative to continuous flow studies for determining anaerobic treatability characteristics of an industrial wastewater. In addition, the toxic effect of the wastewater on the anaerobic microorganisms is easily assessed in a relatively short period of time. As part of a study to screen the anaerobic treatability characteristics of a large number of industrial wastewaters, additional research was carried out to modify the original serum bottle procedures for routine use. Several experiments were completed in which the relative proportions of inoculum, nutrient medium, and wastewater were varied. This permitted assay conditions to be optimized to prevent nutrient deficiencies and excessive lags in inoculum activity, while providing for the testing of a wide range of wastewater concentrations. The effect of changing the total assay volume on the requirement for monitoring biogas accumulation and composition was also investigated.