Isolation and characterisation of Sri Lankan yeast germplasm and its evaluation for alcohol production

Use of inferior yeast cultures represents one of the reasons for low fermentation efficiencies in Sri Lankan alcohol distilleries that use sugarcane molasses. The present study isolated and characterised yeast strains found in natural environments in Sri Lanka and evaluated their performance under laboratory conditions in an effort to select superior strains for industrial fermentations. Yeasts were characterised based on morphological and physiological features such as sugar fermentation and nitrate assimilation. Ethanol production, alcohol tolerance and growth rate of the most promising strains were monitored following laboratory fermentations of molasses. Over a thousand yeast cultures were collected and screened for fermentative activity and a total of 83 yeast isolates were characterised as higher ethanol producers. Most of these belonged to the genus Saccharomyces. Certain strains produced over 10% (v/v) alcohol in molasses media during 72 h laboratory fermentations. Only two strains, SL-SRI-C-102 and 111, showed an appreciable fermentation efficiency of about 90%. The latter strain produced the highest level of ethanol, 11% (v/v) within a 48 h fermentation and exhibited improved alcohol tolerance when compared with the baker's yeast strains currently used in Sri Lankan alcohol distilleries. This study highlights the benefits of exploiting indigenous yeasts for industrial fermentation processes

Electro-extractive fermentation for efficient biohydrogen production

Electrodialysis, an electrochemical membrane technique, was found to prolong and enhance the production of biohydrogen and purified organic acids via the anaerobic fermentation of glucose by Escherichia coli. Through the design of a model electrodialysis medium using cationic buffer, pH was precisely controlled electrokinetically, i.e. by the regulated extraction of acidic products with coulombic efficiencies of organic acid recovery in the range 50–70% maintained over continuous 30-day experiments. Contrary to\ud previous reports, E. coli produced H2 after aerobic growth in minimal medium without inducers and with a mixture of organic acids dominated by butyrate. The selective separation of organic acids from fermentation provides a potential nitrogen-free carbon source for further biohydrogen production in a parallel photofermentation. A parallel study incorporated this fermentation system into an integrated biohydrogen refinery (IBR) for the conversion of organic waste to hydrogen and energy

Dry anaerobic digestion of organic residues on-farm - a feasibility study

Objectives The feasibility study shall answer the following questions: Are there economical and ecological advantages of on-farm dry digestion biogas plants? How the construction and operation parameters of a dry digestion biogas plant influence environment, profit, and sustainability of on-farm biogas production? The aim of the feasibility study is to provide facts and figures for decision makers in Finland to support the development of the economically and environmentally most promising biogas technology on-farm. The results may encourage on-farm biogas plant manufacturers to develop and market dry anaerobic digestion technology as a complementary technology. This technology may be a competitive alternative for farms using a dry manure chain or even for stockless farms. Results Up to now farm scale dry digestion technology does not offer competitive advantages in biogas production compared to slurry based technology as far as only energy production is concerned. However, the results give an over-view of existing technical solutions of farm-scale dry digestion plants. The results also show that the ideal technical solution is not invented yet. This may be a challenge for farmers and entrepreneurs interested in planning and developing future dry digestion biogas plants on-farm. Development of new dry digestion prototype plants requires appropriate compensation for environmental benefits like closed energy and nutrient circles to improve the economy of biogas production. The prototype in Järna meets the objectives of the project since beside energy a new compost product from the solid fraction was generated. On the other hand the two-phase process consumes much energy and the investment costs are high (>2000 € m-3 reactor volume). Dry digestion on-farm offers the following advantages: Good process stability and reliability, no problems like foam or sedimentation, cheap modules for batch reactors, less reactor capacity, reduced transport costs due to reduced mass transfer in respect of the produced biogas quantity per mass unit, compost of solid digestion residues suitable as fertiliser also outside the farm gate, use of on-farm available technology for filling and discharging the reactor, less process energy for heating because of reduced reactor size, no process energy for stirring, reduced odour emissions, reduced nutrient run off during storage and distribution of residues because there is no liquid mass transfer, suitable for farms using deep litter systems. These advantages are compensated by following constraints: Up to 50% of digestion residues are needed as inoculation material (cattle manure does not need inoculation) requiring more reactor capacity and mixing facilities. Retention time of dry digestion is up to three times longer compared to wet digestion requiring more reactor capacity and more process energy, filling and discharging batch reactors is time and energy consuming. We conclude that only farm specific conditions may be in favour for dry digestion technology. Generally, four factors decide about the economy of biogas production on-farm: Income from waste disposal services, compensation for reduction of greenhouse gas emission, compensation for energy production and - most important for sustainable agriculture - nutrient recycling benefits. Evaluation of the results We did not find any refereed scientific paper that includes a documentation of an on-farm dry digestion biogas plant. It seems that we tried first. We also could not find any results about the biogas potential of oat husks, so we may have found these results first. Farm scale production of anaerobically treated solid manure for composting is new. Dry fermentation biogas plants offer the possibility to design solid manure compost by variation of fermentation process parameters. From different scientific publication databases we found about 10 000 references concerning biogas research during the past 10 years. Less than ten are dealing with biogas reactors for non-liquid substrates on-farm. Recent research mainly concentrates on basic research, biogas process research for communal waste, large-scale biogas plants, and research on laboratory level. This mirrors the fact, that production of research papers is rather financed than product development on site. Our conclusion is that it seems worldwide to be very difficult or even impossible to find financial support for on site research, especially for on-farm prototype biogas reactors. We suppose the following reasons for this fact: biogas plant research requires proficiency in many different scientific disciplines, lack of co-operation between engineering and life sciences, high development costs to transfer basic research results into practical technical solutions, low interest of researchers because on site and on-farm research enjoys low appreciation in terms of scientific credits, portability of farm specific design and process solutions is difficult. Our conclusion is that on site and on-farm research has to be supported by funding agencies if integration of biogas and bio energy into the farm organism is considered as an important target within the agricultural policy framework. Future research on both dry fermentation technique and biogas yield of solid organic residues may close present knowledge gaps. Prototype research may offer competitive alternatives to wet fermentation for farms using a solid manure chain and/or energy crops for biogas production. To encourage farmers and entrepreneurs to foster the development of dry fermentation technology support in terms of education and advisory services is also necessary

A two-stage, two-organism process for biohydrogen from glucose

H2 can potentially be produced in a two-stage biological process: the fermentation of glucose by Escherichia coli HD701 and the photofermentation of the residual medium by Rhodobacter sphaeroides O.U. 001. In a typical batch fermentation, E. coli consumed glucose and produced H2, organic end-products and biomass. Organic end-products and residual glucose were removed during subsequent photofermentation by R. sphaeroides, with associated growth and neutralization of pH. However, photoproduction of H2 did not occur during photofermentation of the residual liquor per se due to the presence of fixed nitrogen compounds. Nevertheless, this two-stage approach could be applied to dispose of sugar-containing industrial wastes, H2 being used for on-site power generation

Electricity-assisted production of caproic acid from grass

Background: Medium chain carboxylic acids, such as caproic acid, are conventionally produced from food materials. Caproic acid can be produced through fermentation by the reverse beta-oxidation of lactic acid, generated from low value lignocellulosic biomass. In situ extraction of caproic acid can be achieved by membrane electrolysis coupled to the fermentation process, allowing recovery by phase separation. Results: Grass was fermented to lactic acid in a leach-bed-type reactor, which was then further converted to caproic acid in a secondary fermenter. The lactic acid concentration was 9.36 +/- 0.95 g L-1 over a 33-day semi-continuous operation, and converted to caproic acid at pH 5.5-6.2, with a concentration of 4.09 +/- 0.54 g L-1 during stable production. The caproic acid product stream was extracted in its anionic form, concentrated and converted to caproic acid by membrane electrolysis, resulting in a >70 wt% purity solution. In a parallel test exploring the upper limits of production rate through cell retention, we achieved the highest reported caproic acid production rate to date from a lignocellulosic biomass (grass, via a coupled process), at 0.99 +/- 0.02 g(-)L(-1) h(-1). The fermenting microbiome (mainly consisting of Clostridium IV and Lactobacillus) was capable of producing a maximum caproic acid concentration of 10.92 +/- 0.62 g L-1 at pH 5.5, at the border of maximum solubility of protonated caproic acid. Conclusions: Grass can be utilized as a substrate to produce caproic acid. The biological intermediary steps were enhanced by separating the steps to focus on the lactic acid intermediary. Notably, the pipeline was almost completely powered through electrical inputs, and thus could potentially be driven from sustainable energy without need for chemical input

Effect of ammonium concentration on alcoholic fermentation kinetics by wine yeasts for high sugar content

Kinetics of alcoholic fermentation by Saccharomyces cerevisiae wine strains in a synthetic medium with high sugar content were established for different nitrogen initial content and are presented for 4 strains. The composition of the medium was close to grape must except that the nitrogen source consisted mainly in ammonium and was varied from 120 to 290 mg N/L assimilable nitrogen. The overall nitrogen consumed was also estimated in order to determine nitrogen requirement variability. The effect of assimilable nitrogen was in general greater on sugar consumption rates than on growth and 3 kinds of effect on sugar consumption rates were observed: i) existence of an optimal initial nitrogen level for a maximal sugar consumption rate (inhibition if excess), ii) no effect of nitrogen beyond the intermediary level (saturation), iii) sugar consumption rate proportional to the initial nitrogen level (activation). In all cases, the amount of consumed nitrogen increased with its initial concentration and so did the fructophilic capacity of the strains. The optimal requirement varied from 0.62 to 0.91 mg N per g of sugars according to the different strains. There was no general correlation between the sugar assimilation rates and the nitrogen requirement

Maximizing efficiency of rumen microbial protein production.

Rumen microbes produce cellular protein inefficiently partly because they do not direct all ATP toward growth. They direct some ATP toward maintenance functions, as long-recognized, but they also direct ATP toward reserve carbohydrate synthesis and energy spilling (futile cycles that dissipate heat). Rumen microbes expend ATP by vacillating between (1) accumulation of reserve carbohydrate after feeding (during carbohydrate excess) and (2) mobilization of that carbohydrate thereafter (during carbohydrate limitation). Protozoa account for most accumulation of reserve carbohydrate, and in competition experiments, protozoa accumulated nearly 35-fold more reserve carbohydrate than bacteria. Some pure cultures of bacteria spill energy, but only recently have mixed rumen communities been recognized as capable of the same. When these communities were dosed glucose in vitro, energy spilling could account for nearly 40% of heat production. We suspect that cycling of glycogen (a major reserve carbohydrate) is a major mechanism of spilling; such cycling has already been observed in single-species cultures of protozoa and bacteria. Interconversions of short-chain fatty acids (SCFA) may also expend ATP and depress efficiency of microbial protein production. These interconversions may involve extensive cycling of intermediates, such as cycling of acetate during butyrate production in certain butyrivibrios. We speculate this cycling may expend ATP directly or indirectly. By further quantifying the impact of reserve carbohydrate accumulation, energy spilling, and SCFA interconversions on growth efficiency, we can improve prediction of microbial protein production and guide efforts to improve efficiency of microbial protein production in the rumen

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

Background: The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. Results: An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. Conclusions: An industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates

Molecular and enological characterization of autochthonous <i>Saccharomyces cerevisiae</i> strains isolated from grape-musts and wines Cannonau

Fermentation by autochthonous yeasts may produce wines with enological properties that are unique of a specific area or variety of grape must. In order to identify yeast starter strains for the production of the Sardinian wine Cannonau DOC, 66 Saccharomyces cerevisiae strains, isolated from musts and wines Cannonau of six vitivinicole areas in Sardinia, were subjected to enological characterization and molecular identification. The RFLP-PCR fingerprinting of the ITS region of rRNA (ITS1-5,8S- ITS2) as well as ethanol, foam, and H2S production were analysed

Quantification of nutrient supply in forage-based diets for beef cattle

End of Project ReportIntroduction Cattle rearing systems in Ireland are predominantly grass-based as 80% of agricultural land is dedicated to grassland (silage, hay and pasture) (CSO, 2007). Feed costs represent the largest single variable cost in beef production in Ireland. Grazed grass is generally the cheapest source of food available for beef (and milk) production provided that the environment and management permit high yields of high quality herbage to be utilised (McGee, 2000). Environmental legislation and the rules of environmental schemes such as the European Union (EU) Rural Environmental Protection Scheme are progressively restricting the application of fertilizer Nitrogen (N), and many grazing systems are becoming more extensive. Over 80% of all farms in Ireland make grass silage (O’Kiely et al., 1998) and it accounts for 87% of total grass conserved (Mayne and O’Kiely, 2005). The deficiencies in nutrient supply to beef cattle from grass silage are usually overcome by supplementing with concentrates (McGee, 2005), which are primarily cereal-based (Drennan et al., 2006). However, diverse types of concentrates containing a variety of feed ingredients, particularly non-cereal by-products are available and frequently fed as supplements to grass silage or as highconcentrate diets. The relatively small amount of information available on feeding these contrasting concentrates to beef cattle is inconsistent. Moreover, there has been an increased use of other ensiled forages such as maize and whole-crop cereals. These forages have high intake potential and can reduce the concentrate feeding level, while maintaining or increasing performance of beef cattle (Keady, 2005). With increasing costs of beef production and increasing constraints of environmental regulations, efficient utilisation of consumed nutrients by cattle is imperative in providing sustainable production and income to farmers. Feed evaluation systems are used to match the dietary nutrient supply with animal requirements for a specific level of production (Dijkstra et al., 2007). These systems are important in order to optimise the efficiency of feed utilisation, to improve animal performance and to reduce nutrient losses to the environment (Dijkstra et al., 2007). Although the reticulo-rumen is central to the profile of nutrients available for absorption, yet quantitative knowledge of the rates of passage and the digestion of nutrients in the rumen is limited compared with that on degradation rates (Dijkstra et al., 2007). There is a lack of information that adequately characterises the supply of nutrients from forages and feedstuffs specific to Ireland, especially for fresh grass-based diets of which, there are very few studies reported in the literature. This shortcoming impedes our ability to capitalise on the merits of evolving feeding systems. This project aimed to: 1. Increase the knowledge and advance the understanding on rumen digestion and nutrient flow from the rumen of the main forages / forage-based diets offered to beef cattle in Ireland. 2. Evaluate strategies for optimal utilization of nutrients consumed by cattle