Introduction
Legislatures such as The Renewable Fuel Standard in the U.S.A. and The Renewable Transport Fuel Obligation in the U.K. have driven massive increases in bioethanol production from cereal (first-generation bioethanol). In 2011, the estimated global production was around 113 billion liters (OECD-FAO Agricultural Outlook 2012-2021). The rising volume of bioethanol production also results in increasing quantities of the current coproduct, distiller’s dried grains with solubles (DDGS). Forty-four million tons of DDGS was incorporated into animal feed in 2011; an increase of nearly a quarter on 2010 production (DEFRA 2011).
The use of ethanol fermentation residues as animal feed is not a new concept but while the scale has grown dramatically in recent decades (nearly 200 fold), the technology and the fundamental principles have remained unchanged to a great extent. While in early potable alcohol production, the use of the fermentation residues as animal feed was an incidental benefit to the process, the scale and environmental aims of modern bioethanol production have significantly altered the perception of how the coproducts can be handled and disposed of. Initially, drying the coproduct and marketing it as a feed for livestock was simply a convenient means of removing the excess material from the site of production. A global business has now evolved in marketing coproducts as animal feed, which provides a valuable contribution to the environmental sustainability of both bioethanol and biodiesel production.
The animal feed industry may be loosely divided into two sectors: one sector addressing the requirements of ruminant animals (primarily cattle and sheep) and the other sector addressing nonruminants (primarily fish, pigs, and poultry). The ability of ruminants to digest fiber as an energy source and to utilize nonprotein nitrogen to meet their amino acid requirements means that the fibrous products are predominantly integrated into ruminant diets, while the majority of high protein biorefinery coproducts, such as soya bean meal are directed toward nonruminants. The very high growth rates of commercial strains of pig and poultry render them extremely sensitive to fluctuations in the quality of feed provided and the density of protein and energy in the feed, which limits the inclusion of many coproducts. However, current markets are likely to become saturated as the nutritional quality of DDGS limits its inclusion level in the feed of nonruminant production species.
Production of bioethanol is a mature technology using enzyme liquefaction and saccharification of starch to produce glucose which is then fermented by Saccharomyces cerevisiae yeast to produce ethanol. The leftover mash (whole stillage) is decanted into a fibrous wet grain and a liquid component, thin stillage, which contains the majority of the yeast protein and soluble components. Thin stillage has a very high water content, with only around 60–85 g/kg dry matter (Mustafa et al. 2000), so it is evaporated into a syrup, remixed with the wet grain, and dried to form DDGS. The drying of the coproduct is an energy-demanding, expensive necessity in order remove all the “waste” material not required for the production of ethanol from the distillery which, if not removed, would congest the primary process of ethanol production.
Currently wheat DDGS is successfully used in ruminant nutrition at inclusion levels of between 25% and 35% without affecting nutrient digestibility or growth characteristics of cattle (Li et al. 2011; Yang et al. 2012) but the high fiber content decreases feed intake and limits nutrient utilization in both pigs and chicks (Youssef et al. 2008; McDonnell et al. 2011). While there is potential for incorporating DDGS into cattle feed, the capacity for absorbing bioethanol coproduct into animal feed could be greatly increased through entering the nonruminant sector, as global production of pork (104 million tons) and poultry meat (85 million tons), (USDA Foreign Agricultural Service 2013) is greater than beef production (58 million tons in 2012). If bioethanol fermentation residues cannot be adapted into a coproduct suitable for the larger pig and poultry feed markets, it is likely to saturate the existing beef market and revert to a waste product.
Inclusion of DDGS in animal feed has also been found to deleteriously effect pellet quality; increasing DDGS content has been negatively correlated with pellet durability (Shim et al. 2011) and has been shown to increase the quantity of fines (Loar et al. 2010). Loar et al. (2010) also found that higher DDGS inclusion increased energy use in the condenser due to the viscosity of the mash. Production rate was shown to decrease with 30% DDGS inclusion, which may be due to reduced supplemental rock phosphate, which has a scrubbing effect in the die.
The increases in consumer demand for poultry meat, especially in developing countries will require increasing protein supplies for feed. Beyond the energy component of the diet, protein forms the largest dietary component for pigs, poultry, and fish. The usefulness or quality of a plant protein source for animal feeds depends on three main factors: the volume of protein provided, the availability of that protein to the animal, and the number of antinutritional factors (ANFs) contained with the material. ANFs are components within a plant-derived feed material that invoke a detrimental response within the animal, such as reduced growth or poor excreta quality. Availability of protein depends on how closely the profile of amino acids in the protein source matches the requirements of the growing animal and also how easily each amino acid may be digested. As the digestibility of a protein differs not only between species but also between animals of differing life stages, it is necessary to measure the digestibility of any new feed material in the specific target animal (Kluth and Rodehutscord 2006). The favored method in poultry is to measure the digestibility of each amino acid at the terminal end of the ileum, prior to bacterial fermentation of digesta in the caeca and express the digestible amino acid content as a coefficient of the total amino acid in the protein (Bryden et al. 2009). This is referred to as coefficient of apparent ileal digestibility (CAID); with “apparent” referring to the undetermined potential for endogenous secretions from the bird making a small contribution to the digestible amino acid content.
Yeast has been considered as a protein source in animal feed for many years (Vananuvat 1977), particularly with the drive toward production of more sustainable sources of protein for animal feed. Yeast has been fed successfully to chicks at up to 10% total dietary inclusion (Onol and Yalcin 1995) but higher levels have depressed performance, due to deficiencies in some amino acids and issues with palatability and texture (Caballero-Cordoba and Sgarbieri 2000). In fish, yeast has been used to replace 50% fishmeal, with no significant differences in growth and improved protein conversion (Oliva-Teles and Goncalves 2001). Due to the high ethanol exposure during the process, bioethanol yeast may have a thicker, toughened cell wall, which is more resistant to enzyme proteolysis (Caballero-Cordoba and Sgarbieri 2000).
Yeast (Saccharomyces cerevisiae) is produced in the bioethanol process and is the most valuable component of DDGS. Spencer Martins and van Uden (1977) estimated that 0.071 g yeast is produced for every gram of starch fermented. Thus a 400 million liter bioethanol plant, fermenting approximately 1.1 M tons of wheat per annum would theoretically produce 48,000 tons of yeast, so pooled material from several plants of similar size has the potential to be a very valuable source of supplementary protein. Yeast contains valuable proteins, B vitamins, nucleotides and high inositol and glutamic acid levels (Silva et al. 2010).
Recently, a process has been developed which separates out a protein rich, yeast fraction from the ethanol stillage, which may be more suitable as a feed ingredient for monogastrics than DDGS (Williams et al. 2009). In this method, the traditional process of recombining the fibrous wet grain (whole stillage) with the liquid, yeast containing fraction (thin stillage) heating to drive off excess water is replaced by maintenance of separate product streams. The thin stillage is diverted into a continuous throughput disk stack centrifuge, to produce separate streams containing either the protein rich yeast fraction or a watery syrup containing pentose sugars, while the whole stillage fraction is used to produce a material similar to the traditional DDGS product. This process has been engineered as a modular addition to existing first generation bioethanol plants, so that it may be expeditiously incorporated into both new and existing plants across the globe. The yeast-based product may help improve the credibility of first generation bioethanol by increasing the nutritional value of the coproduct stream. The aim of this study was to characterize a novel protein coproduct stream from bioethanol production and to examine its efficacy as a protein source in poultry diets through determining content of digestible amino acids and subsequent feed conversion ratios (FCR) in broilers.
Material and Methods
Material
For this characterization study, stillage from a wheat-based bioethanol plant operating in France was separated post-distillation in a pilot plant scale facility. A horizontal bowl decanter was used initially to remove the majority of the fiber from the stillage. This separation was controlled by flow rate to obtain a liquid fraction with around 5% dry matter. Spun tests on the separated material showed that this dry matter content did not have substantial fiber contamination. The liquid stream was then dewatered using a continuously operating nozzle centrifuge designed for liquid separation (GEA Westfalia, Oelde, Germany). The solid discharge from this centrifuge was an approximately 40% dry matter yeast cream. The yeast cream was dried to a powder using a commercial spray dryer to produce a fine, free-flowing powder, referred to as a yeast protein concentrate (YPC).
Experimental design
Two broiler chick trials were undertaken: Trial 1 to quantify amino acid-related parameters of YPC and Trial 2 to evaluate bird performance and bone mineral-related parameters. In Trial 1, each protein source (YPC or solvent extracted soya) was fed at graded levels within an otherwise synthetic diet to determine the total content of digestible amino acids in each protein source through linear regression. Trial 2 investigated the effects of direct substitution of YPC for varying proportions of the soya component in a commercially formulated chick starter diet on performance, digesta viscosity, and foot ash. Ethical approval of the trial aims and protocol was sought and gained from the University’s local ethical review body for each trial.
Both trials used day old Ross 308, male broiler chicks, housed in pens of four chicks in a purpose built, poultry house with one pen considered to be one replicate. Temperature, ventilation, and lighting regimes were maintained in line with commercial guidelines and birds were checked at least twice daily to ensure environmental conditions were adequate. Feed and water were available ad libitum at all times.
Trial 1 – Digestible and total amino acid content of YPC
All pens were fed a commercial chick starter crumb from day 1 to day 23 before transferral to experimental diets manufactured in-house. The commercial crumb was wheat–soya based and contained 22% protein and 12.6 MJ/kg of metabolisable energy. Each protein source was fed at three inclusion levels (200, 400, and 600 g/kg diet) to five pens (replicates) per inclusion level as per the format suggested by Short et al. (1999) for determining digestible amino acid content of feed materials in poultry. The two protein sources were incorporated into nutritionally complete mash diets containing vitamins, minerals and oil and titanium dioxide as an inert marker at 5 g/kg, with the remainder made up with an equal mix of dextrose and wheat starch (Table 1). After 3 days of feeding the test diets, the birds were euthanized and digesta collected from the distal end of the small intestine (ileum). Samples were pooled into one pot per pen and frozen at _20°C, prior to freeze drying and grinding.
Samples of digesta and protein sources (YPC and soya) were analyzed for titanium dioxide content by the method of Short et al. (1996). Amino acid content of digesta and protein sources was determined using a Biochrom 30 amino acid analyser based on ion exchange chromatography. Briefly, samples were oxidized with performic acid prior to acid hydrolysis with norleucine added as an internal standard, and then analyzed against prepared standards. The determined apparent digestible amino acid content of each protein source was regressed against rate of inclusion of the protein source and extrapolated to 1000 g/kg to give the apparent digestible content for each amino acid measured. A CAID was then calculated using the total amino acid content of the respective protein source. The two protein sources were also analyzed for protein (Association of Official Analytical Chemists (AOAC) Official method 2001.11 2007), moisture (48 h at 110°C), fat (Association of Official Analytical Chemists (AOAC) Official method 2003.05 2007), and ash (13 h at 650°C). For each amino acid, CAID was compared between the two protein sources using independent sample t-tests. The statistical package used was SPSS v.19 (IBM statistics, Somers, NY).
Trial 2 – Bird Performance trial
Diets were formulated by a commercial feed producer to meet the nutritional needs of the age and strain of birds with the YPC directly replacing solvent-extracted soya at four levels: 0%, 20%, 40%, or 80% of the soya portion of the diet to create diets as shown in Table 2. Each experimental diet was fed to six pens of birds from 0 to 21 days posthatch. Bird performance was evaluated by measuring feed intake, bird weight gain, and (subsequently) FCR on a weekly basis. The birds were weighed on day 1, day 8, day 15, and day 21 posthatch. On day 21, birds were euthanized and digesta was removed from the jejunal section of the gastrointestinal tract and pooled into one pot per pen. The sample was homogenized prior to centrifugation at 5000 rpm for 10 min. A Brookfield DV2 digital cone and plate viscometer at 41°C was used to measure viscosity of the digesta supernatant in duplicate. Finally, the percentage foot ash was determined using the method described by Garcia and Dale (2006). The total foot was removed at the tibial-tarsal joint and dried for 5 days at 105°C, before reweighing and ashing at 650°C for 14 h. Percentage ash was calculated as the ash weight as a percentage of dry weight. All four chicks in a pen were analyzed individually using the left foot of each chick to create a mean value per pen.
After Kolmogorov–Smirnoff (KS) testing to confirm normality, statistical analysis was carried out using one way ANOVA with Duncan post hoc testing to compare the effect of dietary treatment on feed intake, bodyweight gain, FCR, foot ash, and viscosity. The statistical package used was SPSS v19 (IBM statistics).
Pellet quality
Pellets were produced using the commercially formulated chick dietary ingredients containing graded levels of YPC, with the YPC directly replacing oil-extracted soya at four levels: 0%, 37.5%, 62.5%, and 87.5% as shown in Table 3. These diets were pelleted in a small scale commercial facility (Target Feeds, Cheshire, U.K.) to produce 2–3 mm diameter pellets. These pellets were then assessed for quality using three measures. Percentage fines were quantified in five replicate samples by collecting the fines sieved from a weighed quantity of pellets through a 2 mm square hole sieve. Pellet durability index was measured in duplicate by the method of Beyer et al. (2000) with pellets tumbled on a roller mixer for 10 min to mimic handling. Pellet drop tests were carried out using the method of Tumuluru et al. (2010), where single pellets were dropped from 1.85 cm onto a metal plate. Mass retained in the pellet is expressed as a percentage of initial weight and drop tests were repeated 12 times for each diet.
Results
Material proximate analysis
Proximate analysis of YPC compared to soya (Table 4) shows that YPC contains a broadly similar nutrient profile characterized by high dry matter and protein content but notably higher fat and lower mineral content in YPC compared to soya.
Trial 1
The total and apparently digestible amino acid contents of soya and YPC with subsequent CAID are shown in Table 5. While the total amino acid content was numerically higher in YPC than soya for 12 of the 17 amino acids measured, apparently digestible amino acid content was significantly higher in YPC for just five: methionine, glutamate, valine, leucine, and phenylalanine. Conversely, digestible amino acid content of aspartate, arginine and, notably, lysine was significantly higher in soya than YPC. However, CAID was not significantly different between protein sources for any amino acid other than lysine, which was significantly lower in YPC than soya.
Trial 2
Performance
Table 6 shows the effects of replacing increasing portions of dietary soya with YPC on bird performance. There was a significant reduction in body weight gain of 10% or 11%, respectively, in birds fed diets containing 20% or 40% YPC replacement of soya compared to the control diet. Reduction in weight gain was more substantial at the highest rate of YPC inclusion (80% replacement of soya), where it was decreased by 40% compared to the control diet. While weight gain decreased with increasing dietary inclusion of YPC, feed intake was not affected until rate of inclusion reached 80% replacement of soya, when it was significantly reduced. These values translated into numerical but nonsignificant increase (deterioration) in FCR for birds fed diets containing 20% or 40% YPC replacement of soya compared to control and a significant increase in FCR in birds fed diets containing 80% replacement of soya when compared to any other diet.
Viscosity of digesta and skeletal mineral content are determined in Table 7 by quantification of the supernatant of centrifuged digesta and foot ash content, respectively. Digesta supernatant viscosity was significantly increased in birds fed diets containing 40% or 80% YPC as soya replacement compared to either control or 20% YPC replacement of soya. There was no significant difference in foot ash content of birds fed diets containing 20% or 40% YPC replacement of soya compared to control but foot ash content was significantly increased in birds fed 80% YPC as soya replacement.
Pellet quality
The pellet quality measures are all detailed in. The values for pellet durability and pellet drop durability were all very high; although there was significantly lower pellet durability at 87.5% YPC as soya replacement. There were significantly lower fines in the higher two YPC inclusion levels, but all diets had a very low level of fines.
Discussion
The parameters investigated in this study suggest that YPC may be a viable feed material for poultry as it meets the improved physical and nutritional characteristics compared to the traditional bioethanol coproduct, DDGS, that have been cited as necessary to enter the nonruminant feed sector as a partial replacement for soya (Hazzledine et al. 2011).
Pellet quality
Pellet quality is very important for poultry production as pelleted feed with a higher PDI (pellet durability index) is more likely to remain intact during handling prior to feeding. DDGS has been found to deleteriously affect pellet quality; both Shim et al. (2011) and Denstadli et al. (2010) showed that increasing DDGS content was negatively correlated with pellet durability. Although pellet durability in this study was significantly reduced at the highest dietary inclusion level (87.5% YPC replacement of soya), this is unlikely to have practical implications as all values derived are relatively high. Interestingly, the proportion of pellet volume lost to fines generally reduced with increasing dietary inclusion of YPC. It appears from the results in this study that the inclusion of up to 62.5% YPC in place of soya has no negative effect on the durability of the pellet. However, these diets were manufactured in a small scale facility which may not be indicative of the pellet quality from a higher throughput process.
The pellets were also noted to be very hard and durable and it may be that they were over conditioned.
Total amino acid content
Yeast protein concentrate appears to be generally similar to soya in terms of total amino acid content but contains substantially more glutamate. Similarly, high levels of glutamate have also been previously reported in amino acid profiling of yeast-based supplements (Silva et al. 2009). Further comparison of YPC published total amino acid values for wheat and the traditional bioethanol coproduct (wheat DDGS) indicate that YPC is more aligned to material deemed a dietary protein source (soya) than material deemed an energy source (wheat) or DDGS (Bandegan et al. 2009). Also, this study indicates that YPC contains considerably higher levels of indispensable amino acids than wheat DDGS. (Fastinger et al. 2006).This due in part to the increased total protein content: 38% for YPC compared with an average measured protein of 21% for wheat DDGS (Bandegan et al. 2009). It is particularly notable that lysine is 147% higher in YPC than DDGS, as lysine is the first limiting amino acid in DDGS. This difference may be due to Maillard reactions, which have been shown to occur during the drying of DDGS (Pederson and Lindberg 2010) and are known to impair the nutritional content of feed materials and the bioavailability of amino acids (particularly lysine) and proteins. While the observed increase in total amino acid content compared to wheat and DGGS may be predicted, YPC is also higher in total amino acids than previously recorded values for bioethanol yeast (Han and Liu 2010) and is comparable to a commercial purified yeast protein extract (Wang et al. 2009). This may be due in part to the mechanical disruption of the disk stack process, which shears the cell walls of the yeast, making the cell contents more available to the animal for digestion. Without this step, the high ethanol content in bioethanol production has been shown to toughen yeast cell walls making them more resistant to proteolysis (Caballero-Cordoba and Sgarbieri 2000).
Coefficients of apparent ileal digestibility of amino acids
While the total amino acid content of a material provides generic information, the proportion of the amino acid content that is digestible in a given species (CAID) better indicates the value ascribed to a protein feed material. YPC provided significantly more of four indispensable amino acids than soya, including methionine, the first rate limiting amino acid for growing broilers. However, soya provided approximately significantly more of two indispensable amino acids: notably double the volume of apparently digestible lysine, which is the second rate limiting amino acid for growing broilers. Indigestible amino acids are excreted as manure and are a major contributor to nitrogen pollution of land. Therefore, consideration of the CAID is essential in evaluating a feed material. Comparisons to other published values for CAID are compromised by variability in sampling techniques and age of birds used but provide some insight into the relative nutrient value of YPC (Ravindran et al. 2005).
Broiler performance
While digestible amino acid content and CAID of a protein source are key indicators of quality, these measures may not translate directly into improved bird growth and FCR if other attributes of the material invoke a negative response in the animal. The second trial investigated the practical implementation of YPC as a protein source for poultry diets in place of soya and also whether YPC may contribute to dietary available phosphorus. Of all the essential minerals, meeting the dietary requirements for phosphorus is the greatest challenge in poultry nutrition (Skinner and Waldroup 1992), particularly as the majority of phosphorus contained in plant materials is bound up into a ring structure (phytate) which is nutritionally unavailable to nonruminant species, so that costly additional inorganic sources of phosphorus or exogenous enzymes are required to release plant-bound phosphorus (Cowieson et al. 2004). Available phosphorus content is often measured indirectly using bone mineralization: foot ash has been to reflect dietary phosphorus levels (Garcia and Dale 2006). YPC appears to provide a viable substitute for soya but there is a limit to how much may be substituted before negative effects are observed in efficiency of bird performance: FCR was significantly decreased in birds fed the highest YPC inclusion level (80% YPC replacement of oil-extracted soya and bodyweight gain was negatively affected at all YPC inclusion levels. This finding indicates broiler chicks to be more sensitive to dietary inclusion of YPC, as 75% replacement of fishmeal by bioethanol yeast has been reported as optimal for performance of several aquaculture species (Gause and Trushenski 2011a,b). However, other research has shown that while replacing up to 50% of fishmeal protein maintains growth without negative impact on markers of gut health or hepatic function maintaining growth, maximum growth appears at between 15% and 20% replacement of fishmeal (Omar et al. 2012).
The reduction in body weight gain may relate to the fine particle size of the YPC. The YPC used in this study was spray dried, which may affect palatability as spray drying produces a fine, free flowing powder of less than 200 lM which can be difficult for the bird to manipulate, thus increasing feeding time and may also increase digesta viscosity (Yasar 2003). This corresponds with the significant increase in digesta viscosity observed in the current study where birds were fed the two higher YPC inclusion levels. This issue may be resolved as pelleted diets are commonly fed in commercial practice to broilers, as it is generally accepted that high-quality pellets give better performance than low-quality pellets or mash (Amerah et al. 2011). Spray drying is also commercially unattractive due to the relatively high energy requirements of the process compared to ring drying but installation of a ring dryer requires rigorous optimization to ensure variable residence time does not result in product variability issues similar to those found in DDGS production.
Bone mineralization
While this study has focussed on evaluation of YPC as a potential protein source for poultry, YPC also appears able to contribute dietary available phosphorus as foot ash of birds fed diets containing YPC was significantly increased. This suggests that the YPC processing may render phosphorus previously bound up as myo-inositol phosphorus (phytate) in the raw wheat freely available for absorption by poultry.
Global significance
This study gives a strong indication that YPC is a suitable feed source for poultry but only one wheat source has been examined in the study. Different varieties of wheat have been shown to effect DDGS composition (Azarfar et al. 2012) and this may translate to differences in the YPC composition. However, the majority of amino acids present in YPC are derived not from wheat protein itself but derive from a single yeast source; saccharomyces cerevisiae, thereby increasing uniformity of amino acid profile. Nonetheless, processing conditions may also effect coproduct composition (Rausch and Belyea 2006) so further studies are needed to quantify variability of YPC from differing wheat varieties and processing conditions to facilitate its use in feed formulation matrices. Conservative estimates of 4% yeast content in DDGS (Ingledew 1999; Han and Liu 2010) indicate a potential production volume of 140 thousand tons of YPC per annum in the EU and 1.7 million tons in the U.S.A., so further investigation into YPC appears economically warranted.
The current global approach to sustainable agriculture hinges on balancing supply of the 4Fs: feed, fuel, food, and fiber. The incorporation of biorefinery coproducts into animal feeds provides a major conduit for finding balance; excess fiber and feed from production of fuel may be converted into food via animal production. There is also controversy over the environmental credentials of first generation bioethanol which could be mitigated by development of a higher nutritional value coproduct, particularly as much of debate centers around the diversion of cereal crops from animal feed to fuel usage. The narrowing of world ethanol margins is also motivating bioethanol plants to increase efficiency within the plant through energy recycling, and to identify new technologies that will improve profit via multiple high value coproduct streams, rather than relying on profit from bioethanol itself. Product drying is a major contributor to the carbon footprint of the plant. As energy costs rise and the cost of drying increases, alternatives that obviate the need for drying and water removal by remediating water such that it can be reused in the process will become more attractive.
Conclusion
YPC appears to be a viable feed material with the potential to partially replace soya in poultry feed and could be produced in large volumes. This scale of production of sustainable protein could alleviate some of the pressures on other protein sources and mitigate the high proportion of cereal consumed through bioethanol production.
Acknowledgments
Funding for this research was jointly provided by the Engineering and Physical Sciences Research Council and Associated British Agriculture, in the form of a CASE Studentship.
Conflict of Interest
None declared.
This article was originally published in Food and Energy Security 2013; 2(3): 197–206 doi: 10.1002/fes3.30. This is an Open Access article under the terms of the Creative Commons Attribution License.
