Characterization of isolated yeast growth response to methionine analogs

Methionine is one of the first limiting amino acids in poultry nutrition. The use of methionine-rich natural feed ingredients, such as soybean meal or rapeseed meal may lead to negative environmental consequences. Amino acid supplementation leads to reduced use of protein-rich ingredients. The objectives of this study were isolation of potentially high content methionine-containing yeasts, quantification of methionine content in yeasts and their respective growth response to methionine analogs. Minimal medium was used as the selection medium and the isolation medium of methionine-producing yeasts from yeast collection and environmental samples, respectively. Two yeasts previously collected along with six additional strains isolated from Caucasian kefir grains, air-trapped, cantaloupe, and three soil samples could grow on minimal medium. Only two of the newly isolated strains, K1 and C1, grew in minimal medium supplied with either methionine analogs ethionine or norleucine at 0.5% (w/v). Based on large subunit rRNA sequences, these isolated strains were identified as Pichia udriavzevii/Issatchenkia orientalis. P. kudriavzevii/I. orentalis is a generally recognized as a safe organism. In addition, methionine produced by K1 and C1 yeast hydrolysate yielded 1.3 ± 0.01 and 1.1 ± 0.01 mg g^?1 dry cell. Yeast strain K1 may be suitable as a potential source of methionine for dietary supplements in organic poultry feed but may require growth conditions to further increase their methionine content.     

Environmental assessment of the poultry sector of CFPR/TUP programme: experience of BRAC's ultra-poor poultry programme

An environmental assessment of the poultry sector of 'Challenging the Frontiers of Poverty Reduction/Targeting the Ultra Poor Program (CFPR/TUP)' programme of BRAC has been conducted. Special focus was given to health and environmental aspects of poultry rearing, including the housing and diseases of both humans and poultry, the surrounding environment, access to and sources of water, waste management, common property resources, and social conflicts related to the poultry programme. Data were collected through field observations, in-depth interviews, and case studies during August 2003. Poultry rearing was found to be a promising income-generating activity for the ultra poor. A few obstacles related to health, resource use, resource management, and social conflicts were identified. There were some innovative initiatives taken by the TUP poultry members related to waste management, other sources of income and water access. Some steps were suggested to resolve social conflicts and health problems, to improve poultry housing and waste marketing, to increase nutritional intake and initiate intermediate income-generating projects, and to highlight management issues of common property resource.     

Investigation of nitrogen-bearing species in catalytic steam gasification of poultry litter

The production of broiler chickens has become one of the largest sectors in U.S. agriculture, and the growing demand for poultry has led to an annual production growth rate of 5%. With increased demand for poultry, litter management has become a major challenge in the agriculture industry. Although the catalytic steam gasification has been accepted as a possible and feasible method for litter management, concern has been expressed about the presence of nitrogen and phosphorus containing species in the fuel gas and/or in the final solid residue. The possible release of phosphorus as phosphine gas in the fuel gas can have an adverse impact on the environment. Similarly, possible release of ammonia from the nitrogen containing species is also not acceptable. Hence, under partial U.S. Department of Agriculture support, a study was conducted to examine the fate and the environmental impact of the nitrogen- and phosphorus-containing species released during catalytic steam gasification of poultry litter. From various preliminary tests, it was concluded that most (approximately 100%) of the phosphorus would remain in the residue, and some (20-70%) of the nitrogen would end up as ammonia in the fuel gas. The effects of temperature, catalyst loading, and type of catalyst on ammonia liberation were studied in a muffled furnace setup at atmospheric pressure. The fraction of nitrogen released as ammonia was found to decrease with an increase in temperature during pyrolysis and steam gasification. It also decreased with an increase in catalyst loading.     

Anaerobic digestion of livestock and poultry manures spiked with tetracycline antibiotics

Abstract

We investigated the anaerobic degradation of tetracycline antibiotics (tetracycline [TC], oxytetracycline [OTC] and chlortetracycline [CTC]) in swine, cattle, and poultry manures. The manures were anaerobically digested inside polyvinyl chloride batch reactors for 64?days at room temperature. The degradation rate constants and half-lives of the parent tetracyclines were determined following first-order kinetics. For CTC the fastest degradation rate was observed in swine manure (k?=?0.016?±?0.001 d^?1; half-life = 42.8?days), while the slowest degradation rate was observed in poultry litter (k?=?0.0043?±?0.001 d^?1; half-life = 161?days). The half-lives of OTC ranged between 88.9 (cattle manure) and 99.0?days (poultry litter), while TC persisted the longest of the tetracycline antibiotics studied with half-lives ranging from 92.4?days (cattle manure) to 330?days (swine manure). In general, the tetracyclines were found to degrade faster in cattle manure, which had the lowest concentrations of organic matter and metals as compared to swine and poultry manures. Our results demonstrate that tetracycline antibiotics persist in the animal manure after anaerobic digestion, which can potentially lead to emergence and persistence of antibiotic resistant bacteria in the environment when anaerobic digestion byproducts are land applied for crop production.     

Optimal loading rates and economic analyses for anaerobic digestion of poultry waste

Four combinations of litter and carcasses from broiler chickens were examined utilizing a thermophilic, stirred-tank digester of demonstration size of approximately 10,000 gal. Under computed optimal loading rates, litter with paper bedding had the highest daily production of methane over an 8-day retention period. The greatest methane production per lb of volatile solids was achieved over 10 days with litter and paper bedding combined with carcasses. This research found that sufficient poultry litter is generated within 20 mi (32 km) of Moorefield, WV, to support a commercial-sized digester operation. However, anaerobic digestion of poultry waste cannot be financially supported by methane production alone. To be financially viable, anaerobic digestion requires a disposal fee for poultry waste and/or the sale of the digested solid effluent as an organic fertilizer to retail markets.     

Quantification of ionophores in aged poultry litter using liquid chromatography tandem mass spectrometry

Veterinary anticoccidials, biochemically known as ionophores, are widely used in poultry feed at therapeutic levels to treat Coccidiosis and at sub-therapeutic levels for growth- promotion. Commonly used ionophores in the US poultry industry are monensin, salinomycin, lasalocid and narasin. There is an increasing concern regarding the persistence of these anticoccidials in the environment. However, little attention has been directed to methods development for quantitatively measuring ionophores in complex environmental matrices such as poultry litters that are land applied. Here, we describe a rapid and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method developed for simultaneous quantification of monensin, lasalocid, salinomycin, and narasin in aged poultry litter samples. Results show significant level of monensin (97.8 ± 3.2?μg kg(-1)), lasalocid (19.2 ± 6.6?μg kg(-1)), salinomycin (70 ± 2.7?μg kg(-1)) and narasin (57.3 ± 2.6?μg kg(-1)) in poultry litter stored for over three years at < 5°C. Our findings indicate that even after several years of unmanaged storage of poultry litter, ionophores may continue to persist in this matrix, raising the possibility of prolonged release into the environment.     

Food nitrogen footprint reductions related to a balanced Japanese diet

Dietary choices largely affect human-induced reactive nitrogen accumulation in the environment and resultant environmental problems. A nitrogen footprint (NF) is an indicator of how an individual’s consumption patterns impact nitrogen pollution. Here, we examined the impact of changes in the Japanese diet from 1961 to 2011 and the effect of alternative diets (the recommended protein diet, a pescetarian diet, a low-NF food diet, and a balanced Japanese diet) on the food NF. The annual per capita Japanese food NF has increased by 55% as a result of dietary changes since 1961. The 1975 Japanese diet, a balanced omnivorous diet that reportedly delays senescence, with a protein content similar to the current level, reduced the current food NF (15.2 kg N) to 12.6 kg N, which is comparable to the level in the recommended protein diet (12.3 kg N). These findings will help consumers make dietary choices to reduce their impacts on nitrogen pollution.     

Quantification of ionophores in aged poultry litter using liquid chromatography tandem mass spectrometry

Veterinary anticoccidials, biochemically known as ionophores, are widely used in poultry feed at therapeutic levels to treat Coccidiosis and at sub-therapeutic levels for growth- promotion. Commonly used ionophores in the US poultry industry are monensin, salinomycin, lasalocid and narasin. There is an increasing concern regarding the persistence of these anticoccidials in the environment. However, little attention has been directed to methods development for quantitatively measuring ionophores in complex environmental matrices such as poultry litters that are land applied. Here, we describe a rapid and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method developed for simultaneous quantification of monensin, lasalocid, salinomycin, and narasin in aged poultry litter samples. Results show significant level of monensin (97.8 ± 3.2 μg kg^?1), lasalocid (19.2 ± 6.6 μg kg^?1), salinomycin (70 ± 2.7 μg kg^?1) and narasin (57.3 ± 2.6 μg kg^?1) in poultry litter stored for over three years at < 5°C. Our findings indicate that even after several years of unmanaged storage of poultry litter, ionophores may continue to persist in this matrix, raising the possibility of prolonged release into the environment.     

Does monensin in chicken manure from poultry farms pose a threat to soil invertebrates?

Monensin is a carboxylic polyether ionophore used in the poultry industry as a coccidiostat. It enters the environment via manure from broiler farms. In spite of its potential presence in the environment, information concerning monensin residues in manure and soil and its toxicity to soil organisms are insufficient. In the present study, two beneficial soil invertebrate species, earthworms (Eisenia andrei) and woodlice (Porcellio scaber), were used to assess the toxicity of monensin. Animals were exposed to a range of monensin concentrations via soil or food. Earthworm reproduction was found to be the most susceptible endpoint (NOEC=3.5 mg kg(-1) dry soil; EC(50)=12.7 mg kg(-1) dry soil), while no adverse effects were recorded in isopods (NOEC?849mgkg(-1) dry soil, NOEC?357mgkg(-1) dry food). The obtained toxicity data were compared with potential concentrations of monensin in soil. In view of this, manure from broiler chickens treated with monensin at a poultry farm was sampled. According to monensin and nitrogen concentrations in the chicken manure and the degradation time of monensin, the predicted environmental concentration (PEC) was calculated. PEC of monensin is around 0.013 mg kg(-1) soil if manure is used after 3 months of composting and 0.05 mg kg(-1) soil if used without storage. Data for earthworm reproduction was used to estimate the predicted no-effect concentration (PNEC). If fresh chicken manure is applied to terrestrial ecosystems, the risk quotient (PEC/PNEC ratio) is above 1, which indicates that monensin might pose an environmental risk under certain conditions. To prevent this, it is strongly recommended to compost chicken manure for several months before using it as fertiliser.     

Diffusion pollution from livestock and poultry rearing in the Yangtze Delta,China

BACKGROUND: The Yangtze Delta is one of the most developed regions in China and includes Shanghai, eight cities in Jiangsu province and eight cities in Zhejiang province. Meat consumption in this region has increased with economic growth, and most of the consumed meat is produced locally. The water quality of surface waters has deteriorated in recent years. An example was the huge blue-green algae bloom in Tai Lake in late May 2007, which affected millions of people's daily drinking water. However, animal husbandry is considered to be one of the main pollution sources. METHODS: Pollutants (NH3-N, total phosphorus (TP), and total nitrogen (TN)) excreted by livestock and poultry, and the resultant COD (chemical oxygen demand) and BOD (biochemical oxygen demand), were estimated using two different methods based on different data sets. RESULTS: The number of livestock and poultry has remained stable in the Yangtze Delta over the four years from 1999 to 2002, with the average number of pigs, cattle, sheep and poultry being 21.1 M, 0.4 M, 7.7 M and 597.6 M, respectively. Pollutants in livestock and poultry excreta estimated by Method I were: 0.12 Mt NH3-N, 0.11 Mt TP and 0.29 Mt TN, resulting in COD and BOD of 1.34 Mt and 1.30 Mt, respectively, while the estimations based on Method II were: 0.18 Mt NH3-N, 0.15 Mt TP and 0.40 Mt TN, resulting in COD and BOD of 1.95 Mt and 1.80 Mt, respectively. DISCUSSION: Pollutants excreted annually by livestock and poultry in the Yangtze Delta are estimated to be: 0.17 Mt NH3-N, 0.16 Mt TP and 0.42 Mt TN, giving rise to a COD of 1.86 Mt and a BOD of 1.72 Mt. Approximately 25% of this pollution was estimated to enter water bodies, which means that the annual pollutant load is 43,700 t NH3-N, 39,400 tTP, 104,600t TN with a COD of 465,000 tand a BOD of 430,100 t. Pollutants from animal husbandry were similar in magnitude to those from industrial wastewater. Pigs produced the most pollution, followed by poultry, cattle and sheep. The pollution load from animal husbandry in the Yangtze Delta is about twice the average level of the whole of China. CONCLUSIONS: Domestic wastewater was the main pollution source in the Yangtze Delta, followed by pollution from raising livestock and poultry and from industrial wastewater. The pollution load in Shanghai and Jiaxing were the greatest, followed by 7 cities of Jiangsu province (except Suzhou) and other cities of Zhejiang province and Suzhou. Pigs and poultry produced about 90% of the total pollutants from animal husbandry. RECOMMENDATIONS AND PERSPECTIVES: The local governments, especially in Shanghai and Jiaxing, should focus their attention on the pollution produced by livestock and poulrry. Controlling pollution from pigs and poultry will have the greatest impact in this region. Control of pollution will be facilitated by the development of large-scale livestock and poultry farming units and a shift away from small scale husbandry.     

Comparison of arsenic content in pelletized poultry house waste and biosolids fertilizer

Managers of human biosolids have been incorporating the practice of waste pelletization for use as fertilizer since the mid 1920s, and waste pelletization has recently been embraced by some poultry producers as a way to move nutrients away from saturated agricultural land. However, the presence of arsenic in pelletized poultry house waste (PPHW) resulting from the use of organoarsenical antimicrobial drugs in poultry production raises concerns regarding additional incremental population exposures. Arsenic concentrations were determined in PPHW and pelletized biosolids fertilizer (PBF) samples. Pellets were processed using strong acid microwave digestion and analyzed by graphite furnace atomic absorption spectroscopy. The mean arsenic concentration in PPHW (20.1 ppm) fell within the lower part of the range of previously report arsenic concentrations in unpelletized poultry house waste. Arsenic concentrations in PBF, the source of which is less clear than for PPHW, were approximately a factor of 5 times lower than those in PPHW, with a mean concentration of 4.1 ppm. The pelletization and sale of these biological waste fertilizers present new pathways of exposure to arsenic in consumer populations who would otherwise not come into contact with these wastes. Arsenic exposures in humans resulting from use of these fertilizer pellets should be quantified to avoid potential unintended negative consequences of managing wastes through pelletization.     

Spatial variability of ammonium and nitrate in soils near a poultry farm

This paper assesses the distribution of ammonium (NH4+) and nitrate (NO3-) nitrogen deposition in native bushland soil adjacent to an open ventilated poultry farm. The farm is located in Thirlmere 150 km south west of Sydney, Australia. A total of 104 geographically referenced soil cores were obtained from the study area. Soil pH, electrical conductivity (EC), NH4(+) - and NO3(-)-nitrogen concentrations were analysed for variable trends at three depths, i.e. 0-30, 30-60, and 60-90 cm. Significantly higher concentrations of the two nitrogen forms and EC were observed nearer to the farm in the surface soil samples (0-30 cm). The distribution of NH4+, NO3- and EC were all correlated in surface samples throughout the study area. There was no indication of NH4+ and NO3- leaching within soil profile sampled and it appears that weeds and native vegetation had utilised the accumulated nitrogen. The level of N deposition adjacent to the poultry farm decreased downwind due to dispersion of plume and to the buffering effects from the bushland.     

Co-composting of distillery wastes with animal manures: carbon and nitrogen transformations in the evaluation of compost stability

The aim of this work was to study the viability of recycling the solid wastes generated by the winery and distillery industry by means of co-composting with animal manures, as well as to evaluate the quality of the composts obtained. Two piles, using exhausted grape marc and cattle manure or poultry manure, respectively (at ratios, on a fresh weight basis, of 70:30), were composted by the Rutgers static pile composting system. Throughout the composting process, a number of parameters were monitored, such as pH, electrical conductivity, organic matter, water-soluble carbon, water-soluble polyphenols, different forms of nitrogen (organic nitrogen, ammonium and nitrate) and humification indices (humification ratio, humification index, percentage of humic acid-like C, polymerisation ratio and cation exchange capacity), as well as the germination index. Organic matter losses followed first-order kinetics equation in both piles, the highest organic matter mineralisation rate being observed with exhausted grape marc and cow manure. On the other hand, the mixture with the lowest C/N ratio, using exhausted grape marc and poultry manure, showed the highest initial ammonium contents, probably due to the higher and more labile N content of poultry manure. The increase in the cation exchange capacity revealed the organic matter humification during composting. In contrast, other humification parameters, such as the humification ratio and the humification index, did not show the expected evolution and, thus, could not be used to assess compost maturity. Composting produced a degradation of the phytotoxic compounds, such as polyphenols, to give composts without a phytotoxic character. Therefore, composting can be considered as an efficient treatment to recycle this type of wastes, due to composts presented a stable and humified organic matter and without phytotoxic effects, which makes them suitable for their agronomic use.     

Bench-scale biofilter for removing ammonia from poultry house exhaust

A bench-scale biofilter was evaluated for removing ammonia (NH3) from poultry house exhaust. The biofilter system was equipped with a compost filter to remove NH3 and calcium oxide (CaO) filter to remove carbon dioxide (CO2). Removal of NH3 and CO2 from poultry house exhaust could allow treated air with residual heat to be recirculated back into the poultry house to conserve energy during winter months. Apart from its use as a plant nutrient, NH3 removal from poultry house exhaust could lessen the adverse environmental impacts of NH3 emissions. Ammonia and CO2 were measured daily with gas detector tubes while temperatures in the poultry pen and compost filter were monitored to evaluate the thermal impact of the biofilter on treated air. During the first 37 days of the 54-day study, exhaust air from 33 birds housed in a pen was treated in the biofilter; for the final 17 days, NH3-laden exhaust, obtained by applying urea to the empty pen was treated in the biofilter. The biofilter system provided near-complete attenuation of a maximum short-term NH3 concentration of 73 ppm. During the last 17 days, with a mean influent NH3 concentration of 26 ppm, the biofilter provided 97% attenuation. The CaO filter was effective in attenuating CO2. Compared with a biofilter sized only for NH3 removal, an oversized biofilter would be required to provide supplemental heat to the treated air through exothermic biochemical reactions in the compost. The biofilter could conserve energy in poultry production and capture NH3 for use as plant nutrient. Based on this study, a house for 27,000 broilers would require a compost filter with a volume of approximately 34 m3.     

Export of nitrogen from catchments: a worldwide analysis

This study reviews nitrogen export rates from 946 rivers of the world to determine the influence of quantitative (runoff, rainfall, inhabitant density, catchment area, percentage of land use cover, airborne deposition, fertilizer input) and qualitative (dominant type of forest, occurrence of stagnant waterbodies, dominant land use, occurrence of point sources, runoff type) environmental factors on nitrogen fluxes. All fractions (total, nitrate, ammonia, dissolved organic and particulate organic) of nitrogen export showed a left-skewed distribution, which suggests a relatively pristine condition for most systems. Total nitrogen export showed the highest variability whereas total organic nitrogen export comprised the dominant fraction of export. Nitrogen export rates were only weakly explained by our qualitative and quantitative environmental variables. Our study suggests that the consideration of spatial and temporal scales is important for predicting nitrogen export rates using simple and easy-to-get environmental variables. Regionally based modelling approaches prove more useful than global-scale analyses.     

Dephosphorylation and quantification of organic phosphorus in poultry litter by purified phytic-acid high affinity Aspergillus phosphohydrolases

Extracellular phosphohydrolases mediate the dephosphorylation of phosphoesters and influence bioavailability and loss of agricultural P to the environment to pose risks of impairment of sensitive aquatic ecosystems. Induction and culture of five strains of Aspergillus were conducted to develop a source of high-affinity and robust phosphohydrolases for detecting environmental P and quantifying bioactive P pools in heterogeneous environmental specimens. Enzyme stability and activity against organic P in poultry litter were evaluated in 71 samples collected across poultry producing regions of Arkansas, Maryland, and Oklahoma of the US Differences existed in strains' adaptability to fermentation medium as they showed a wide range of phytate-degrading activity. Phosphohydrolases from Aspergillus ficuum had highest activity when the strain was cultured on a primarily chemical medium, compared to Aspergillus oryzae which preferred a wheat bran-based organic medium. Kinetics parameters of A. ficuum enzymes (K(m)=210 microM; V(max) of 407 nmol s(-1)) indicated phytic acid-degrading potential equivalent to that of commercial preparations. Purified A. ficuum phosphohydrolases effectively quantified litter bioactive P pools, showing that organic P occurred at an average of 54 (+/-14)% of total P, compared to inorganic phosphates, which averaged 41 (+/-12)%. Litter management and land application options must consider the high water-extractable and organic P concentrations and the biological availability of the organic enzyme-labile P pool. Robustness of A. ficuum enzymes and simplicity of the in situ ligand-based enzyme assay may thus increase routine assessment of litter bioactive P composition to sense for on-farm accumulation of such environmentally-sensitive P forms.     

畜禽养殖业清洁生产的必要性及实施对策研究--以浙江省为例

集约化畜禽养殖业的发展在提高人民生活水平的同时也给环境带来了巨大的压力。清洁生产是基于污染预防的一种环境保护的战略措施,它体现了废弃物的减量化、资源化、无害化原则。实施畜禽养殖业清洁生产是保证畜禽养殖业健康可持续发展的根本途径。分析了畜禽养殖业实施清洁生产的必要性．并根据当前畜禽养殖业清洁生产进展中存在的问题,提出了相应的对策。     

家禽养殖场废弃物处理技术的研究进展

家禽养殖场的废弃物长期以来对环境构成了极大的污染并威胁着畜禽和人类的健康 ,因而对家禽废弃物进行资源化处理势在必行。综述了近 10年来的 3种主要的可供选择的家禽垃圾处理的技术路线的研究进展。介绍了最优化堆肥、厌氧发酵和直接焚烧的试验研究结果     

Fate of arsenic compounds in poultry litter upon land application

The use of the organic As compound, roxarsone, as an antibiotic additive to poultry feed continues to raise concern over potential negative environmental impacts. Total As concentration in poultry litter can reach >40 mg kg⁻¹ and both roxarsone and its mineralization product As(V) have been identified in poultry litters (PL). To investigate the fate of these As species upon land application of PL we conducted two studies. In the first, an Orangeburg soil (Ultisol from the Atlantic Coastal Plain) was spiked with either 20 mg kg⁻¹ As(V) or roxarsone and incubated at 10% moisture content for 4 months. Exchangeable As was determined periodically by extraction with 0.1 M PO₄. Both As(V) and roxarsone displayed similar desorption; initially, approximately 70% of added As was ligand exchangeable and this decreased to 35% after 4 months incubation, presumably due to either slow sorption reactions or a change in solid phase speciation of As to less exchangeable forms. In the second study, various manipulations of two PL samples were applied to the Orangeburg soil at realistic field application rates. The treatments were wet to 10% moisture content and water soluble As, Cu and organic carbon (DOC) was measured over 30 days. Arsenic and Cu solubility were highest from the dried litter samples. Ashing of the PLs decreased soluble As and Cu, presumably because of the loss of organic matter from the ashed litter and subsequent decrease in DOC. Application of leachates from either PL resulted in higher concentrations of soluble As and Cu than when the soil was amended with equivalent concentrations of soluble As and Cu dissolved in DI H₂O. We hypothesize that the increased levels of DOC from the PL treatments enhance As and Cu solubility through competitive sorption and complexation, respectively. In fact, As and Cu solubility was correlated to DOC levels in the amended soil extracts. Even though land application of PL introduced relatively low concentrations of As and Cu to soil it appeared that other soluble constituents of PL significantly enhanced As and Cu solubility.     

Spatial relationships between nitrogen status and pitch canker disease in slash pine planted adjacent to a poultry operation

Pitch canker disease (Fusarium circinatum Nirenberg & O'Donnell) causes serious shoot dieback, reduced growth and mortality in pines found in the southern and western USA, and has been linked to nutrient imbalances. Poultry houses with forced-air ventilation systems produce nitrogen (N) emissions. This study analyzed spatial correlations between pitch canker disease and foliar, forest floor, soil, and throughfall N in a slash pine (Pinus elliottii var. elliottii Engelm.) plantation adjacent to a poultry operation in north Florida, USA. Tissue and throughfall N concentrations were highest near the poultry houses and remained elevated for 400 m. Disease incidence ranged from 57-71% near the poultry houses and was spatially correlated with N levels. Similarly, stem mortality ranged from 41-53% in the most heavily impacted area, and declined to 0-9% at distances greater than 400 m. These results suggest that nutritional processes exacerbate changes in disease susceptibility and expression in slash pine.