Bacterial Degradation of Vegetable Oils

Following initial experiments presented elsewhere (2IOPS), the bacterial degradation of two vegetable oils was investigated in some detail. the number of aerobic and anaerobic heterotrophic bacteria, oil degrading and sulphate reducing bacteria were quantified during simulated spills on a salt marsh. the sediment fatty acid composition was also studied using GC-MS analysis. Degradation of linseed and sunflower oils was concomitant with an increase in the numbers of aerobic and anaerobic bacteria. Fatty acids analysis revealed preferential degradation of the principal components of the oils (18:3ω3 for linseed oil and 18:2ω6 for sunflower oil). the presence of several isomers of the usual polyunsaturated fatty acids was also detected. the identification of some of these new fatty acids has been carried out. Possible pathways of degradation of these vegetable oils are suggested.     

Fatty-acid composition of ovulated eggs from wild and cultured turbot (Scophthalmus maximus) in relation to yolk and oil globule lipids

The fatty-acid composition of lipids from ovulated eggs of wild and cultured turbot was investigated in order to estimate the nutritional requirements during embryonic and early larval development. Lipid comprised 13.8±0.5% (n=5) and 13.2±0.7% (n=7) of the egg dry weight in wild and cultured turbot, respectively. Polyunsaturated fatty acids (PUFA) of the (n-3) series accounted for 39% of total fatty acids in total lipid of both wild and cultured fish. The predominant (n-3) FUFA was docosahexaenoic acid (22:6 n-3), which also was the most abundant fatty acid in turbot eggs and comprised 24 and 23% of the total egg fatty acids in wild and cultured fish, respectively. Phospholipids, triacylglycerols and cholesterol-wax esters of turbot eggs all exhibited a specific fatty-acid profile distinctly different from that of total lipid. The general pattern of the fatty-acid distribution in lipids of eggs from wild and cultured turbot was similar, but the relative amount of 18:2(n-6) was considerably higher and 20:1(n-9) slightly higher in cultured fish. These differences were extended to all lipid classes and probably reflect the dietary intake of certain vegetable and marine fish oils. Calculations based on light microscopical studies showed that 55 to 60% of the total lipids in cultured turbot eggs are confined to the oil globule. The size of the oil globule remained constant during embryogenesis, and a reduction in size occurred first after hatching and mainly after yolk depletion. This implies that the total amount of lipids utilised during the embryonic development is considerably less than the total lipids present in ovulated turbot eggs. Comparison of the fatty-acid composition of total lipids from eggs and vitellogenin of wild turbot reveals that egg lipids contained a lower level of saturated and a higher level of monounsaturated fatty acids. Eggs also contained wax esters, which were not detected in vitellogenin, suggesting that vitellogenin is not the only source of lipids for turbot eggs.     

硝化作用微生物的分子生物学研究进展

回顾了硝化作用微生物的分子生物学研究进展 ,主要介绍了硝化作用微生物的种类 ,包括氨氧化菌、亚硝酸氧化菌、异养氨氧化菌和厌氧氨氧化菌 .这些微生物的系统发育分析表明亚硝化菌的系统发育相对简单 ,而硝化细菌则要复杂许多 .还介绍了在硝化作用微生物生态学研究中应用的分子生物学技术 ,如PCR、变性梯度胶电泳 (DGGE)、原位荧光杂交 (FISH)等 ,以及不同类群细菌中与硝化作用相关的酶类及其基因 .文章的最后还提出了一些研究展望 .图 3参 35     

土壤中两种手性有机氯农药的选择性降解研究

为了更深入的了解和阐释手性有机氯农药在土壤中的转化和环境归趋,采用实验室室内避光培养方法,研究了o,p＇-DDT及o,p＇-DDD 2种手性有机氯农药的外消旋体在水稻土厌氧培养体系和菜园土好氧培养体系中的选择性降解情况。为了更好的利用土壤中土著微生物的活性,我们选择了厌氧微生物比较丰富的水稻土和好氧微生物丰度比较高的菜园土来做培养实验。结果表明实验体系中o,p＇-DDT及o,p＇-DDD在水稻土和菜园土中的降解均没有明显的对映体选择性。这一结果与2种手性化合物不同对映体在自然环境中的含量普遍具有差异性有所不同,说明野外环境条件和室内模拟实验条件的差异会影响手性化合物的降解选择性。在2种体系中,o,p＇-DDT的降解速率均高于o,p＇-DDD的降解速率。这与以前的研究报道一致,DDD比DDT更难降解。通过2个体系的比较,发现DDT的降解速率在厌氧体系中高于好氧体系,而DDD的降解速率与之相反,好氧体系高于厌氧体系。这应该与DDT和DDD 2种化合物的化学结构及2种土壤中微生物群落的差异有关。对于厌氧体系中的2种水稻土,采自中山的水稻土中DDT的降解速率高于江门的水稻土,这应该与中山水稻土有机质含量高于江门的水稻土有关。有机质含量的高低直接反映了土壤中微生物的多少,进而会影响污染物的降解速率。研究发现本实验所用水稻土和菜园土总有机碳含量偏低可能是影响2种化合物的对映体无降解选择性的因素之一。此外,由于本研究采用缓冲溶液将pH 调控在中性,因此本文的土壤 pH 对降解选择性的影响仍有待进一步研究。本研究中好氧体系和厌氧体系对目标物的降解选择性无明显差异。     

西南部分地区麻疯树种子油的理化性质及脂肪酸组成分析

从西南6个地区采集了麻疯树种子并分析比较了其生物学性状和种子油的理化性质及脂肪酸组成.所有样品的百粒重范围为41.80~71.39g,出仁率60.02%~81.43%,种仁含油率51.76%~57.93%,种子含油率31.07%~47.17%,不饱和脂肪酸含量74.89%~79.68%.其中永胜产麻疯树的种子品质最好,其出仁率为81.43%,种仁含油率57.93%,种子含油率47.17%,不饱和脂肪酸含量79.68%.攀枝花和宁南产麻疯树的种子品质次之.双柏和罗甸麻疯树的种子油酸值为0.81,在所有样品(0.81~9.46)中最低.对麻疯树种子及种子油性质的相关数据进行方差分析,结果表明,产地不同,麻疯树种子的生物学性质、种子油理化性质和脂肪酸组成都有一些差异.图2表3参18     

Increased docosahexaenoic acid levels in total and polar lipid of European sea bass (Dicentrarchus labrax) postlarvae fed vegetable or animal phospholipids

A 6-week feeding trial was conducted with 44-d-old European sea bass (Dicentrarchus labrax L.) in order to examine the effect of various dietary phospholipid (PL) sources on the incorporation of n-3 highly unsaturated fatty acids (HUFA) in tissue lipids. From weaning onwards the fish received diets prepared by coating different lipid fractions (7.5% diet) on an extruded basal diet (92.5% diet). The two PL-free control diets contained 0.5 and 2% of an emulsifier blend, respectively. Seven other diets contained 2% PL, differing by their purity and origin (vegetable or animal). All diets were rendered isolipidic by the addition of hydrogenated coconut oil. Feeding the PL-supplemented diets, except the diet containing hydrolyzed soybean PL (lyso PL), resulted in a higher survival and a 10 to 30% better growth as compared to the PL-free diets. No difference according to the PL origin was observed. The sea bass final lipid content increased with increasing body weight. Also the lipid class composition of the fish was clearly correlated with the final weight gain. Total neutral lipid increased from 51% of total lipid (initial fish) to 76% for fish fed the PL-free diets, and up to 88% for fish fed the sunflower PL. Weaning the fish on the experimental diets induced important changes in their fatty acid profiles characterized by a decrease in 18:3n-3, 20:5n-3 and 20:4n-6 and an increase in saturated fatty acids and 22:6n-3 (DHA). According to the fatty acid composition of both total and polar lipid, the weaned fish could be divided into three groups reflecting the dietary fatty acids: a group fed the vegetable PL, a group fed the animal PL and a PL-deprived group. An effect of dietary PL on the incorporation of dietary n-3 HUFA, more particularly DHA, was noticed. For a similar supply of DHA through the neutral lipids in the diet, fish fed PL-supplemented diets (except for the lyso PL diet) had 10 to 25% higher DHA levels in total and polar lipid than PL-deprived fish. This PL effect was already clear at the end of the weaning and was not related to the presence of n-3 HUFA in the PL source, as suspected in a previous study when feeding egg yolk PL. A better incorporation of DHA was not obtained by replacing the PL by an emulsifier or by lyso PL with higher emulsifying properties. Present results confirm a role of dietary PL in the absorption of dietary neutral lipids, by a mechanism other than emulsification. Received: 27 May 1997 / Accepted: 30 June 1997     

磺胺嘧啶在水中的微生物降解研究

为了探明磺胺嘧啶在水中的环境行为,通过室内模拟降解实验分别研究了磺胺嘧啶在湖水和猪场废水中的好氧和厌氧微生物降解,考察了供氧方式和有机质含量对磺胺嘧啶微生物降解的影响。结果表明:磺胺嘧啶在猪场废水中厌氧微生物降解速率高于其好氧组,而磺胺嘧啶在湖水中厌氧微生物降解速率低于其好氧组。磺胺嘧啶在湖水和猪场废水中的好氧或厌氧微生物降解均较缓慢,这可能与其较强的抑菌性和微生物的营养状况有关。通过微生物培养还研究了好氧降解时磺胺嘧啶对湖水中微生物种群生长的影响,数据显示:磺胺嘧啶对湖水和猪场废水中细菌的生长具有一定的刺激作用,而对真菌和放线菌的生长影响不明显。     

Collembola gut passage shapes microbial communities in faecal pellets but not viability of dietary algal cells

Microarthropods are known as vectors for soil microorganisms, predominantly fungi. This laboratory study uses the widespread unicellular green algae Chlorella vulgaris as model to assess the role of Collembola in algal dispersal and to determine the effects of gut passage on propagation. Living algal cells were observed in 70 % of the faecal pellets of Folsomia candida, Heteromurus nitidus and Protaphorura fimata. Moreover, marker fatty acids for green algae, i.e. 16:2ω6,9 and 16:3ω3,6,9, were consistently detected in the pellets. Compared to the algal diet, the high content of methyl-branched total lipid fatty acid (TLFA) with hydroxyl substitution indicated microbial colonisation during gut passage. The TLFA profile of faeces revealed no species-specific differences but similar changes in microbial communities over the duration of feeding, indicating comparable indigenous bacteria and colonisation mechanisms during gut passage. In sum, faecal pellets of soil microarthropods such as Collembola can act as a vector for both dietary algae and specific gut-associated microorganisms, with the latter likely involved in resource degradation inside and outside the gut habitat.     

腐熟堆肥接种对蔬菜废物中高温好氧降解过程的影响

通过不同比例腐熟堆肥接种,进行蔬菜废物高温好氧降解试验,研究接种对蔬菜废物好氧降解过程的影响.结果表明:当接种率为5%,3%和1%时,增加腐熟堆肥接种比例有利于提高有机物的好氧降解率,试验末期有机物降解率分别为53.3%,50.0%和43.7%,糖类和纤维素类降解的差别是其降解率差距的主要来源;腐熟堆肥接种对堆肥过程中微生物演化规律影响明显,但接种率达到一定水平后,接种对生物相的影响会随堆肥过程而逐步消失;通过对微生物相演变与生物质降解过程的相关性分析,表明微生物种群增殖与特定生物质类型降解存在明显的相关性,从而提示了采用按堆肥过程中生物质分类的降解状况分段接种微生物的可能性.     

Trophic specialisation of metazoan meiofauna at the Håkon Mosby Mud Volcano: fatty acid biomarker isotope evidence

We report the results of a detailed investigation on the trophoecology of two dominant meiofaunal species at the Håkon Mosby Mud Volcano (HMMV), a deep-sea cold methane-venting seep. Analyses of fatty acids (FAs) and their stable carbon isotopes were used to determine the importance of chemosynthetic nutritional pathways for the dominant copepod species (morphologically very similar to Tisbe wilsoni) inhabiting the volcano’s centre and the abundant nematode Halomonhystera disjuncta from the surrounding microbial mats. The strong dominance of bacterial biomarkers (16:1ω7c, 18:1ω7c and 16:1ω8c) coupled with their individual light carbon isotopes signatures (δ¹³C ranging from ?52 to ?81‰) and the lack of symbiotic relationships with prokaryotes (as revealed by molecular analyses and fluorescent in situ hybridisation) indicated that chemosynthetically derived carbon constitutes the main diet of both species. However, the copepod showed a stronger reliance on the utilisation of methanotrophic bacteria and contained polyunsaturated FAs of bacterial origin (20:5ω3 and 22:6ω3 with isotope signatures δ¹³C < ?80‰). Instead, the FA profiles of H. disjuncta suggested that sulphide-oxidising bacteria constituted the main diet of this nematode. Therefore, HMMV can be regarded as a persistent deep-sea cold seep, allowing a chemosynthesis-based trophic specialisation by the dominant meiofaunal species inhabiting its sediments. The present investigation, through the determination of the fatty acid profiles, provides the first evidence for trophic specialisation of meiofauna associated with sub-habitats within a cold seep.     

Application of heterogeneous catalysis to biodiesel synthesis using microalgae oil

• Microalgae oil application for biodiesel synthesis is discussed. • Catalytic effectiveness of ferment preparations and chemical catalyst is disputed. • Application of heterogeneous catalysts for biodiesel synthesis is reviewed. • Possibilities of catalyst regeneration is shown. Recently, there is a growing interest in the use of microalga in various fields. Microalgae have properties such as rapid reproduction and high biomass accumulation, and under certain conditions, some are able to accumulate a large amount of oil. However, microalgae oil often contains more free fatty acids than the vegetable oil and is therefore unsuitable for biodiesel synthesis using alkaline catalysts. For this reason, some authors suggest the application of heterogeneous catalysis. A particular interest in the use of immobilized enzymes has developed. Other solid substances can also be used as heterogeneous catalysts are usually metal oxides, carbonates or zeolites. The use of these catalysts results in simpler biodiesel synthesis, especially purification processes, a cleaner end product and a less polluted environment. The molar ratio of alcohol to oil is lower during enzymatic transesterification, and more than 90% ester yield is obtained using a molar ratio of alcohol to oil of 3:1 to 4.5:1. The alcohols do not have a negative effect on the effectiveness of chemical catalysts, so it is possible to use alcohols in molar ratio from 4:1 to 12:1. The optimal temperature of enzymatic process is 30℃‒50℃. An ester yield of more than 95% was obtained in 12‒48 h. Using chemical catalysts, greater than a 95% yield of esters was obtained at higher temperatures in a shorter time. Material costs of enzymatic catalysis can be reduced by reusing the catalysts directly or after regeneration.     

Studies on the nutrition of marine flatfish. The effect of dietary fatty acids on the tissue fatty-acids of the plaice Pleuronectes platessa

The ratios of polyunsaturated fatty acids in the lipids of hatchery-reared plaice [Pleuronectes platessa (L.)] maintained on a diet containing corn oil and freeze-dried cod muscle accurately reflect ratios of polyunsaturated fatty acids in the diet. Such plaice have much greater levels of triglycerides in both their livers and extrahepatic tissues than wild plaice of comparable size; the total amount of 22:6 3 acid in triglycerides of the two groups is, however, very similar. Transfer of experimental plaice to a fat-free diet caused a marked depletion of triglycerides from both livers and extrahepatic tissues with almost all fatty acids being reduced in amount. Addition of 12:0 and 14:0 ethyl esters (4% of each) to the fat-free diet caused some accumulation of triglycerides in extrahepatic tissues. Further addition of 18:2 6 and 18:3 3 acids (both at levels of 0.4%) to the diet caused an accumulation of triglycerides in the liver. These triglycerides had increased levels of saturated and monounsaturated fatty acids up to C 18 but the levels of higher polyunsaturated fatty acids were not inereased. There was relatively little change in the fatty acids of phospholipids throughout these experimental dietary regimes. The levels of phospholipids were constant and very similar to those of wild plaice. The results indicate: (1) plaice fed a diet relatively low in 22:6 3 acid may accumulate relatively large amounts of triglyceride in order to store adequate amounts of 22:6 3 acid; (2) plaice are capable of synthesising saturated and mono-unsaturated fatty acids up to C 18 in the presence of dietary 12:0 and 14:0 acids; (3) 18:2 6 and 18:3 3 acids in the diet do not undergo chain elongation and further desaturation in plaice with the dietary regimes we used.     

Effect of Polymeric Coatings On Phytoplankton Standing Crop

Some polymeric marine coatings based on vinylcopolymer and linseed oil have been prepared in the laboratory and their efffect on phytoplankton standing crop was tested. the coating is prepared either from vinylcopolymer or linseed oil or a mixture of both binders with 1:1, 1:2 and 2:1 ratios in the presence or absence of a soluble resin. the coatings were applied to glass slides and tested in a sea water medium. Also, correlations between the weight of the coating film and numbers of phytoplankton species were calculated. the results showed no correlation between the weight of films of either vinylite or oil binders and the total number of all phytoplankton species, but significant correlation was found for some phytoplankton species, such as Thalassiosira decipiens. the weight of the film coating of vinylcopolymer in the presence of soluble resin with 1:1, 1:2 and 2:1 ratios is significantly correlated to the total number of phytoplankton and some other dominant species. Formulations of linseed oil and a soluble resin with 1:1, 1:2 and 2:1 ratios are correlated to the numbers of Chaetoceros affinis, Bellorochea malleus and Skeletonema costatum.     

微生物法在油气勘探中的应用研究

石油天然气的微生物勘探法是通过测定土壤中以微渗漏轻烃为食物来源的微生物菌落来圈定烃类渗漏的范围.本文探讨了甲烷氧化菌、厌氧纤维素分解菌和硫酸盐还原菌在油气勘探中的意义,并将其作为指示微生物,以川西孝泉地区已知天然气田为例,对微生物油气勘查技术的试验效果进行研究.结果发现,在气田上方指示微生物存在明显异常现象,即在气田上方甲烷氧化菌、厌氧纤维素分解菌数量较少,硫酸菌还原菌较高;气田外缘甲烷氧化菌、厌氧纤维素分解菌数量较高,而硫酸菌还原菌较低.研究表明,微生物地表油气勘探技术用于油气预测是有效的.图2参12     

Inter-specific and geographical variations in the fatty acid composition of mangrove leaves: implications for using fatty acids as a taxonomic tool and tracers of organic matter

Fatty acid compositions of the leaves of six species of mangroves were studied to ascertain their use as biomarkers for determining the fate of mangrove organic matter in the habitat and as taxonomic tool. Mangrove leaves were collected from three locations in the western Pacific Ocean: Moreton Bay (MB) (Australia), Hong Kong (China) and Okinawa (Japan). In MB, samples were collected from two sites separated by 15 km: Logan River Estuary (LRE) and Jabiru Island. In addition, along the LRE, leaves were collected from five stations at ∼2–3 km apart. Results show that the analysis of the entire fatty acid profiles of the mangrove leaves is a promising taxonomic tool as the profiles of most species were sufficiently different to be separated in an non-metric multidimensional scaling plot. In addition, geographically separated populations of the same species could also be identified by their fatty acid profiles. In most cases, two non-ubiquitous groups of fatty acids dominated in the mangrove leaves: the polyunsaturated 18:2ω6 and 18:3ω3 and the long chain fatty acids (≥24:0). With respect to the relative contributions of these fatty acids, three groups of species were identified, in which one or both groups of fatty acids may potentially be used as markers of the mangrove organic matter in the estuarine environment.     

Vegetable Oil Spills - Pollution Or Over-Cautiousness?

Vegetable oil spills are relatively uncommon compared to those of mineral oil in the marine environment. Evidence from the spillages that have occurred indicate that they can cause the death of animals in the vicinity through suffocation, inhibition of feeding and other non-specific toxic effects. Smothering of the intertidal area may lead to the development of anoxic conditions with associated changes in species diversity. Polymerisation appears to be a key process in determining the persistence of these oils in the intertidal region. Experiments have indicated that indigenous bacteria are capable of degrading the oil until it polymerises and work is being directed towards determining mechanisms to aid in the degradation process. in conclusion, vegetable oils are pollutants although the risk to the marine environment is small but that does not mean we should not be prepared for such events.     

Carbon substrate usage by zooplankton-associated bacteria,phytoplankton-associated bacteria,and free-living bacteria under aerobic and anaerobic conditions

Mesozooplankton provide oxic and anoxic microhabitats for associated bacteria, whose carbon substrate usage activities complement those of the ambient bacteria. The metabolic profiles of bacterial communities associated with the calanoid copepod Acartia tonsa under aerobic and anaerobic conditions were examined in comparison with phytoplankton-associated bacteria. Carbon substrate usage by phytoplankton-associated bacteria was significantly different than that of copepod-associated bacteria in both aerobic and anaerobic conditions. Substrate utilization by copepod-associated bacteria was more dependent upon oxygen condition than whether the bacteria were located on the copepod exoskeleton or within the gut. Results suggest that gut bacteria were responsible for a large portion of anaerobic substrate usage by copepod-associated bacteria. The metabolic profiles of bacteria associated with six common zooplankton groups and free-living bacteria collected in July 2012 from the York River estuary, Virginia, (37°14′50.36″N, 76°29′58.03W) were also compared, and there were significant differences in their substrate utilization patterns between aerobic and anaerobic incubations, and among the different zooplankton groups. Through trophic interactions, phytoplankton-associated or free-living bacteria may be introduced to the anoxic zooplankton gut and its associated bacterial community. Inclusion of these anaerobic microenvironments and their microbial inhabitants increased the total number of substrates used by 57 % over what was used by aerobic phytoplankton-associated bacteria alone, and by 50 % over what was used by aerobic free-living bacteria in the York River. Therefore, the presence of zooplankton-associated microhabitats and their bacteria expanded the functionality of aquatic microbial communities and led to a more comprehensive substrate usage.     

Acetate incorporation into the lipids of the anemone Anthopleura elegantissima and its associated zooxanthellae

Anthopleura elegantissima containing zooxanthellae, as well as isolated zooxanthellae, incubated with acetate-1-¹⁴C under both light and dark conditions readily incorporate radioactivity into their total lipid pools. In both cases, the specific activity was greatly increased in the light. Dark-incubated anemones and isolated zooxanthellae incorporate activity predominantly into polar lipid; the remainder being present principally in the triglyceride moiety. Light-incubated organisms, however, show a dramatic redistribution of isotope towards greatly increased triglyceride and was ester incorporation, with a concomitant drop in polar lipid. onder light conditions, 70 to 75% of the radioactivity found in the fatty acids of the total zooxanthellae lipid was present in hexadecanoic (16:0) and octadecenoic (18:1) fatty acids. These are also the two major fatty acids by mass. Octadecanoic acid (18:0) is less than 5% by mass. Isotope incorporation patterns suggest that octadecenoic acids arise by elongation of hexadecenoic acids and that this conversion is blocked in the dark. Isotope incorporation patterns for anemones suggest that fatty acids, primarily in the form of saturated or monoenoic fatty acids, are translocated from algal to animal cells. No activity was found in either octadecadienoic (18:2) or octadecatrienoic (18:3) acids. The significance of these data is discussed.     

Nitrifying and denitrifying bacteria in aerobic granules formed in sequencing batch airlift reactors

The purpose of this study was to investigate nitrifying bacteria and denitrifying bacteria isolated from aerobic granules. Aerobic granules were formed in an internal-circulate sequencing batch airlift reactor (SBAR) and biodegradation of NH₃ ^?-N was analyzed in the reactor. Bacteria were isolated and determined from aerobic granules using selected media. The growth properties and morphology of bacteria colonies were observed by controlling aerobic or anaerobic conditions in the culture medium. It was found that bacteria in aerobic granules were diverse and some of them were facultative aerobes. The diversity of bacteria in aerobic granules was a premise of simultaneous nitrification and denitrification.     

Larval growth and settlement of the European oyster (Ostrea edulis) as a function of food quality measured as fatty acid composition

Larval growth rate and settlement of the European flat oyster Ostrea edulis were experimentally studied as a function of the composition of dietary fatty acids. Diets differing in fatty acid composition were composed by mixtures of the microalgae Isochrysis galbana, Pavlova lutheri and Chaetoceros calcitrans. Fatty acid content in the tissue of the feeding larvae, analyzed by gas chromatography and mass spectrometry, reflected the composition in the diet. Larval growth rate was significantly correlated to the three omega-3 polyunsaturated fatty acids (PUFA) C18:3, C18:4 and C22:6, with minor differences for neutral and polar lipids. No relation between growth rate and the omega-3 PUFA C20:5 was detected, a PUFA often implied as essential for bivalves. It is suggested that naturally occurring variability in fatty acid composition may constrain larval growth. In settlement experiments in both still water and flume flow little substrate selectivity was found for some contrasting substrates. It is concluded that differences in dietary fatty acids may explain as much of settlement success as the variability of substrates. Received: 12 October 1998 / Accepted: 6 April 1999