Isolation of a thermotolerant bacterium producing medium-chain-length polyhydroxyalkanoate

Aims:  The aim of this study was to isolate a thermotolerant micro‐organism that produces polyhydroxyalkanoates (PHAs) composed of medium‐chain‐length (mcl) HA units from a biodiesel fuel (BDF) by‐product as a carbon source. Methods and Results:  We successfully isolated a thermotolerant micro‐organism, strain SG4502, capable to accumulate mcl‐PHA from a BDF by‐product as a carbon source at a cultivation temperature of 45°C. The strain could also produce mcl‐PHA from acetate, octanoate and dodecanoate as sole carbon sources at cultivation temperatures up to 55°C. Taxonomic studies and 16S rRNA gene sequence analysis revealed that strain SG4502 was phylogenetically affiliated with species of the genus Pseudomonas. This study is the first report of PHA synthesis by a thermotolerant Pseudomonas. Conclusions:  A novel thermotolerant bacterium capable to accumulate mcl‐PHA from a BDF by‐product was successfully isolated. Significance and Impact of the Study:  A major issue regarding industrial production of microbial PHAs is their much higher production cost compared with conventional petrochemical‐based plastic materials. Especially significant are the cost of a fermentative substrate and the running cost to maintain a temperature suitable for microbial growth. Thus, strain SG4502, isolated in this study, which assimilates BDF by‐product and produces PHA at high temperature, would be very useful for practical application in industry.     

Isolation and characterization of Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent

We examined a single, non-spore-forming, aerobic, thermophilic strain that was isolated from a deep-sea hydrothermal vent in the Guaymas Basin at a depth of 2000 m and initially placed in a phenetic group with Thermus scotoductus (X-1). We identified this deep-sea isolate as a new strain belonging to Thermus thermophilus using several parameters. DNA–DNA hybridization under stringent conditions showed 74% similarity between the deep-sea isolate and T. thermophilus HB-8^T (T = type strain). Phenotypic characteristics, such as the utilization of carbon sources, hydrolysis of different compounds, and antibiotic sensitivity were identical in the two strains. The polar lipids composition showed that strain Gy1211 belonged to the genus Thermus. The fatty acids composition indicated that this strain was related to the marine T. thermophilus strain isolated from the Azores. The new isolate T. thermophilus strain Gy1211 grew optimally at 75°C, pH 8.0, and 2% NaCl. A hydrostatic pressure of 20 MPa, similar to the in situ hydrostatic pressure of the deep-sea vent from which the strain was isolated, had no effect on growth. Strain HB-8^T, however, showed slower growth under these conditions. Received: November 26, 1997 / Accepted: May 20, 1999     

A Novel,Highly Viscous Polysaccharide Excreted by an <Emphasis Type="Italic">Alteromonas</Emphasis> Isolated from a Deep-Sea Hydrothermal Vent Shrimp

A deep-sea, mesophilic, aerobic, and heterotrophic microorganism, able to produce an extracellular polysaccharide, was isolated from a shrimp collected near an active hydrothermal vent of the Mid-Atlantic Ridge. On the basis of phenotypic and phylogenetic analyses and DNA/DNA relatedness, this strain could be assigned to the species Alteromonas macleodii as a variant of the fijiensis subspecies. It was selected for its ability to exhibit a swarming mucoid phenotype on specific media. The bacterium secreted, under laboratory conditions, an extremely viscous exopolysaccharide consisting of glucose, galactose as neutral sugars, and glucuronic, galacturonic acids as uronic acids, along with pyruvate and acetate as main substituents. Received: 10 July 2002 / Accepted: 13 August 2002     

Effects of Dissolved Sulfide, pH, and Temperature on Growth and Survival of Marine Hyperthermophilic Archaea

The ability of metabolically diverse hyperthermophilic archaea to withstand high temperatures, low pHs, high sulfide concentrations, and the absence of carbon and energy sources was investigated. Close relatives of our study organisms, Methanocaldococcus jannaschii, Archaeoglobus profundus, Thermococcus fumicolans, and Pyrococcus sp. strain GB-D, are commonly found in hydrothermal vent chimney walls and hot sediments and possibly deeper in the subsurface, where highly dynamic hydrothermal flow patterns and steep chemical and temperature gradients provide an ever-changing mosaic of microhabitats. These organisms (with the possible exception of Pyrococcus strain GB-D) tolerated greater extremes of low pH, high sulfide concentration, and high temperature when actively growing and metabolizing than when starved of carbon sources and electron donors/acceptors. Therefore these organisms must be actively metabolizing in the hydrothermal vent chimneys, sediments, and subsurface in order to withstand at least 24 h of exposure to extremes of pH, sulfide, and temperature that occur in these environments.     

In situ electrosynthetic bacterial growth using electricity generated by a deep-sea hydrothermal vent

Electroautotrophic microorganisms have attracted great attention since they exhibit a new type of primary production. Here, in situ electrochemical cultivation was conducted using the naturally occurring electromotive forces at a deep-sea hydrothermal vent. The voltage and current generation originating from the resulting microbial activity was observed for 12 days of deployment, with fluctuation in response to tidal cycles. A novel bacterium belonging to the genus Thiomicrorhabdus dominated the microbial community specifically enriched on the cathode. Metagenomic analysis provided the draft genome of the bacterium and the gene repertoire indicated that the bacterium has the potential for thio-autotrophic growth, which is a typical physiological feature of the members of the genus, while the bacterium had a unique gene cluster encoding multi-heme cytochrome c proteins responsible for extracellular electron transfer. Herein, we propose this bacterium as a new species, specifically enriched during electricity generation, as ‘Candidatus Thiomicrorhabdus electrophagus’. This finding suggests the natural occurrence of electrosynthetic microbial populations using the geoelectricity in deep-sea hydrothermal environments.Subject terms: Soil microbiology, Biogeochemistry     

A novel mcl PHA-producing bacterium, Pseudomonas guezennei sp. nov., isolated from a 'kopara' mat located in Rangiroa, an atoll of French Polynesia

Aims: The aim of the present study was to describe an aerobic, mesophilic and heterotrophic bacterium, designated RA26, able to produce a medium‐chain‐length polyhydroxyalkanoate (PHA). It was isolated from a French Polynesian bacterial mat located in the atoll of Rangiroa. Methods and Results: This micro‐organism, on the basis of the phenotypical features and genotypic investigations can be clearly assigned to the Pseudomonas genus and the name of Pseudomonas guezennei is proposed. Optimal growth occurs between 33 and 37°C, at a pH between 6·4 and 7·1 and at ionic strength of 15 g l^?1 of sea salts. The G+C content of DNA is 63·2%. Under laboratory conditions, this bacterium produced a novel, medium‐chain‐length PHA, mainly composed of 3‐hydroxydecanaote (64 mol.%) and 3‐hydroxyoctanoate (24 mol.%) (GC‐MS, NMR) from a single nonrelated carbon substrate, i.e. glucose. Conclusions: The bacterium P. guezennei produces a novel PHA mcl with elastomeric properties. Significance and Impact of the Study: PHAs share physical and material properties that recommend them for application in various areas, and are considered as an alternative to nonbiodegradable plastics produced from fossil oils. In this study, we describe a new bacteria with the capability to synthesize a novel PHA with promising biotechnological applications.     

Insights into the functional role of fungi in deep-sea hydrothermal vents through the analysis of stable isotopes of carbon,nitrogen, and sulfur

The functional diversity of fungi remains poorly explored in the deep-sea, particularly in hydrothermal vents. Here, we approached this gap through the analysis of stable isotopes of carbon (δ¹³C), nitrogen (δ¹⁵N), and sulfur (δ³⁴S) of fourteen isolates obtained from three deep-sea vent systems of the southern Gulf of California. The δ¹³C results indicated that 60% of the isolates relied on mixed carbon sources fixed by the Calvin-Benson-Bassham and the reductive Tricarboxylic Acid (rTCA) cycles, whereas 40% relied exclusively on rTCA carbon. The δ¹⁵N and δ³⁴S values suggested a dependence on local and external nitrogen sources and the assimilation of chemosynthetic and photosynthetic inputs. Fungal δ¹³C and δ¹⁵N overlapped with those of primary and secondary vent macroconsumers, implying the assimilation of bacterial and invertebrate necromass and their ecological role as parasites. These findings provide insights into the unexplored trophic versatility of fungi in chemosynthetic ecosystems, highlighting their importance in deep-sea trophic dynamics.     

Growth and Phylogenetic Properties of Novel Bacteria Belonging to the Epsilon Subdivision of the Proteobacteria Enriched from Alvinella pompejana and Deep-Sea Hydrothermal Vents

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45°C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.     

Zobellella denitrificans strain MW1, a newly isolated bacterium suitable for poly(3-hydroxybutyrate) production from glycerol

Aims: To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol. Methods and Results: Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram‐negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98·5% and 96·2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41°C and pH 7·3 and accumulated PHB up to 80·4% (w/w) of cell dry weight. PHB accumulation was growth‐associated. Although it was not an absolute requirement, 20 g l^?1 sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol. Conclusions: A new PHB‐accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry. Significance and Impact of the Study: This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.     

The biodegradation of poly-3-hydroxyalkanoates,PHAs, with long alkyl substitutents byPseudomonas maculicola

Utilizing a quantitative clear zone technique, the activity of an extracellular depolymerase system fromPseudomonas maculicola was investigated. Polymer degradation was influenced by the amount and availability of secondary carbon sources, with a simultaneous utilization of both sources. The initial carbon source in the liquid preculture also affected the eventual colony growth and polymer degradation. The enzyme solution was determined to readily degrade poly-3-hydroxyalkanoates (PHAs) with relatively long alkyl substituents at the 3 position: poly-3-hydroxyoctanoate (PHO), poly-3-hydroxynonanoate (PHN), and their copolymers (P[HO-co-HN]) and poly-3-hydroxyundecanoate (PHU). However, the system was unable to degrade either PHAs with shorter alkyl groups, including poly-3-hydroxybutyrate (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-co-HV]) or PHAs with unusual substituents such as poly(3-hydroxy-5-phenylvaleric acid) (PHPV). It is proposed that degradation of these more bulky side chain polymers was prevented by the inability of the bacteria to assimilate their monomeric components, which inhibited the successful utilization of secondary carbon sources and thus inhibited colony growth.     

Bacteria dominate the ammonia-oxidizing community in a hydrothermal vent site at the Mid-Atlantic Ridge of the South Atlantic Ocean

Ammonia oxidation is the first and rate-limiting step of nitrification, which is carried out by two groups of microorganisms: ammonia-oxidizing bacteria (AOB) and the recently discovered ammonia-oxidizing archaea (AOA). In this study, diversity and abundance of AOB and AOA were investigated in five rock samples from a deep-sea hydrothermal vent site at the Mid-Atlantic Ridge (MAR) of the South Atlantic Ocean. Both bacterial and archaeal ammonia monooxygenase subunit A (amoA) gene sequences obtained in this study were closely related to the sequences retrieved from deep-sea environments, indicating that AOB and AOA in this hydrothermal vent site showed typical deep ocean features. AOA were more diverse but less abundant than AOB. The ratios of AOA/AOB amoA gene abundance ranged from 1/3893 to 1/242 in all investigate samples, indicating that bacteria may be the major members responding to the aerobic ammonia oxidation in this hydrothermal vent site. Furthermore, diversity and abundance of AOA and AOB were significantly correlated with the contents of total nitrogen and total sulfur in investigated samples, suggesting that these two environmental factors exert strong influences on distribution of ammonia oxidizers in deep-sea hydrothermal vent environment.     

南大西洋深海热液区可培养硫氧化微生物多样性及其硫氧化特性

【目的】探索南大西洋热液环境中的硫氧化细菌多样性并研究其硫氧化特性。【方法】通过富集培养和分离纯化获得硫氧化细菌,利用变性梯度凝胶电泳(DGGE)分析富集菌群组成结构,采用离子色谱法对获得的硫氧化细菌硫氧化特性进行检测。【结果】从南大西洋深海环境样品中共分离到48株菌,分属于alpha-Proteobacteria(28株,58.3%)、Actinobacteria(11株,22.9%)和gama-Proteobacteria(9株,18.8%)共3个门,其中Thalassospira、Martelella和Microbacterium为优势属。DGGE结果表明深海热液环境样品中微生物多样性丰富且不同站位存在差异。硫氧化特性研究结果表明,约60%的分离菌株具有硫氧化能力,可以氧化S_2O_3~(2–)生成SO_4~(2–)。获得一株硫氧化能力较强的潜在新种L6M1-5,在实验条件下可高效氧化S_2O_3~(2–),最大氧化速率可达0.56 mmol/(L·h)。【结论】南大西洋深海热液环境中可培养硫氧化细菌多样性丰富,为研究热液环境中的硫循环过程提供了实验材料和理论参考;同时高效硫氧化菌的获得,为工业化含硫废水的处理提供了良好的菌种资源。     

Phylogenetic Diversity of Nitrogenase (nifH) Genes in Deep-Sea and Hydrothermal Vent Environments of the Juan de Fuca Ridge

The subseafloor microbial habitat associated with typical unsedimented mid-ocean-ridge hydrothermal vent ecosystems may be limited by the availability of fixed nitrogen, inferred by the low ammonium and nitrate concentrations measured in diffuse hydrothermal fluid. Dissolved N₂ gas, the largest reservoir of nitrogen in the ocean, is abundant in deep-sea and hydrothermal vent fluid. In order to test the hypothesis that biological nitrogen fixation plays an important role in nitrogen cycling in the subseafloor associated with unsedimented hydrothermal vents, degenerate PCR primers were designed to amplify the nitrogenase iron protein gene nifH from hydrothermal vent fluid. A total of 120 nifH sequences were obtained from four samples: a nitrogen-poor diffuse vent named marker 33 on Axial Volcano, sampled twice over a period of 1 year as its temperature decreased; a nitrogen-rich diffuse vent near Puffer on Endeavour Segment; and deep seawater with no detectable hydrothermal plume signal. Subseafloor nifH genes from marker 33 and Puffer are related to anaerobic clostridia and sulfate reducers. Other nifH genes unique to the vent samples include proteobacteria and divergent Archaea. All of the nifH genes from the deep-seawater sample are most closely related to the thermophilic, anaerobic archaeon Methanococcus thermolithotrophicus (77 to 83% amino acid similarity). These results provide the first genetic evidence of potential nitrogen fixers in hydrothermal vent environments and indicate that at least two sources contribute to the diverse assemblage of nifH genes detected in hydrothermal vent fluid: nifH genes from an anaerobic, hot subseafloor and nifH genes from cold, oxygenated deep seawater.     

Geochemical and microbiological evidence for a hydrogen-based,hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) beneath an active deep-sea hydrothermal field

Subsurface microbial communities supported by geologically and abiologically derived hydrogen and carbon dioxide from the Earths interior are of great interest, not only with regard to the nature of primitive life on Earth, but as potential analogs for extraterrestrial life. Here, for the first time, we present geochemical and microbiological evidence pointing to the existence of hyperthermophilic subsurface lithoautotrophic microbial ecosystem (HyperSLiME) dominated by hyperthermophilic methanogens beneath an active deep-sea hydrothermal field in the Central Indian Ridge. Geochemical and isotopic analyses of gaseous components in the hydrothermal fluids revealed heterogeneity of both concentration and carbon isotopic compositions of methane between the main hydrothermal vent (0.08 mM and –13.8 PDB, respectively) and the adjacent divergent vent site (0.2 mM and –18.5 PDB, respectively), representing potential subsurface microbial methanogenesis, at least in the divergent vent emitting more ¹³C-depleted methane. Extremely high abundance of magmatic energy sources such as hydrogen (2.5 mM) in the fluids also encourages a hydrogen-based, lithoautotrophic microbial activity. Both cultivation and cultivation-independent molecular analyses suggested the predominance of Methanococcales members in the superheated hydrothermal emissions and chimney interiors along with the other major microbial components of Thermococcales members. These results imply that a HyperSLiME, consisting of methanogens and fermenters, occurs in this tectonically active subsurface zone, strongly supporting the existence of hydrogen-driven subsurface microbial communities.     

Deep-sea hydrothermal vent Epsilonproteobacteria encode a conserved and widespread nitrate reduction pathway (Nap)

Despite the frequent isolation of nitrate-respiring Epsilonproteobacteria from deep-sea hydrothermal vents, the genes coding for the nitrate reduction pathway in these organisms have not been investigated in depth. In this study we have shown that the gene cluster coding for the periplasmic nitrate reductase complex (nap) is highly conserved in chemolithoautotrophic, nitrate-reducing Epsilonproteobacteria from deep-sea hydrothermal vents. Furthermore, we have shown that the napA gene is expressed in pure cultures of vent Epsilonproteobacteria and it is highly conserved in microbial communities collected from deep-sea vents characterized by different temperature and redox regimes. The diversity of nitrate-reducing Epsilonproteobacteria was found to be higher in moderate temperature, diffuse flow vents than in high temperature black smokers or in low temperatures, substrate-associated communities. As NapA has a high affinity for nitrate compared with the membrane-bound enzyme, its occurrence in vent Epsilonproteobacteria may represent an adaptation of these organisms to the low nitrate concentrations typically found in vent fluids. Taken together, our findings indicate that nitrate reduction is widespread in vent Epsilonproteobacteria and provide insight on alternative energy metabolism in vent microorganisms. The occurrence of the nap cluster in vent, commensal and pathogenic Epsilonproteobacteria suggests that the ability of these bacteria to respire nitrate is important in habitats as different as the deep-sea vents and the human body.     

Metabolism of the thermophilic bacterium <Emphasis Type="Italic">Oceanithermus profundus</Emphasis>

The metabolism of the novel facultatively anaerobic thermophilic bacterium Oceanithermus profundus was studied during growth on maltose, acetate, pyruvate, and hydrogen. The utilization of carbohydrates was shown to proceed via the glycolytic pathway. Under microaerobic growth conditions, the metabolism of O. profundus grown on maltose depended on the substrate concentration. At an initial maltose concentration of 1.4 mM, O. profundus carried out oxygen respiration, and in the presence of 3.5 mM maltose, facilitated fermentation occurred, with the formation of acetate and ethanol and limited involvement of oxygen. The use of pyruvate and acetate occurred via the TCA cycle. In cells grown on acetate, the activity of glyoxylate pathway enzymes was revealed. Depending on the energy-yielding process providing for growth (oxygen respiration or nitrate reduction), cells contained cytochromes a and c or b, respectively. The results obtained demonstrate the plasticity of the metabolism of O. profundus, which thus appears to be well-adjusted to the rapidly changing conditions in deep-sea hydrothermal vents.     

The complete genome sequence of Thermococcus onnurineus NA1 reveals a mixed heterotrophic and carboxydotrophic metabolism

Members of the genus Thermococcus, sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of Thermococcus onnurineus NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis, T. onnurineus NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to CO₂, thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for T. onnurineus NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in Thermococcus.     

Microbial diversity of a sulfide black smoker in main endeavour hydrothermal vent field,Juan de Fuca Ridge

Submarine hydrothermal vents are among the least-understood habitats on Earth but have been the intense focus of research in the past 30 years. An active hydrothermal sulfide chimney collected from the Dudley site in the Main Endeavour vent Field (MEF) of Juan de Fuca Ridge was investigated using mineralogical and molecular approaches. Mineral analysis indicated that the chimney was composed mainly of Fe-, Zn-and Cu-rich sulfides. According to phylogenetic analysis, within the Crenarchaeota, clones of the order Desulfurococcales predominated, comprising nearly 50% of archaeal clones. Euryarchaeota were composed mainly of clones belonging to Thermococcales and deep-sea hydrothermal vent Euryarchaeota (DHVE), each of which accounted for about 20% of all clones. Thermophilic or hyperthermophilic physiologies were common to the predominant archaeal groups. More than half of bacterial clones belonged to ɛ-Proteobacteria, which confirmed their prevalence in hydrothermal vent environments. Clones of Proteobacteria (γ-, δ-, β-), Cytophaga-Flavobacterium-Bacteroides (CFB) and Deinococcus-Thermus occurred as well. It was remarkable that methanogens and methanotrophs were not detected in our 16S rRNA gene library. Our results indicated that sulfur-related metabolism, which included sulfur-reducing activity carried out by thermophilic archaea and sulfur-oxidizing by mesophilic bacteria, was common and crucial to the vent ecosystem in Dudley hydrothermal site.     

Structure of the exopolysaccharide of Vibrio diabolicus isolated from a deep-sea hydrothermal vent

The structure of the exopolysaccharide produced under laboratory conditions by Vibrio diabolicus, a bacterium recovered from a deep-sea hydrothermal vent, has been investigated using sugar and methylation analysis and NMR spectroscopy. The polysaccharide consists of a linear tetrasaccharide repeating unit with the following structure. -->3)-beta-D-Glcp Nac-(1-->4)-beta-D-Glcp A-(1-->4)-beta-D-Glcp A-(1-->4)-alpha-D-Galp NAc-(1-->