Speed bumps and barricades in the carbon cycle: substrate structural effects on carbon cycling

The observation that a fraction of organic matter produced in marine systems evades the concerted efforts of microbial communities and is buried in sediments suggests that there are ‘speed bumps’ in carbon degradation pathways that impede microbially driven remineralization processes. The initial step in degradation of macromolecules, extracellular enzymatic hydrolysis, is often stated to be ‘the’ rate-limiting step in carbon remineralization. Experimental investigations described here, however, demonstrate that at least in certain cases, microbes produce extracellular enzymes on time scales of hours to tens of hours in response to substrate addition, and hydrolysis is extremely rapid. If enzymatic hydrolysis can be rapid, what factors slow or stop organic matter degradation? A lack of the correct inducer to initiate enzyme production, and/or a lack of the correct organism to produce the required enzyme, may result in a complete lack of hydrolysis in certain environments—a barricade, rather than a speed bump. Preliminary evidence supporting this hypothesis includes a comparison of polysaccharide hydrolysis in seawater and sediments, which demonstrates that the spectrum of enzymes active in seawater and sediments are fundamentally different. Furthermore, a survey of enzyme activities in surface waters from a range of locations suggests that pelagic microbial communities also differ widely in their abilities to express specific extracellular enzymes. Trans-membrane transport through porins is yet another potential location of structure-related selectivity.Our efforts to identify speed bumps and barricades are hampered by our inability to structurally characterize in sufficient detail the macromolecular structures present in marine systems. Furthermore, assessments of organic matter ‘quality’ from a chemical perspective do not necessarily accurately reflect the availability of organic carbon to microbial communities. For these communities, in fact, ‘quality’ may be a variable, which depends on the enzymatic and uptake capabilities of community members. To begin to assess substrate structure and quality from a microbial perspective, we will have to combine specific knowledge of macromolecular structures with detailed investigations of the enzymatic and transport capabilities of heterotrophic marine microbes.     

盐藻多糖提取工艺的研究

探讨盐藻D.peircei多糖的提取工艺.采用正交实验确定了提取盐藻粗多糖的最佳工艺条件,通过酶解方法脱除其蛋白.实验结果表明,提取盐藻粗多糖的最佳工艺条件为100℃,提取10h,固液比1∶16,pH=9,通过中性蛋白酶对其酶解,可脱除其部分蛋白,使糖含量达到29.01%,初步确定此多糖为含有硫酸基和己糖醛酸的蛋白多糖.     

气相色谱法分析海洋沉积物中多糖的组成

碳水化合物是海洋有机质的主要组成部分，海洋中的碳水化合物主要以聚合物的形式存在，其分子组成信息对研究它们的来源及其在生物地球化学循环中的作用具有重要意义。作者应用毛细管气相色谱法分析了海洋沉积物中多糖的组成。利用2．0mo1／L三氟乙酸，在80℃水解8h，把沉积物中的多糖水解成单糖，从而把单糖萃取到水相中。水解液在60℃旋转蒸发除去三氟乙酸，用等量的阴离子和阳离子交换树脂混合柱脱除残留离子，以Milli—Q水洗脱。洗脱液经蒸发、干燥后，用吡啶溶解，在0．2％LiClO4吡啶溶液催化下，于60℃经48h达到互变异构平衡。平衡异构体经Regisil试剂衍生化成三甲基硅醚(1MS)，用HP5890气相色谱仪进行分析。使用长30m、内径0．32mm的HP—5石英毛细管柱，配FID检测器，进样口和检测室温度保持在300℃，采用无分流进样，以N2作载气，流速为2．0ml/min，柱箱在140℃保留4min，然后以2℃／min的速度升温到220℃，保留10min。以每种单糖的相对保留时间定性；选择每种单糖中分离效果好、组成占优势的异构体峰面积作为单糖定量的依据，采用内标标准曲线法定量。应用这一方法分析了厦门港沉积物样品，确定了沉积物中7种主要中性单糖的组成。方法的样品加标准回收率为77．0％一115％，相对标准偏差为1．8％一11％；空白加标准回收率在93．8％一113％之间，相对标准偏差为0．6％一9．5％；当信噪比为3时，各单糖的检出限为0．02一0．06mg／L;内标戊五醇的全程回收率为79．8％(n＝6，RSD=5．2％)。     

三种螺旋藻及其蛋白质、多糖和脂类结合硒的研究

于1992年9月—1995年1月将极大、钝顶、盐泽等三种螺旋藻在加4—100mg／L硒浓度的培养基内培养，研究藻细胞及其蛋白质、多糖和脂类结合硒的量，探讨硒的结合机理。结果表明，极大螺旋藻累积的硒随外加硒的浓度而增加，但累积系数接近平均值2．184；在同样的硒浓度（8mg／L）条件下，盐泽螺旋藻对硒的累积远大于极大和钝顶两种螺旋藻的，高达696．968×10－6；极大螺旋藻中蛋白质和脂类结合的硒分别占藻细胞含硒量的14．63％和16．05％，两者均高于其它两种藻中相对应的量；三种藻细胞多糖结合硒的能力均很弱，但胞外多糖结合硒的能力较强。根据实验结果推测，螺旋藻累积硒的机理一方面是大分子化合物的吸附作用，另一方面是通过生化过程使硒与蛋白质和脂类结合形成大分子化合物。     

High concentrations of exopolymeric substances in Arctic winter sea ice: implications for the polar ocean carbon cycle and cryoprotection of diatoms

Exopolymeric substances (EPS) produced by microorganisms play important roles in various aquatic, porous, and extreme environments. Only recently has their occurrence in sea ice been considered. We used macroscopic and microscopic approaches to study the content and possible ecological role of EPS in wintertime fast ice near Barrow, Alaska (71°20′ N, 156°40′ W). Using Alcian blue staining of melted ice samples, we observed high concentrations of EPS in all samples examined, ranging from 0.79 to 7.71 mg xanthan gum equivalents (XGEQV) l⁻¹. Areal conversions to carbon equivalents yielded 1.5−1.9 g C m⁻² ice in March and 3.3−4.0 g C m⁻² in May (when the ice was thicker). Although EPS did not correlate with macronutrient or pigment data, the latter analyses indicated ongoing or recent biological activity in the ice within temperature horizons of −11°C to −9°C and warmer. EPS correlated positively with bacterial abundance (although no functional relationship could be deduced) and with dissolved organic carbon (DOC) concentrations. Ratios of EPS/DOC decreased at colder temperatures within the core, arguing against physical conversion of DOC to EPS during freezing. When sea-ice segments were maintained at representative winter temperatures (−5°C,−15°C and −25°C) for 3−14 months, the total EPS content increased significantly at rates of 5−47 μg XGEQV l⁻¹ d⁻¹, similar to published rates of EPS production by diatoms. Microscopic images of ice-core sections at these very cold temperatures, using a recently developed non-invasive method, revealed diatoms sequestered in spacious brine pockets, intact autofluorescent chloroplasts in 47% of the (pennate) diatoms observed, and indications of mucus in diatom-containing pores. The high concentrations of EPS detected in these winter ice cores represent a previously unrecognized form of organic matter that may contribute significantly to polar ocean carbon cycles, not only within the ice but after springtime release into the water column. The EPS present in very high concentrations in the brine of these microhabitats appear to play important buffering and cryoprotectant roles for microorganisms, especially diatoms, against harsh winter conditions of high salinity and potential ice-crystal damage.     

Brown algae overproduce cell wall polysaccharides as a protection mechanism against the heavy metal toxicity

Brown algae are often used as heavy metal biomonitors and biosorbents because they can accumulate high concentrations of metals. Cation-exchange performed by cell wall polysaccharides is pointed out as the main chemical mechanism for the metal sequestration. Here, we biochemically investigated if the brown alga Padina gymnospora living in a heavy metal contaminated area would modify their polysaccharidic content. We exposed non-living biomass to Cd and Pb and studied the metals adsorption and localization. We found that raw dried polysaccharides, sulfate groups, uronic acids, fucose, mannose, and galactose were significantly higher in contaminated algae compared with the control ones. Metal concentrations adsorbed by non-living biomass were rising comparatively to the tested concentrations. Electron microscopy showed numerous granules in the cell walls and X-ray microanalysis revealed Cd as the main element. We concluded that P. gymnospora overproduces cell wall polysaccharides when exposed to high metal concentrations as a defense mechanism.     

Polycyclic Aromatic Hydrocarbons and Polysaccharides in River Sediments from the Odra Basin after the 1997 Flood

Fourteen polycyclic aromatic hydrocarbons (PAH), six monosaccharides (SA) formed following the hydrolysis of polysaccharides, as well as water and organic matter contents were determined in river sediments sampled in thirteen points in the Odra basin after the catastrophic flood of 1997. The water content is related to the water absorbing capacity of the soil in the Odra catchment area. The PAH content increases together with the increase in the organic matter (OM) content which suggests that these species are mainly of anthropogenic origin. On the other hand, SA and OM contents decrease with increasing water content, which implies that both enhance biological life. The PAH content tends to decrease when the SA content increases. This relation goes hand in hand with the quality of water resources, which is greater when the quantity of polysaccharides is higher.     

化学融雪剂对小球藻生长和生理特性的影响

寒冷地区道路表面除雪化冰使用的化学融雪剂随着地表径流进入水体,将影响水生态系统中的水生生物正常生长,并破坏水生态系统平衡。为研究化学融雪剂对水生生物的毒性效应,分析了不同浓度有机融雪剂对小球藻生长特征、藻细胞光和色素、蛋白质及多糖含量的影响。结果表明：浓度为2 g·L^-1时有机融雪剂对小球藻生长无明显影响,当融雪剂浓度为4 g·L^-1时,小球藻细胞生长表现出明显抑制效应,且抑制效应随融雪剂浓度增加呈显著上升趋势(P<0.01);当融雪剂浓度小于4 g·L^-1时,有机融雪剂对小球藻细胞内叶绿素a合成有明显促进作用,但随着有机融雪剂处理浓度升高,藻细胞内叶绿素a含量逐渐下降。当融雪剂的处理浓度大于4 g·L^-1的时候,藻细胞内蛋白质和多糖含量与对照组相比呈显著下降趋势(P<0.01)。这说明融雪剂浓度高于4 g·L^-1时会抑制水体中小球藻正常生长繁殖,破坏藻体细胞,最终导致水生态系统平衡被破坏。     

Surface associations of enzymes and of organic matter: Consequences for hydrolytic activity and organic matter remineralization in marine systems

The extent to which marine organic matter is associated with surfaces and the consequences of such associations for organic matter remineralization are the focus of considerable attention. Since extracellular enzymes operating outside microbial cells are required to hydrolyze organic macromolecules to sizes sufficiently small for substrate uptake, the effects of surface interactions–on enzymes as well as on substrates–for hydrolytic activity also require investigation. We used a simplified laboratory system consisting of a free (dissolved) polysaccharide (pullulan) and the same polysaccharide tethered to agarose beads to restrict mobility, plus the corresponding free enzyme (pullulanase) and the same enzyme sorbed to montmorillonite (Mte), to investigate systematically the consequences of surface associations of enzymes and of substrates on hydrolytic activity. Changes in substrate molecular weight were monitored with time to measure the course of enzymatic hydrolysis. Although hydrolysis of free substrate was nearly complete after 2 min incubation with the free enzymes, the sorbed enzymes also effectively hydrolyzed free substrate, and the data suggest that they retained activity longer in solution compared to the free enzymes. Sorbed enzymes progressively hydrolyzed the free substrate from > 50 kD to lower molecular weights during a 24 h incubation, with a final product distribution on average showing only 1.4% and 10.3% of substrate still in the > 50 kD and 10 kD size classes, while 46.6%, 29.3%, and 12.5% of substrate was in the 4 kD, monomer, and free tag size classes, respectively. This product distribution was very similar to that of the free substrate/free enzyme experiment. Tethering the substrate to agarose beads led to lower substrate release (2–3% of total substrate after 98 h incubation) into solution compared to the free substrate case. For tethered substrates, the state of the enzyme (free or sorbed) measurably affected the molecular weight distribution of the hydrolysis products, with free enzymes producing a higher fraction of high molecular weight hydrolysis products (28.7 ± 5.4% of substrate > 50 kD at the end of the incubation) compared to sorbed enzymes (11.6 ± 2.8% of substrate > 50 kD at the end of the incubation.) Tethered substrates were also hydrolyzed in a sediment slurry from surface sediments from Cape Lookout Bight, North Carolina; 0.1% of total substrate was released by enzymes naturally present in 1 cm³ of sediment after 144 h incubation, demonstrating that the enzymes naturally present in marine sediments are also capable of accessing tethered substrates. These investigations suggest that surface associations of enzymes in marine systems may extend the active lifetime of such enzymes, providing an opportunity for hydrolysis over longer periods of time and producing a different size spectrum of hydrolysis products relative to free enzymes. Furthermore, in well-mixed systems, surface-associated enzymes can hydrolyze substrates whose mobility is restricted, highlighting the importance of processes such as resuspension and bioturbation on organic matter remineralization.     

分子修饰在多糖构效关系研究中的应用

分子修饰可改变多糖结构和生物活性,是多糖构效关系及其药物研究的有效手段;而构效关系研究结果又指导着多糖分子修饰的方向,为其药物设计、研究和开发提供理论支持。本文综述了分子修饰在多糖构效关系研究中的作用,并对分子修饰在多糖类药物研究中的应用前景进行了展望。