Monitoring <Emphasis Type="Italic">ALS1</Emphasis> and <Emphasis Type="Italic">ALS3</Emphasis> Gene Expression During In Vitro <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Formation Under Continuous Flow Conditions

ALS1 and ALS3 encode cell-surface associated glycoproteins that are considered to be important for Candida albicans biofilm formation. The main goal of the present study was to monitor ALS1 and ALS3 gene expression during C. albicans biofilm formation (on silicone) under continuous flow conditions, using the Centers for Disease Control biofilm reactor (CDC reactor). For ALS1, we found few changes in gene expression until later stages of biofilm formation (72 and 96 h) when this gene appeared to be downregulated relative to the gene expression level in the start culture. We observed an induction of ALS3 gene expression in the initial stages of biofilm formation (0.5, 1, and 6 h), whereas at later stages, this gene was also downregulated relative to the gene expression level in the start culture. We also found that biofilms of an als3/als3 deletion mutant contained less filaments at several time points (1, 6, 24, and 48 h), although filamentation as such was not affected in this strain. Together, our data indicate an important role for ALS3 in the early phases of biofilm formation in the CDC reactor, probably related to adhesion of filaments, while the role of ALS1 is less clear.     

Modelling of cometabolic transformation ofortho-xylene in a denitrifying biofilm system

A model describing the cometabolic biotransformation ofo-xylene with toluene as primary carbon source in a continuously fed fixed biofilm reactor is presented. The model is based on the concept of competitive inhibition betweeno-xylene and toluene. The proposed model simulated successfully the transformation ofo-xylene and the associated by-products formation, as well as the toluene degradation. However, it appears that an accurate measurement of active biomass density and distribution in the biofilm is needed, since these factors dramatically affects the modelling. The modelling of various kinetic experiments indicates that the active biomass (or toluene degraders) is accumulated on the top of the biofilm, leading to the conclusion that only a minor part of the biofilm thickness was active. The calibrated model is able to predict the removal of toluene ando-xylene for concentrations ranging from 0 to 30 mg/L. For higher concentrations toxicity phenomena may decrease the accuracy of the model.     

The accumulation of fixed biomass increases the observed growth yield of a nitrifying biofilm

The observed growth yield (Y _obs) of a nitrifying biofilm metabolizing ammonia in a continuous flow reactor was constant below a fixed biomass concentration of 40–50 g COD-biomass cm^–2. Beyond this range, an increased Y _obs with the additional accumulation of fixed biomass could be due to a considerable accumulation of inactive materials within the nitrifying biofilm.     

Biofilm formation and partial biodegradation of polystyrene by the actinomycete <Emphasis Type="Italic">Rhodococcus ruber</Emphasis>

Polystyrene, which is one of the most utilized thermoplastics, is highly durable and is considered to be non-biodegradable. Hence, polystyrene waste accumulates in the environment posing an increasing ecological threat. In a previous study we have isolated a biofilm-producing strain (C208) of the actinomycete Rhodococcus ruber that degraded polyethylene films. Formation of biofilm, by C208, improved the biodegradation of polyethylene. Consequently, the present study aimed at monitoring the kinetics of biofilm formation by C208 on polystyrene, determining the physiological activity of the biofilm and analyzing its capacity to degrade polystyrene. Quantification of the biofilm biomass was performed using a modified crystal violet (CV) staining or by monitoring the protein content in the biofilm. When cultured on polystyrene flakes, most of the bacterial cells adhered to the polystyrene surface within few hours, forming a biofilm. The growth of the on polystyrene showed a pattern similar to that of a planktonic culture. Furthermore, the respiration rate, of the biofilm, exhibited a pattern similar to that of the biofilm growth. In contrast, the respiration activity of the planktonic population showed a constant decline with time. Addition of mineral oil (0.005% w/v), but not non-ionic surfactants, increased the biofilm biomass. Extended incubation of the biofilm for up to 8 weeks resulted in a small reduction in the polystyrene weight (0.8% of gravimetric weight loss). This study demonstrates the high affinity of C208 to polystyrene which lead to biofilm formation and, presumably, induced partial biodegradation.     

Evaluating scaling of capillary photo-biofilm reactors for high cell density cultivation of mixed trophies artificial microbial consortia

Capillary biofilm reactors (CBRs) are attractive for growing photoautotrophic bacteria as they allow high cell-density cultivation. Here, we evaluated the CBR system's suitability to grow an artificial consortium composed of Synechocystis sp. PCC 6803 and Pseudomonas sp. VBL120. The impact of reactor material, flow rate, pH, O₂, and medium composition on biomass development and long-term biofilm stability at different reactor scales was studied. Silicone was superior over other materials like glass or PVC due to its excellent O₂ permeability. High flow rates of 520 μL min⁻¹ prevented biofilm sloughing in 1 m capillary reactors, leading to a 54% higher biomass dry weight combined with the lowest O₂ concentration inside the reactor compared to standard operating conditions. Further increase in reactor length to 5 m revealed a limitation in trace elements. Increasing trace elements by a factor of five allowed for complete surface coverage with a biomass dry weight of 36.8 g m⁻² and, thus, a successful CBR scale-up by a factor of 25. Practical application : Cyanobacteria use light energy to upgrade CO₂, thereby holding the potential for carbon-neutral production processes. One of the persisting challenges is low cell density due to light limitations and O₂ accumulation often occurring in established flat panel or tubular photobioreactors. Compared to planktonic cultures, much higher cell densities (factor 10 to 100) can be obtained in cyanobacterial biofilms. The capillary biofilm reactor (CBR) offers good growth conditions for cyanobacterial biofilms, but its applicability has been shown only on the laboratory scale. Here, a first scale-up study based on sizing up was performed, testing the feasibility of this system for large-scale applications. We demonstrate that by optimizing nutrient supply and flow conditions, the system could be enlarged by factor 25 by enhancing the length of the reactor. This reactor concept, combined with cyanobacterial biofilms and numbering up, holds the potential to be applied as a flexible, carbon-neutral production platform for value-added compounds.     

On-line monitoring of antifouling and fouling-release surfaces using bioluminescence and fluorescence measurements during laminar flow

A laminar flow biofilm-monitoring system was used to determine the efficacies of three antifouling (AF) coatings and five fouling-release (FR) coatings againstVibrio harveyi attachment. On-line measurements of tryptophan fluorescence and bioluminescence from each coating, normalized to an upstream stainless steel coupon, were used to determine the effects of AF and FR surfaces on biofilm formation. The AF coatings consisted of 5, 10, and 35 wt% Sea Nine 211 (C9211) incorporated into a vinyl copolymer. Both the 10 and 35 wt% coatings significantly inhibited biofilm biomass development measured by tryptophan fluorescence compared to the stainless steel control.V. harveyi bioluminescence was significantly greater than tryptophan fluorescence in cells attached to these coatings, suggesting that bioluminescence expression may be a marker for cellular stress or toxicity in biofilms. Five different polydimethylsiloxane (PDMS) FR coatings did not inhibit biofilm formation under low flow conditions. However, four PDMS coatings demonstrated decreased biomass levels compared to stainless steel after exposure to a shear stress of 330 dynes cm^–2. There was no toxic additive in these coatings; bioluminescence and tryptophan fluorescence were proportional.     

Performance of a flat plate,air-lift reactor for the growth of high biomass algal cultures

A flat plate, multi-pass air lift reactor (FPALR) for the culture of photosynthetic organisms was constructed from twin wall acrylic sheet and its performance characterised. When operated at an air input of 2.01 min⁻¹ the multi-pass system had a Reynolds number of 5200 indicating fully turbulent flow. Chlorella vulgaris 211/11c was found to have a stationary phase biomass of 1.48 g 1⁻¹ when grown in the flat plate air lift reactor (FPALR) at 100 μmol m⁻²s⁻¹ compared to 1.11 g 1⁻¹ when cultured in the continually stirred tank reactor (CSTR) at the same PFD (photon flux density). The same organism cultured at 200 μmol m⁻²s⁻¹ achieved a stationary phase biomass of 1.71 g 1⁻¹ in the FPALR. In contrast, Scenedesmus sp. produced a stationary phase biomass of 2.27 g1⁻¹ and 1.27 g1⁻¹, when cultured at 100 μmol m⁻²s⁻¹ in the FPALR and the CSTR respectively. The growth rates of both organisms were also higher in the PFALR.     

干旱胁迫对蒙古黄芪生长及根部次生代谢物含量的影响

以山西道地药材黄芪一年生幼苗为试验材料,设置常规水分条件(CK)、轻度干旱胁迫(A1)、中度干旱胁迫(A2)、重度干旱胁迫(A3)4个不同处理,研究土壤干旱胁迫对黄芪生长及生理的影响。结果表明:黄芪茎叶快速生长集中在出苗后80—120d,以后生长减缓;当茎叶枯萎时,根中生物量短期快速积累。与常规水分条件相比,干旱胁迫处理显著降低了黄芪苗高及茎叶生物量,但对抗氧化能力、根系生长及次生代谢物积累产生了不同的影响。轻度干旱胁迫下SOD、POD、CAT 3种抗氧化酶活性升高,丙二醛(MDA)含量和细胞膜透性降低,同时根长与根生物量增加、多糖与皂苷两种次生代谢物积累增多,黄芪药材的质量得到显著提高(P0.05);胁迫上升到中度、重度时,SOD酶活性逐渐降低,重度胁迫下低于对照,而POD及CAT酶活性、MDA含量、细胞膜透性均随胁迫增强而升高,相反,根长、根生物量、多糖与皂苷含量降低,导致黄芪药材的质量显著降低(P0.01)。综上表明,干旱胁迫下,SOD酶表现较为敏感,可能在清除活性氧中起主要作用;轻度水分胁迫能有效启动黄芪体内抗氧化酶系统和次生代谢,它们相互协作共同对抗胁迫对细胞产生的伤害,通过降低地上部分的生长,将营养物质优先运往根部,促进根产量及药材质量的提高。这一结论,可在黄芪多糖和皂苷次生代谢物定向培育的水分管理中加以利用。     

On-line estimation of O<Subscript>2</Subscript> production,CO<Subscript>2</Subscript> uptake,and growth kinetics of microalgal cultures in a gas-tight photobioreactor

Growth of the green algae Chlamydomonas reinhardtii and Chlorella sp. in batch cultures was investigated in a novel gas-tight photobioreactor, in which CO₂, H₂, and N₂ were titrated into the gas phase to control medium pH, dissolved oxygen partial pressure, and headspace pressure, respectively. The exit gas from the reactor was circulated through a loop of tubing and re-introduced into the culture. CO₂ uptake was estimated from the addition of CO₂ as acidic titrant and O₂ evolution was estimated from titration by H₂, which was used to reduce O₂ over a Pd catalyst. The photosynthetic quotient, PQ, was estimated as the ratio between O₂ evolution and CO₂ up-take rates. NH₄ ⁺, NO₂ ⁻, or NO₃ ⁻ was the final cell density limiting nutrient. Cultures of both algae were, in general, characterised by a nitrogen sufficient growth phase followed by a nitrogen depleted phase in which starch was the major product. The estimated PQ values were dependent on the level of oxidation of the nitrogen source. The PQ was 1 with NH₄ ⁺ as the nitrogen source and 1.3 when NO₃ ⁻ was the nitrogen source. In cultures grown on all nitrogen sources, the PQ value approached 1 when the nitrogen source was depleted and starch synthesis became dominant, to further increase towards 1.3 over a period of 3–4 days. This latter increase in PQ, which was indicative of production of reduced compounds like lipids, correlated with a simultaneous increase in the degree of reduction of the biomass. When using the titrations of CO₂ and H₂ into the reactor headspace to estimate the up-take of CO₂, the production of O₂, and the PQ, the rate of biomass production could be followed, the stoichiometrical composition of the produced algal biomass could be estimated, and different growth phases could be identified.     

The effect of acetic acid concentration on the growth and production of gamma-linolenic acid byMucor circinelloides CBS 203.28 in fed-batch culture

The effect of different initial acetic acid concentrations on the growth of and lipid and gamma-linolenic acid (GLA) production byMucor circinelloides CBS 203.28 was determined in a 14 litre stirred tank reactor operated in a fedbatch, pH-stat mode with acetic acid as carbon source and pH titrant. Increased acetic acid concentrations in the culture resulted in a significant increase in the crude oil content of the biomass. By contrast, all the other parameters such as the biomass concentration, GLA and oil yield on acetic acid, the GLA content of the biomass and oil, the growth rate and volumetric rate of GLA production decreased with an increase in acetic acid concentration. The best results were obtained with acetic acid at 2 g/1, which gave 39.8 mg GLA/g biomass and 15.6% GLA in the neutral lipid fraction, amounting to 340 mg GLA/1 culture. A decrease in the glyco- and phospho-lipid fractions during the cultivation coincided with an increase in the neutral lipid fraction. The GLA content of the biomass remained within rather narrow limits of 3.5% to 4% of the biomass, irrespective of the oil content of the biomass. The fatty acid profile was not greatly affected by the acetic acid concentration. The hyphae of the fungus were characterized by the accumulation of large intracellular oil droplets and some septa delimited the hyphae.     

The effect of harvesting on biomass production and nutrient removal in phototrophic biofilm reactors for effluent polishing

An increasing number of wastewater treatment plants require post-treatment to remove residual nitrogen and phosphorus. This study investigated various harvesting regimes that would achieve consistent low effluent concentrations of nitrogen and phosphorus in a phototrophic biofilm reactor. Experiments were performed in a vertical biofilm reactor under continuous artificial lighting and employing artificial wastewater. Under similar conditions, experiments were performed in near-horizontal flow lanes with biofilms of variable thickness. It was possible to maintain low nitrogen and phosphorus concentrations in the effluent of the vertical biofilm reactor by regularly harvesting half of the biofilm. The average areal biomass production rate achieved a 7 g dry weight m^?2 day^?1 for all different harvesting frequencies tested (every 2, 4, or 7 days), corresponding to the different biofilm thicknesses. Apparently, the biomass productivity is similar for a wide range of biofilm thicknesses. The biofilm could not be maintained for more than 2 weeks as, after this period, it spontaneously detached from the carrier material. Contrary to the expectations, the biomass production doubled when the biofilm thickness was increased from 130 μm to 2 mm. This increased production was explained by the lower density and looser structure of the 2 mm biofilm. It was concluded that, concerning biomass production and labor requirement, the optimum harvesting frequency is once per week.     

Heterogeneous biofilm activity in a segregated anaerobic fluidized bed bioreactor

Bed segregation in a fluidized bed bioreactor profoundly influenced biofilm thickness and microbial activities of the biofilm along the bed height. Bioparticles coated with a thin biofilm, observed at the bottom of the reactor, had a higher specific activity in propylene glycol and n-propanol degradation than in thick biofilms developed at the top of the reactor. Although no significant difference was observed in specific activity for propionate and acetate along the reactor flow axis, more total propionate and acetate conversion occurred in regions of thicker biofilm accumulation.     

二年生三七农艺和质量性状对环境光强的响应特征

为探究光照强度对二年生三七(Panax notoginseng)农艺性状和质量性状的影响,采用人工遮荫的方法,对三七植株的农艺性状、解剖结构、生物量和皂苷含量进行研究。结果表明,三七生物量积累以透光率为13.5%时最大;三七总皂苷含量在透光率9.2%时最高,透光率为13.5%时单株皂苷含量较大。当透光率降低,三七的叶片和茎部生物量增加;透光率增加时,三七通过增加叶片上表皮厚度、海绵组织厚度、栅栏组织厚度和叶片厚度来减少光捕获。因此,在透光率为9.2%~13.5%时对三七的生长、生物量及皂苷的积累有促进作用。     

铁基纳米酶对鼠伤寒沙门菌生物被膜的影响

运用结晶紫染色定量法、生物被膜形态观察、生物被膜干重称量法、活菌定量计数法和细菌内活性氧检测法,评估氧化铁纳米酶和硫化铁纳米酶对鼠伤寒沙门菌生物被膜的影响及其机制.结果显示:鼠伤寒沙门菌S025株与这两类铁基纳米酶共孵育48h后,其生物被膜结晶紫染色吸光度值(A)、生物被膜厚度、生物被膜干重和活菌数量与未处理组相比均显著下降,活性氧水平显著上升,其中硫化铁纳米酶效果优于四氧化三铁纳米酶;在生物被膜形成后,加入铁基纳米酶处理0.5h、2h和12h,生物被膜结晶紫染色A值、生物被膜厚度、生物被膜干重和活菌数量与未处理组相比均显著下降,活性氧水平显著上升,硫化铁纳米酶效果同样优于四氧化三铁纳米酶.以上结果表明,铁基纳米酶通过调控鼠伤寒沙门菌胞内活性氧水平,不仅可以预防该菌的生物被膜形成,而且可以破坏已形成的生物被膜,本研究将有助于预防和治疗鼠伤寒沙门菌生物被膜引起的相关疾病.     

Flowcell culture of Porphyromonas gingivalis biofilms under anaerobic conditions

We have developed an anaerobic biofilm culture system. The system is inexpensive, simple to use and, unlike an anaerobic glovebox, requires no dedicated space. As a test of the system, Porphyromonas gingivalis was cultured under low oxygen (1–2 ppm) and under anaerobic conditions (≤0.1 ppm O₂). In the presence of small amounts of oxygen, the organism attached and formed an initial biofilm over the course of 4 h, but the biofilm was unable to maintain its growth and had lost biomass after 18 h. Also, ambiguous results were obtained when the biofilm was stained with a viability stain. Under anaerobic conditions, the biofilm was able to continue growth — biomass was greater after 18 h than after 4 h, and the anaerobic biofilm had a less ambiguous staining pattern than did the low-O₂-grown biofilm.     

Immobilization of blue green microalgae on loofa sponge to biosorb cadmium in repeated shake flask batch and continuous flow fixed bed column reactor system

Summary An indigenous strain of blue green microalga, Synechococcus sp., isolated from wastewater, was immobilized onto loofa sponge discs and investigated as a potential biosorbent for the removal of cadmium from aqueous solutions. Immobilization has enhanced the sorption of cadmium and an increase of biosorption (21%) at equilibrium was noted as compared to free biomass. The kinetics of cadmium biosorption was extremely rapid, with (96%) of adsorption within the first 5 min and equilibrium reached at 15 min. Increasing initial pH or initial cadmium concentration resulted in an increase in cadmium uptake. The maximum biosorption capacity of free and loofa immobilized biomass of Synechococcus sp. was found to be 47.73 and 57.76 mg g⁻¹ biomass respectively. The biosorption equilibrium was well described by Langmuir adsorption isotherm model. The biosorbed cadmium was desorbed by washing the immobilized biomass with dilute HCl (0.1 M) and desorbed biomass was reused in five biosorption–desorption cycles without an apparent decrease in its metal biosorption capacity. The metal removing capacity of loofa immobilized biomass was also tested in a continuous flow fixed-bed column bioreactor and was found to be highly effective in removing cadmium from aqueous solution. The results suggested that the loofa sponge-immobilized biomass of Synechococcus sp. could be used as a biosorbent for an efficient removal of heavy metal ions from aqueous solution.     

Heterogeneity of biofilms in rotating annular reactors: Occurrence, structure, and consequences

A rotating annular reactor (Roto Torque) was used for qualitative and quantitative studied on biofilm heterogeneity. In contrast to the classic image of biofilms as smooth, homogeneous layers of biomass on a substratum, studies using various pure and mixed cultures consistently revealed more-dimensional structures that resembled dunes and ridges, among others. These heterogeneities were categorized and their underlying causes analyzed. Contrary to expectations, motility of the microorganisms not a decisive factor in determining biofilm homogeneity. Small Variations in substratum geometry homogeneity. Small variations in substratum geometry and flow patterns were clearly reflected in the biofilm pattern. Nonhomogeneous flow and shear patterns in the reactor, together with inadequate mixing resulted in significant, position-dependent differences in surface growth. It was therefore not possible to take representative samples of the attached biomass. Like many other types of reactors, the Roto Torque reactor is valuable for qualitative and morphological biofilm experiments but less suitable for quantitative physiological and kinetics studies using attached microorganisms. (c) 1994 John Wiley & Sons, Inc.     

Characterisation of bacterial adhesion and removal in a flow chamber by micromanipulation measurements

Flow device analyses and micromanipulation were used to assess the adhesive and cohesive integrity of the immobilised bacterial populations (biomass) of Pseudomonas fluorescens, which were harvested at different growth times and applied to a substrate made of stainless steel subsequently accommodated in a specially designed flow chamber. After the biomass was exposed to a fluidic environment for a period of time, the biomass samples were removed from the flow chamber and the apparent adhesion and cohesion of the remaining biomass was measured using a micromanipulation technique. The surface area of the substrate covered by the biomass exposed to the fluid flow was monitored by a digital camera and then quantified by image analysis. The results indicate a strong correlation between micromanipulation measurements and flow chamber experiments. The micromanipulation data show that the apparent adhesive strength of the biomass increased with the growth time. Moreover, the apparent adhesive strength was found to be stronger than the bacterial cohesive strength. The data was used to interpret the removal behaviour of the biomass from the flow chamber. Using these techniques, specific mechanisms of biomass detachment from a surface and optimised cleaning strategies can be postulated.     

低磷胁迫对雷公藤幼苗形态指标及生物量的影响

采用土培的方法,研究了磷(P)胁迫对雷公藤(Tripterygium wilfordii Hook.f.)1年生和3年生幼苗在一个生长季内形态指标及生物量的影响。结果表明:低P处理显著抑制了雷公藤幼苗地上部分的增长,而对地下部分的影响较小,导致根冠比的增加,在相同P水平处理下,3年生雷公藤的根冠比高于1年生的;低P胁迫显著影响了雷公藤的生物量积累及分配,雷公藤幼苗总生物量及1年生雷公藤细根生物量占根系生物量的比例下降,根系生物量在总生物量中的比例及3年生雷公藤细根生物量在根系生物量中的比例上升。这表明3年生雷公藤对P逆境有更好的适应机制,对低P林地套种雷公藤的苗龄选择有参考价值;雷公藤幼苗的株高、最长枝均与生物量呈显著的正相关,可作为耐低P雷公藤良种选育中优良性状的指标。中轻度的P胁迫不显著影响根系生物量的积累,维持较大比例的根系是雷公藤适应低P胁迫的主要策略。     

Production of eleutherosides in in vitro regenerated embryos and plantlets of <Emphasis Type="BoldItalic">Eleutherococcus chiisanensis</Emphasis>

High frequency somatic embryogenesis of Eleutheorcoccus chiisanensis was achieved through suspension culture of embryogenic cells in hormone-free Murashige and Skoog liquid medium supplemented with 30 g sucrose l⁻¹. Cotyledonary somatic embryos were germinated and converted into plantlets using 20 μM gibberellic acid which were then grown in a 10 l airlift bioreactor. HPLC analysis revealed the accumulation of eleutheroside B, E and E1 in the embryos and plantlets. Thus mass production of embryos and plantlets of E. chiisanensis can be achieved in liquid cultures and the biomass produced may become an alternative source of eleutherosides.