THE FISH POND EXPERIMENT: CHAPTER TWO

A tubular photobioreactor for outdoor cultivation of Spirulina platensis was successfully operated for the last two years. The reactor was made of transparent 2.4-cm diameter tubes with a total length of approximately 101-m and a volume of 124-liter. Flow was induced using an airlift pumping system. To optimize the system further, a larger tube diameter was also tested. Preliminary results have suggested that a larger tube diameter might provide increased output with reduced surface area requirements. Results of experiments comparing the productivity of the same culture volume in tubes of 2.4 cm and 5.0 cm will be presented which show a small decrease in productivity by volume, but a large increase in areal productivity. This suggests that the larger tube diameter would be an appropriate choice for larger scale systems. Additionally, data will be presented demonstrating the effectiveness of an on-line surface scatter turbidimeter for accurate measurement of Spirulina density when correlated to manual dry weight measurements.     

Variation in Photosynthetic Performance Relative to Thallus Microhabitat Heterogeneity in Lithothamnion australe (Rhodophyta,Corallinales) Rhodoliths

Rhodoliths are free‐living, coralline algae that create heterogeneous structure over sedimentary habitats. These fragile ecosystems are threatened by anthropogenic disturbances that reduce their size and three‐dimensional structural complexity. We investigated how physical disturbance from boat moorings affects photosynthetic performance in the rhodolith Lithothamnion australe. Photosynthetic parameters were measured for intact rhodoliths and crushed rhodolith fragments of two sizes (ca. 1 and 2 cm diameter), while chlorophyll fluorescence was measured at the surface of rhodoliths of these two sizes, between the interior branches of the larger rhodoliths, and at the surface of 52 various sized (0.4–3.5 cm diameter) rhodoliths. Gross productivity and net productivity were 15% and 36% higher, respectively, in the smaller L. australe, while respiration was 10% higher in the larger individuals. Thallus crushing reduced gross productivity by 20% and 41%, and net productivity by 9% and 14% in the smaller and larger rhodoliths, respectively. It also reduced respiration by 33% and 60% in the smaller and larger rhodoliths, respectively. Fluorescence parameters were all greater at the surface of the larger L. australe than the smaller individuals, and greater at the surface than in the interior parts of the larger individuals. Across a range of rhodolith sizes, surface fluorescence parameters were at their maxima in 1.54 to 2.32 cm diameter individuals. These results show that L. australe’s complex structure creates heterogeneity in photosynthesis and respiration between their surface and interior parts and among rhodolith sizes. This information can help predict how rhodoliths may respond to disturbance and environmental stressors.     

Monitoring of tube‐nesting bees and wasps with bamboo tube nest traps of different types in two types of forests in temperate Japan

Tube‐nesting bees and wasps were collected with bamboo tube nest traps in a cherry blossom forest and an old secondary forest in Tama Forest Science Garden in the western suburbs of Tokyo. Curtain type traps, in which bamboo tubes were vertically arranged, attracted more bees and wasps to nests than bundle type traps, in which bamboo tubes were bundled haphazardly. The attractiveness of small tubes was similar between small tube traps, which were only composed of small tubes (about 4 mm in diameter), and size mixture traps, which set small tubes together with larger tubes, indicating that the presence of the larger species did not affect the nesting of small species. In the cherry forest, which was characterized by the sparse distribution of deciduous cherry trees with a thin canopy layer and rich growth of weeds, Megachilidae, Colletidae, Sphecidae and Eumenidae were common. In the shady secondary forest, where evergreen trees (Abies firma Sieb. et Zucc., Quercus glauca Thunb.) and shrubs (Aucuba japonica Thunb., Eurya japonica Thunb., Camellia japonica L.) were prevalent, Pompilidae was abundant. Among five species of Crabronidae recorded in this study, four were collected mostly in the cherry forest, whereas the most abundant, Trypoxylon malaisei Gussakovskij, was also common in the secondary forest. Likewise, Auplopus carbonarius (Scopoli) (Pompilidae) was common in the secondary forest and not rare in the cherry forest. These results suggest that the tube‐nesting bees and wasps can be a good bioindicator for monitoring environment.     

Mixing time and oxygen transfer characteristics of double draft tube airlift fermentor

Despite the increasing importance of airlift fermentors, very little published information is available on how the geometric configurations of the draft tubes and the air-sparging system affect the mixing and oxygen transfer characteristics of the fermentor. A 14-L air-lift fermentor was designed and build with a fixed liquid height to diameter ratio of 1.5 utilizing four equally spaced air jets at the bottom. Two jet orifice sizes were used, 1.27 and 3.81 mm i.d., and for each jet size the following four geometric configurations were used: Single inner concentric draft tube, single outer concentric draft tube, two concentric draft tubes, and no draft tubes where the fermentor was operated as a shallow bubble column. It was found that the presence of draft tubes stabilized liquid circulation patterns and gave systemically higher mixing times than those obtained in the absence of draft tubes. In addition, the double draft tube geometry resulted in higher mixing times than the single draft tubes. For the power unit volume range 20 to about 250 W/m³ the larger 3.81-mm orifices gave systemically higher k_L a values than the smaller 1.27-mm i.d. orifices. At 200 W/m³ the use of a single outer draft tube with the 3.81-mm orifices resulted in 94% increase in k_L a values over that obtained with no draft tubes. However, the effect of draft tube geometry on k_L a values when the 1.27-mm orifices were used was not significant. The air bubble formation characteristics at the jet orifices were found to be different, which reflected the differences observed in mass transfer and mixing characteristics. The power economy for oxygen transfer was found to be depend strongly on the orifice size and less on the geometric configuration of draft tubes.     

Tomato root distribution, yield and fruit quality under subsurface drip irrigation

Tomato rooting patterns were evaluated in a 2-year field trial where surface drip irrigation (R0) was compared with subsurface drip irrigation at 20 cm (RI) and 40 cm (RII) depths. Pot-transplanted plants of two processing tomato, `Brigade' (C1) and `H3044' (C2), were used. The behaviour of the root system in response to different irrigation treatments was evaluated through minirhizotrons installed between two plants, in proximity of the plant row. Root length intensity (L _a), length of root per unit of minirhizotron surface area (cm cm^–2) was measured at blooming stage and at harvest. For all sampling dates the depth of the drip irrigation tube, the cultivar and the interaction between treatments did not significantly influence L _a. However differences between irrigation treatments were observed as root distribution along the soil profile and a large concentration of roots at the depth of the irrigation tubes was found. For both surface and subsurface drip irrigation and for both cultivars most of the root system was concentrated in the top 40 cm of the soil profile, where root length density ranged between 0.5 and 1.5 cm cm^–3. Commercial yields (t ha^–1) were 87.6 and 114.2 (R0), 107.5 and 128.1 (RI), 105.0 and 124.8 (RII), for 1997 and 1998, respectively. Differences between the 2 years may be attributed to different climatic conditions. In the second year, although no significant differences were found among treatments, slightly higher values were observed with irrigation tubes at 20 cm depth. Fruit quality was not significantly affected by treatments or by the interaction between irrigation tube depth and cultivar.     

Transport processes during sterilization of vertical and 5 degree horizontal dead-legs

Experimental temperature profiles and biological kill data from dead-ended tubes of various diameters and lengths commonly used in biotechnology applications were compared to those predicted by a finite element model for steam-in-place (SIP) sterilization at 122°C. Diameter was shown experimentally and numerically to have a significant effect with larger diameter tubes exhibiting greater buoyant driven convective flow and more rapid sterilization. The overall Grashof number was shown to be the significant parameter relating magnitude of convective flow to tube diameter and varied as the diameter cubed. Analysis of air/steam mixture flow patterns showed air displacement from 0.4 cm diameter tubes to be due primarily to molecular diffusion, whereas 1.0 and 1.7 cm tubes showed a two-stage convective flow pattern. There exists a critical diameter of 0.4 cm below which SIP sterilization due to buoyancy driven flow does not occur and steam bleeders should be used.     

In situ microscopy reveals reversible cell wall swelling in kelp sieve tubes: one mechanism for turgor generation and flow control?

Kelps, brown algae (Phaeophyceae) of the order Laminariales, possess sieve tubes for the symplasmic long‐distance transport of photoassimilates that are evolutionarily unrelated but structurally similar to the tubes in the phloem of vascular plants. We visualized sieve tube structure and wound responses in fully functional, intact Bull Kelp (Nereocystis luetkeana [K. Mertens] Postels & Ruprecht 1840). In injured tubes, apparent slime plugs formed but were unlikely to cause sieve tube occlusion as they assembled at the downstream side of sieve plates. Cell walls expanded massively in the radial direction, reducing the volume of the wounded sieve elements by up to 90%. Ultrastructural examination showed that a layer of the immediate cell wall characterized by circumferential cellulose fibrils was responsible for swelling and suggested that alginates, abundant gelatinous polymers of the cell wall matrix, were involved. Wall swelling was rapid, reversible and depended on intracellular pressure, as demonstrated by pressure‐injection of silicon oil. Our results revive the concept of turgor generation and buffering by swelling cell walls, which had fallen into oblivion over the last century. Because sieve tube transport is pressure‐driven and controlled physically by tube diameter, a regulatory role of wall swelling in photoassimilate distribution is implied in kelps.     

Growth and development of conifer pollen tubes

Conifer pollen tubes are an important but underused experimental system in plant biology. They represent a major evolutionary step in male gametophyte development as an intermediate form between the haustorial pollen tubes of cycads and Ginkgo and the structurally reduced and faster growing pollen tubes of flowering plants. Conifer pollen grains are available in large quantities, most can be stored for several years, and they grow very well in culture. The study of pollen tube growth and development furthers our understanding of conifer reproduction and contributes towards our ability to improve on their productivity. This review covers taxonomy and morphology to cell, developmental, and molecular biology. It explores recent advances in research on conifer pollen and pollen tubes in vivo, focusing on pollen wall structure, male gametophyte development within the pollen wall, pollination mechanisms, pollen tube growth and development, and programmed cell death. It also explores recent research in vitro, including the cellular mechanisms underlying pollen tube elongation, in vitro fertilization, genetic transformation and gene expression, and pine pollen tube proteomics. With the ongoing sequencing of the Pinus taeda genome in several labs, we expect the use of conifer pollen tubes as an experimental system to increase in the next decade.     

Mass cultivation of Nannochloropsis sp. in annular reactors

A study was made on the mass cultivation of Nannochloropsis sp. in newly designed annular reactors operated under natural, artificial or combined illumination. The annular reactor consists of two 2-m-high Plexiglas cylinders of different diameter placed vertically one inside the other so as to form an annular culture chamber. Artificial illumination is supplied by lamps placed inside the inner cylinder. Two annular reactors of different diameter (50 and 91 cm), light path (4.5 and 3.0 cm) and illuminated surface area (5.3 and 9.3 m²) were experimented with. The effect of two different artificial light sources (fluorescent tubes and metal halide lamps) on culture productivity was investigated in both systems. The highest productivity on a per reactor basis (about 34 g (d. wt) reactor^–1 24 h^–1) was achieved with the larger reactor illuminated by a 400-W metal halide lamp. From February to May a 91-cm reactor illuminated only with natural light was operated in parallel with a 91-cm reactor subjected to combined illumination. Under natural illumination productivity increased from 16.6 g (d. wt) reactor^–1 d^–1 in February to 34.1 g (d. wt) reactor^–1 d^–1 in May. Under combined illumination productivity was 41.3 g (d. wt) reactor^–1 d^–1 in February and increased up to 48.3 g (d. wt) reactor^–1 d^–1 in May. Although the culture exposed to combined illumination always attained higher yields, the productivity gap between the two cultures decreased gradually along the season as solar radiation and minimum night temperatures increased. A 1200-L plant made of ten 50-cm annular reactors was set up and operated for two years with combined illumination yielding an average of 270 g of dry Nannochloropsis sp. biomass per day. More than 2000 L of concentrate suspension (50 g (d. wt) L^–1) of Nannochloropsis sp. were produced and successfully used by fish hatcheries as live feed for rotifers and for rearing seabream larvae with the green-water technique. This study indicates that the annular reactor can be profitably used for long-term cultivation of Nannochloropsis in temperate climates. Besides reliability and ease of operation, the main advantage of the system is that it can be used under natural illumination, yet artificial light can be also supplied to maintain high productivity levels in winter or on cloudy days.     

The air‐lift photobioreactors with flow patterning for high‐density cultures of microalgae and carbon dioxide removal

A photobioreactor containing microalgae is a highly efficient system for converting carbon dioxide (CO₂) into biomass. Using a microalgal photobioreactor as a CO₂ mitigation system is a practical approach to the problem of CO₂ emission from waste gas. In this study, a marine microalga, Chlorella sp. NCTU‐2, was applied to assess biomass production and CO₂ removal. Three types of photobioreactors were designed and used: (i) without inner column (i.e. a bubble column), (ii) with a centric‐tube column and (iii) with a porous centric‐tube column. The specific growth rates (μ) of the batch cultures in the bubble column, the centric‐tube and the porous centric‐tube photobioreactor were 0.180, 0.226 and 0.252 day^?1, respectively. The porous centric‐tube photobioreactor, operated in semicontinuous culture mode with 10% CO₂ aeration, was evaluated. The results show that the maximum biomass productivity was 0.61 g/L when one fourth of the culture broth was recovered every 2 days. The CO₂ removal efficiency was also determined by measuring the influent and effluent loads at different aeration rates and cell densities of Chlorella sp. NCTU‐2. The results show that the CO₂ removal efficiency was related to biomass concentration and aeration rate. The maximum CO₂ removal efficiency of the Chlorella sp. NCTU‐2 culture was 63% when the biomass was maintained at 5.15 g/L concentration and 0.125 vvm aeration (volume gas per volume broth per min; 10% CO₂ in the aeration gas) in the porous centric‐tube photobioreactor.     

Apical F‐actin‐regulated exocytic targeting of NtPPME1 is essential for construction and rigidity of the pollen tube cell wall

In tip‐confined growing pollen tubes, delivery of newly synthesized cell wall materials to the rapidly expanding apical surface requires spatial organization and temporal regulation of the apical F‐actin filament and exocytosis. In this study, we demonstrate that apical F‐actin is essential for the rigidity and construction of the pollen tube cell wall by regulating exocytosis of Nicotiana tabacum pectin methylesterase (NtPPME1). Wortmannin disrupts the spatial organization of apical F‐actin in the pollen tube tip and inhibits polar targeting of NtPPME1, which subsequently alters the rigidity and pectic composition of the pollen tube cell wall, finally causing growth arrest of the pollen tube. In addition to mechanistically linking cell wall construction and apical F‐actin, wortmannin can be used as a useful tool for studying endomembrane trafficking and cytoskeletal organization in pollen tubes.     

Non-destructive measurement of wheat roots in large undisturbed and repacked clay soil cores

Summary Non-destructive observations of root growth and distribution can be obtained from counting root intercepts with observation tubers inserted in the root zone. This paper describes the technique of inserting clear acrylic tubes horizontally into large undisturbed and repacked soil cores. Counts of roots intersecting scribed lines on the sides of the tubes were made with a fibrescope. Comparison was made between observation root tubes of different diameter (25 and 38.5 mm).The r² values for the relationships between root intercept counts and destructively determined values of root length density (RLD) ranged from 0.78 to 0.96. The larger diameter tubes had higher r² values. Theoretical calibration of the technique does not appear to be possible since analysis indicated that fewer roots intersected the scribed lines on the observation tube than would have been expected from a non-disturbed, randomly distributed root system. It is not known if this discrepancy is due to non-randomness or to an artifact associated with the insertion of the observation tube. Roots were not more prolific at the edge of the soil cores. Comparison of values of root length per unit soil surface area, rates of downward root growth and water uptake rates were within the ranges previously reported for wheat roots of field crops grown on clay soils. Observed root growth and distribution was found to be sensitive to four soil and water treatments imposed. It is concluded that the technique will allow quantitative analysis of root growth and distribution in undisturbed soil cores.     

Radiocarbon dating and metal analyses of `fossil' and living tubes of Protula (Annelida: Polychaeta)

Since the initiation of benthic and trawling studies in southern California over 50 years ago, empty calcareous tubes, measuring up to 36 cm in length and 2.1 cm in diameter, have been collected and presumed to belong to the serpulid genus Protula. ¹⁴C dating of these `fossil' tubes, ranged from 631 ±34 to 3987 ±42 yrs. In connection with monitoring programs, living specimens of Protula superba Moore, 1909 were collected from the benthos off southern California. A comparison of the elemental profile of the ancient and extant tubes was conducted to ascertain the effects of time and the recent increase in various anthropogenically mobilized metals on the incorporation of Al, As, Ba, Cd, Ce, Co, Cr, Fe, Mo, Ni, Pb, U, V and Zn in the calcareous tube. Surprisingly, inductively coupled plasma mass spectroscopy (ICP-MS) analysis of the digested tubes showed the concentrations of the elements were generally higher in `fossil' tubes than those obtained from living animals. However, the concentration of elements was higher in `fossil' tubes collected from the vicinity of municipal outfall discharges compared to `fossil' tubes collected from near an offshore island. Laser ablation ICP-MS analyses across transverse sections of `fossil' and living tubes showed spatial heterogeneity of elements in the structure. Elevated signals for Pb, Gd, Ce, La, Pr, Sm, Eu and Nd were observed on both internal and external surfaces on all tubes. Elevated responses for Fe, Zn, Sr, Mn, Tb, Dy, Ho, Er and Bi were primarily noted on the outer surfaces. It is hypothesized that the higher concentrations of metals in the `fossil' tubes may be the result of surface contamination facilitating elemental adsorption or substitution over the years. It is concluded that the use of biomineralized structures as biomonitors of water quality should involve rigorous chelating procedures to remove contaminating metals from lattice bound elements locked in the mineral matrix.     

Tube-building behavior in Grandidierella,and two species of Cerapus

Individuals of Grandidierella bonnieroides and Cerapus sp. R commence tube formation by enrolling themselves in a blanket of detritus and gluing together clumps of the material with strands of silk in a few minutes. The initial tubes are very ragged. G. bonnieroides then expands the initial tube by simply dragging in more detritus. In contrast, Cerapus sp. R. commences building an architectured tube outward from the ragged initial tube using very fine detritus collected either by grasping nearby benthic material or by filter feeding particles from the water column. The diameter of the nearly circular architectured tube is tailored very exactly to the height of the body for which we propose a formula. The more advanced Cerapus sp. K skips the initial phase of blanket-tube formation and only makes short architectured tubes as long as the body, carries these about in a fashion analogous to hermit crabs, and attaches them temporarily to epfloral-faunal substrates well above the sediment surface; therefore, no detritus masses are available to Cerapus sp. K. Tube-building in amphipods is roughly classified into 12 kinds having many subdivisions. Advancement of, or specialization in tube building, lacks apparent correlation with morphological advancement or systematic and evolutionary deployment.     

Chiral separation performance of micrometer‐sized monodispersed silica spheres with high protein loading

Micrometer‐sized monodispersed silica spheres (～360 μm) with high loading of bovine serum albumin (BSA) (～450 mg/g) were prepared as an adsorbent for large‐scale chiral separation. The new adsorbent was characterized with scanning electron microscopy (SEM), nitrogen adsorption‐desorption isotherms, the mercury intrusion method, infrared spectroscopic analysis, and elementary analysis. The extent of chiral separation was tested with rac‐tryptophan (rac‐Trp) and rac‐phenylalanine (rac‐Phe) as solutes. The results showed that the absorbent exhibited a high surface area, large pore volume, and bimodal macromesoporous structures, enabling fast mass transfer and high separation efficiency. A fast adsorption rate, reaching equilibrium in less than 6 min, and a high degree of chiral separation, with the enantiomer excess (e.e.) value reaching as high as 100% in the first 10 min, was observed in a small (5 cm in height, 0.46 cm in internal diameter) packed column that could be regenerated with a pH 5.0 HAc‐NaAc buffer. The results show that monodispersed silica spheres with a high BSA loading have great potential for applications in large‐scale chiral separation processes. Chirality, 2009. © 2008 Wiley‐Liss, Inc.     

Disturbance and investment: developmental responses of tropical lotic midges to repeated tube destruction in the juvenile stages

Abstract. 1. Investment in silken tubes may give tubicolous chironomid larvae more to lose than non‐tube building taxa when physical disturbances or the actions of other organisms induce dispersal. In this study, two chironomid species from flood‐prone Australian tropical streams were subjected to repeated experimental destruction of their larval and pupal tubes in laboratory experiments, with life‐history parameter responses recorded. 2. When subjected to the tube destruction treatments, Echinocladius martini (Orthocladiinae), whose larvae construct branched loose silken tubes in fast flow, showed no reduction in survivorship to adult, whereas Polypedilum australotropicus (Chironominae), which builds compact tightly woven tubes incorporating detrital material within pool leaf packs, showed reduced survivorship of about 25%. 3. For both species, tube destruction reduced pupal duration, regardless of whether destruction occurred in the larval stage only, or in both the larval and pupal stages. This may be a response to the risks of exposure outside tubes in nature, particularly for pupae, which are incapable of spinning silk. 4. Tube destruction caused little or no reduction in adult size, longevity, or female fecundity (oocyte number) for either species, but oocyte development and size was retarded in E. martini females, reflecting the energetic and nutritive costs of the tube destruction treatments. 5. Contrasts in the responses of the two species reflect differences in tube‐building behaviour and in situ habitat use. These results demonstrate that tube loss can affect the fitness of individual chironomids, though both species proved generally resilient, which may reflect their long‐term exposure to variable tropical lotic conditions.     

Histochemical evidence of β‐chitin in parapodial glandular organs and tubes of Spiophanes (Annelida,Sedentaria: Spionidae), and first studies on selected Annelida

A generic character of the genus Spiophanes (Annelida, Sedentaria: Spionidae) is the presence of parapodial glandular organs. Parapodial glandular organs in Spiophanes species include secretory cells with cup‐shaped microvilli, similar to those present in deep‐sea inhabiting vestimentiferans and frenulate Siboglinidae. These cells are supposed to secrete β‐chitin for tube‐building. In this study, transverse histological and/or ultrathin sections of parapodial glandular organs and tubes of Spiophanes spp. as well as of Glandulospio orestes (Spionidae) and Owenia fusiformis (Oweniidae) were examined. Fluorescent markers together with confocal laser scanning microscopy, and Raman spectroscopy were used to detect chitin in the parapodial glandular organs of Spiophanes and/or in the glands of Owenia and Glandulospio. Tubes of these taxa were tested for chitin to elucidate the use of it for tube‐building. The examinations revealed a distinct labelling of the gland contents. Raman spectroscopy documented the presence of β‐chitin in both gland types of Spiophanes. The tubes of Spiophanes were found to have a grid‐like structure that seems to be built with this β‐chitin. Tests of tubes of Dipolydora quadrilobata (Spionidae) for chitin were negative. However, the results of our study provide strong evidence that Spiophanes species, O. fusiformis and probably also G. orestes produce chitin and supposedly use it for tube‐building. This implies that the production of chitin and its use as a constituent part of tube‐building is more widespread among polychaetes as yet known. The histochemical data presented in this study support previous assumptions inferring homology of parapodial glandular organs of Spionidae and Siboglinidae based on ultrastructure. Furthermore, transmission electron microscopy‐based evidence of secretory cells with nail‐headed microvilli in O. fusiformis suggests homology of parapodial grandular organs across annelids including Sipuncula. J. Morphol. 276:1433–1447, 2015. © 2015 Wiley Periodicals, Inc.     

Aristolochia bhamoensis sp. nov. (Aristolochiaceae) and a diagnostic key to all known A. subgen. Siphisia species from Myanmar

Aristolochia bhamoensis from Myanmar is here described and illustrated. This new species is morphologically similar to A. faviogonzalezii (confined to northern Vietnam) and A. cathcartii (growing in the Himalayan region) but is distinguished from the latter two species by having a cream‐white upper part of the perianth tube, with visible purple ridges, a trumpet‐shaped limb, 5.0–5.5 cm diameter, inner surface of limb lobes densely covered with dark‐purple bristles, a nearly circular mouth, 3.2–3.5 cm wide, upper half of throat being dark‐purple to blackish, without striations or dots, and lower half purple, with conspicuous white striation. Morphological characters such as a 3‐lobed gynostemium and a 3‐lobed limb support a placement of the new species in the subgenus Siphisia. A diagnostic key is provided to the seven Siphisia species known from Myanmar.