A Novel Algicidal Bacterium, Microbulbifer sp. YX04, Triggered Oxidative Damage and Autophagic Cell Death in Phaeocystis globosa, Which Causes Harmful Algal Blooms.

Phaeocystis globosa causes severe marine pollution by forming harmful algal blooms and releasing hemolytic toxins and is therefore harmful to marine ecosystems and aquaculture industries. In this study, Microbulbifer sp. YX04 exerted high algicidal activity against P. globosa by producing and secreting metabolites. The algicidal activity of the YX04 supernatant was stable after exposure to different temperatures (-80 to 100°C) and pH values (4 to 12) for 2 h, suggesting that algicidal substances could temporarily be stored under these temperature and pH value conditions. To explore the algicidal process and mechanism, morphological and structural changes, oxidative stress, photosynthesis, autophagic flux, and global gene expression were investigated. Biochemical analyses showed that the YX04 supernatant induced reactive oxygen species (ROS) overproduction, which caused lipid peroxidation and malondialdehyde (MDA) accumulation in P. globosa. Transmission electron microscopy (TEM) observation and the significant decrease in both maximum photochemical quantum yield (Fv/Fm) and relative electron transfer rate (rETR) indicated damage to thylakoid membranes and destruction of photosynthetic system function. Immunofluorescence, immunoblot, and TEM analyses indicated that cellular damage caused autophagosome formation and triggered large-scale autophagic flux in P. globosa. Transcriptome analysis revealed many P. globosa genes that were differentially expressed in response to YX04 stress, most of which were involved in photosynthesis, respiration, cytoskeleton, microtubule, and autophagosome formation and fusion processes, which may trigger autophagic cell death. In addition to P. globosa, the YX04 supernatant showed high algicidal activity against Thalassiosira pseudonana, Thalassiosira weissflogii, Skeletonema costatum, Heterosigma akashiwo, and Prorocentrum donghaiense. This study highlights multiple mechanisms underlying YX04 supernatant toxicity toward P. globosa and its potential for controlling the occurrence of harmful algal blooms. IMPORTANCEP. globosa is one of the most notorious harmful algal bloom (HAB)-causing species, which can secrete hemolytic toxins, frequently cause serious ecological pollution, and pose a health hazard to animals and humans. Hence, screening for bacteria with high algicidal activity against P. globosa and studies on the algicidal characteristics and mechanism will contribute to providing an ecofriendly microorganism-controlling agent for preventing the occurrence of algal blooms and reducing the harm of algal blooms to the environment. Our study first reported the algicidal characteristic and mechanism of Microbulbifer sp. YX04 against P. globosa and demonstrated that P. globosa shows different response mechanisms, including movement ability, antioxidative systems, photosynthetic systems, gene expression, and cell death mode, to adapt to the adverse environment when algicidal compounds are present.

Fe2P-decorated N,P Codoped Carbon Synthesized via Direct Biological Recycling for Endurable Sulfur Encapsulation.

In spite of the great potential in leading next-generation energy storage technology, Li-S batteries suffer rapid capacity decay arising from the shuttling effect of lithium polysulfides (LiPSs), a major concern that must be addressed before commercialization can be realized. To tackle this challenge, we demonstrate a facile approach to fabricate a hierarchically structured composite of Fe2P@nitrogen, phosphorus codoped carbon (Fe2P@NPC) by direct biological recycling of iron metal from electroplating sludge using bacteria. This material, featuring uniform dispersion of Fe2P nanoparticles (NPs) in porous NPC matrix, effectively adapts volume variation of sulfur upon cycling and simultaneously provides multiple channels for efficient lithium ion transport. In addition, Fe2P NPs with strong adhesion properties of tightly anchored soluble LiPSs formed during discharge can significantly facilitate the decomposition of Li2S during the subsequent charging process. The Li-S cell built on this cathode architecture delivers high specific capacity (1555.7 mAh g-1 at 0.1 C), appreciable rate capability (679.7 mAh g-1 at 10 C), and greatly enhanced cycling performance (761.9 mAh g-1 at 1.0 C after 500 cycles).

Construction of heterostructured NiFe2O4-C nanorods by transition metal recycling from simulated electroplating sludge leaching solution for high performance lithium ion batteries.

NiFe2O4 has been regarded as one of the promising candidates for lithium-ion battery (LIB) anode materials due to its high theoretical specific capacity. However, the large volume expansion and pulverization of NiFe2O4 during the charge/discharge process result in severe capacity fading. Herein, heterostructured NiFe2O4-C nanorods have been successfully fabricated by recovering transition metals from simulated electroplating sludge leaching solution. The constructed NiFe2O4-C heterointerface plays a vital role in accommodating volume change, stabilizing the reaction products and providing rapid electron and Li+ ion transportation ability, resulting in a high and stable Li+ accommodation performance. The fabricated NiFe2O4-C nanorods demonstrate a high specific capacity (889.9 mA h g-1 at 100 mA g-1), impressive rate capability (861.5, 704.5, 651.4, 579.6 and 502.1 mA h g-1 at 0.2, 0.6, 1.0, 2.0 and 5.0 A g-1) and cycling stability (650.2 mA h g-1 at 2 A g-1 after 500 cycles). This work exemplifies a facile and effective approach for the fabrication of high performance LIB electrode materials by recycling metals from electroplating sludge in an application-oriented manner.

Effective harvesting of the marine microalga Thalassiosira pseudonana by Marinobacter sp. FL06.

In this study, Marinobacter sp. FL06 was used to effectively harvest the energy-producing microalga Thalassiosira pseudonana through direct flocculation. Strain FL06 showed 92.7% flocculating efficiency against T. pseudonana, and no metal ion was added for the flocculation process, resulting in a more environmentally friendly process. The flocculation efficiency of FL06 was stable over a wide range of pH values and temperatures, indicating that the application of this bacteria has potential advantages under various conditions. Strain FL06 also exhibited flocculation activity against different microalgae, indicating that the strain can be used to harvest multiple types of microalgae. Strain FL06 showed high chemotactic ability toward algal cells, suggesting that chemotaxis is important for flocculation. This study provides the first demonstration that the Marinobacter genus could be used to harvest T. pseudonana biomass. In summary, the results showed that FL06 has the potential for effective harvesting of microalgal biomass.

Fast-growing algicidal Streptomyces sp. U3 and its potential in harmful algal bloom controls.

To find the potential algicidal microorganisms and apply them to prevent and terminate harmful algal blooms (HABs), we isolated an actinomycete U3 from Mangrove, which had a potent algicidal effect on the harmful alga Heterosigma akashiwo. It could completely lyse the algal cells by producing active compounds, which were highly sensitive to high temperature and strong alkaline, but resistant to acid. One µg/mL of crude extract of the fermentation supernatant could kill 70% of H. akashiwo cells in 3 d. Unlike most of the other known algicidal Streptomyces, U3 showed strong ability of proliferation with the algal inclusion as the nutrient source. The washed mycelial pellets also gradually exhibited significant algicidal effect during the visible growth in the algal culture. It suggests that U3 could efficiently absorb nutrients from algal culture to support its growth and produce algicidal compounds that might cause the autophagy of algal cells. Therefore, applying U3, as a long-term and environmentally friendly bio-agent to control the harmful blooms of H. akashiwo, would be effective and promising. And the decrease of bioavailable DOM and increase of bio-refractory DOM during the algicidal process of U3 provided new insights into the ecological influence of algicial microorganisms on marine ecosystem.

The algicidal mechanism of prodigiosin from Hahella sp. KA22 against Microcystis aeruginosa.

In recent years, Microcystis aeruginosa blooms have occurred throughout the world, causing huge economic losses and destroying aquatic ecosystems. It is necessary to develop effective and ecofriendly methods to control M. aeruginosa blooms. Here, we report a high algicidal activity of prodigiosin (PG) against M. aeruginosa as well as the algicidal mechanism. PG showed high algicidal activity against M. aeruginosa, with a 50% lethal dose (LD50) of 5.87 µg/mL in 72 h. A combination of methods, including propidium iodide and Annexin V-fluorescein staining assays and light and electron microscopy indicated the existence of two modes of cell death with features similar to those in eukaryotic programmed cell death: necrotic-like and apoptotic-like. Biochemical and physiological analyses showed that PG generates reactive oxygen species (ROS), which induce lipid peroxidation, damage the membrane system and destroy the function of the photosystem. A proteomics analysis revealed that many proteins were differentially expressed in response to PG stress and that most of these proteins were involved in important metabolic processes, which may trigger necrotic-like or apoptotic-like cell death. The present study sheds light on the multiple toxicity mechanisms of PG on M. aeruginosa and its potential for controlling the occurrence of M. aeruginosa blooms in lakes.

Rhizobium albus sp. nov., Isolated from Lake Water in Xiamen, Fujian Province of China.

A Gram-stain-negative, aerobic bacterial strain, designated Y21T, was isolated from surface lake water in Xiamen, Fujian Province of China. Growth was observed at temperatures from 4 to 37 °C, at salinities from 0 to 7.0 % and at pH from 6.0 to 10.0. Optimum growth was observed at 28 °C, at pH 7.0 and with 1.5-2.0 % (w/v) NaCl. The highest similarity of 16S rRNA gene sequence between strain Y21T and the other strains was 96.9 %. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain was a member of the genus Rhizobium, forming a distinct lineage with R. subbaraonis KCTC 23614T. The dominant fatty acids were summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c), C18:1 ω7c 11-methyl, which accounted for 78.1 %. The G+C content of the chromosomal DNA was 60.9 mol%. The predominant respiratory quinone was ubiquinone-10. The polar lipids of strain Y21T were found to consist of five unidentified phospholipids and three unidentified aminolipids. According to its morphology, physiology, fatty acid composition and 16S rRNA sequence data, strain Y21T should be regarded as a new species of the genus Rhizobium, for which Rhizobium albus sp. nov. is proposed (type strain Y21T = MCCC 1F01210T = KCTC 42252T).

A comprehensive insight into functional profiles of free-living microbial community responses to a toxic Akashiwo sanguinea bloom.

Phytoplankton blooms are a worldwide problem and can greatly affect ecological processes in aquatic systems, but its impacts on the functional potential of microbial communities are limited. In this study, a high-throughput microarray-based technology (GeoChip) was used to profile the functional potential of free-living microbes from the Xiamen Sea Area in response to a 2011 Akashiwo sanguinea bloom. The bloom altered the overall community functional structure. Genes that were significantly (p < 0.05) increased during the bloom included carbon degradation genes and genes involved in nitrogen (N) and/or phosphorus (P) limitation stress. Such significantly changed genes were well explained by chosen environmental factors (COD, nitrite-N, nitrate-N, dissolved inorganic phosphorus, chlorophyll-a and algal density). Overall results suggested that this bloom might enhance the microbial converting of nitrate to N2 and ammonia nitrogen, decrease P removal from seawater, activate the glyoxylate cycle, and reduce infection activity of bacteriophage. This study presents new information on the relationship of algae to other microbes in aquatic systems, and provides new insights into our understanding of ecological impacts of phytoplankton blooms.

First Evidence of Altererythrobacter sp. LY02 with Indirect Algicidal Activity on the Toxic Dinoflagellate, Alexandrium tamarense.

Alexandrium tamarense is a toxic harmful algal blooms (HABs) causing species, which poses great threat to human health and marine economy. In this study, we isolated an algicidal bacterium Altererythrobacter sp. LY02 towards to A. tamarense and later investigated the algicidal activity, algicidal mode, characteristics of algicidal active substance and algicidal procedure. The results indicated that the cell-free filtrate of strain LY02 showed high algicidal effect on algal growth, however, bacterial cells almost lost algicidal activity. The algicidal active substance was temperature- and pH-stability, and its molecular weight was less than 1000 Da, and was a non-proteinaceous material or non-polysaccharide, mid-polar substance. Under the algicidal effect of active substance, the morphology and structure of A. tamarense cells were seriously damaged as well as organelles. Our study confirmed that the algicidal active substance could be used as an excellent bio-agent for controlling HABs caused by A. tamarense.

Chitinase producing bacteria with direct algicidal activity on marine diatoms.

Chitinase producing bacteria can involve extensively in nutrient cycling and energy flow in the aquatic environment through degradation and utilization of chitin. It is well known that diatoms cells are encased by box-like frustules composed of chitin. Thus the chitin containing of diatoms shall be a natural target of chitinase producing bacteria, however, the interaction between these two organismic groups has not been studied thus far. Therefore, in this study, the algicidal mechanism of one chitinase producing bacterium (strain LY03) on Thalassiosira pseudonana was investigated. The algicidal range and algicidal mode of strain LY03 were first studied, and then bacterial viability, chemotactic ability and direct interaction characteristic between bacteria and diatom were also confirmed. Finally, the characteristic of the intracellular algicidal substance was identified and the algicidal mechanism was determined whereby algicidal bacterial cells showed chemotaxis to algal cells, fastened themselves on algal cells with their flagella, and then produced chitinase to degrade algal cell walls, and eventually caused algal lysis and death. It is the first time to investigate the interaction between chitinase producing bacteria and diatoms, and this novel special interaction mode was confirmed in this study, which will be helpful in protection and utilization of diatoms resources.

Lysing bloom-causing alga Phaeocystis globosa with microbial algicide: An efficient process that decreases the toxicity of algal exudates.

Algicidal microbes could effectively remove the harmful algae from the waters. In this study, we were concerned with the ecological influence of an algicide extracted from Streptomyces alboflavus RPS, which could completely lyse the Phaeocystis globosa cells within two days. In microcosms, 4 µg/mL of the microbial algicide could efficiently remove P. globosa cells without suppressing other aquatic organisms. Bioluminescent assays confirmed that the toxicity of microbial algicide at this concentration was negligible. Interestingly, the toxicity of P. globosa exudates was also significantly reduced after being treated with the algicide. Further experiments revealed that the microbial algicide could instantly increase the permeability of the plasma membrane and disturb the photosynthetic system, followed by the deformation of organelles, vacuolization and increasing oxidative stress. The pre-incubation of N-acetyl cysteine (NAC) verified that the rapid damages to the plasma membrane and photosynthetic system caused the algal death in the early phase, and the increasing oxidative stress killed the rest. The late accumulation and possible release of CAT also explained the decreasing toxicity of the algal culture. These results indicated that this microbial algicide has great potential in controlling the growth of P. globosa on site.

Photoinhibition of Phaeocystis globosa resulting from oxidative stress induced by a marine algicidal bacterium Bacillus sp. LP-10.

Harmful algal blooms caused by Phaeocystis globosa have resulted in staggering losses to coastal countries because of their world-wide distribution. Bacteria have been studied for years to control the blooms of harmful alga, however, the action mechanism of them against harmful algal cells is still not well defined. Here, a previously isolated algicidal bacterium Bacillus sp. LP-10 was used to elucidate the potential mechanism involved in the dysfunction of P. globosa algal cells at physiological and molecular levels. Our results showed Bacillus sp. LP-10 induced an obvious rise of reactive oxygen species (ROS), which was supposed to be major reason for algal cell death. Meanwhile, the results revealed a significant decrease of photosynthetic physiological indexes and apparent down-regulated of photosynthesis-related genes (psbA and rbcS) and protein (PSII reaction center protein D1), after treated by Bacillus sp. LP-10 filtrates, suggesting photoinhibition occurred in the algal cells. Furthermore, our results indicated that light played important roles in the algal cell death. Our work demonstrated that the major lethal reason of P. globosa cells treated by the algicidal bacterium was the photoinhibition resulted from oxidative stress induced by Bacillus sp. LP-10.

The death mechanism of the harmful algal bloom species Alexandrium tamarense induced by algicidal bacterium Deinococcus sp. Y35.

Harmful algal blooms (HABs) cause a variety of deleterious effects on aquatic ecosystems, especially the toxic dinoflagellate Alexandrium tamarense, which poses a serious threat to marine economic and human health based on releasing paralytic shellfish poison into the environment. The algicidal bacterium Deinococcus sp. Y35 which can induce growth inhibition on A. tamarense was used to investigate the functional mechanism. The growth status, reactive oxygen species (ROS) content, photosynthetic system and the nuclear system of algal cells were determined under algicidal activity. A culture of strain Y35 not only induced overproduction of ROS in algal cells within only 0.5 h of treatment, also decrease the total protein content as well as the response of the antioxidant enzyme. Meanwhile, lipid peroxidation was induced and cell membrane integrity was lost. Photosynthetic pigments including chlorophyll a and carotenoid decreased along with the photosynthetic efficiency being significantly inhibited. At the same time, photosynthesis-related gene expression showed down-regulation. More than, the destruction of cell nuclear structure and inhibition of proliferating cell nuclear antigen (PCNA) related gene expression were confirmed. The potential functional mechanism of the algicidal bacterium on A. tamarense was investigated and provided a novel viewpoint which could be used in HABs control.

Effective harvesting of the microalgae Chlorella vulgaris via flocculation-flotation with bioflocculant.

In this study, bioflocculant from Cobetia marina L03 could be used for effective harvesting of the microalgae Chlorella vulgaris via flocculation-flotation. A flotation efficiency of 92.7% was observed when 20 mg L(-1) bioflocculant was tested for flocculating the microalgal cells with 5mM CaCl2. The bioflocculant was stable at wide ranges of pH and temperature, which is advantageous for its application under various conditions. Chemical analysis of the bioflocculant indicated that it is composed of 31.6% total sugar and 0.2% protein (w/w). This bioflocculant has potential for the high-efficiency harvesting of microalgae and may be useful in reducing one of the barriers to microalgal biofuel production.

Phaeodactylibacter luteus sp. nov., isolated from the oleaginous microalga Picochlorum sp.

A Gram-staining-negative, orange-pigmented, non-motile, aerobic bacterial strain, designated GYP20T, was isolated from a culture of the alga Picochlorum sp., a promising feedstock for biodiesel production, which was isolated from the India Ocean. Growth was observed at temperatures from 20 to 37 °C, salinities from 0 to 3% and pH from 5 to 9.Mg2+ and Ca2+ ions were required for growth. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain was a member of the genus Phaeodactylibacter, which belongs to the family Saprospiraceae. Strain GYP20T was most closely related to Phaeodactylibacter xiamenensis KD52T (95.5% sequence similarity). The major fatty acids were iso-C15 : 1 G, iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3. The predominant respiratory quinone was menaquinone-7 (MK-7). The polar lipids of strain GYP20T were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four unidentified glycolipids, two unidentified phospholipids and three unidentified aminolipids. According to its morphology, physiology, fatty acid composition and 16S rRNA sequence data, the novel strain most appropriately belongs to the genus Phaeodactylibacter, but can readily be distinguished from Phaeodactylibacter xiamenensis GYP20T. The name Phaeodactylibacter luteus sp. nov. is proposed with the type strain GYP20T ( = MCCC 1F01222T = KCTC 42180T).

Erythrobacter luteus sp. nov., isolated from mangrove sediment.

A Gram-staining-negative, orange-pigmented, aerobic bacterial strain, designated KA37T, was isolated from a mangrove sediment sample collected from Yunxiao mangrove National Nature Reserve, Fujian Province, China. Growth was observed at 4-37 °C, 0-3% (w/v) NaCl and pH 5-10. Mg2+ ions were required for growth. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the isolate was a member of the genus Erythrobacter, which belongs to the family Erythrobacteraceae. Strain KA37T was most closely related to Erythrobacter gangjinensis KCTC 22330T (96.9% sequence similarity), followed by Erythrobacter marinus KCTC 23554T (96.8%); similarity to other members of the genus was below 96.6%. The major fatty acids were C17 : 1ω6c, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). Strain KA37T did not produce bacteriochlorophyll a. The predominant respiratory quinone was ubiquinone 10 (Q-10). The polar lipids of strain KA37T were sphingoglycolipid, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, five unknown lipids and one unidentified phospholipid. According to its morphology, physiology, fatty acid composition and 16S rRNA sequence, the novel strain most appropriately belongs to the genus Erythrobacter, but can be distinguished readily from species of the genus Erythrobacter with validly published names. The name Erythrobacter luteus sp. nov. is proposed, with strain KA37T ( = MCCC 1F01227T = KCTC 42179T) as the type strain.

The first evidence of deinoxanthin from Deinococcus sp. Y35 with strong algicidal effect on the toxic dinoflagellate Alexandrium tamarense.

Harmful algal blooms (HABs) could be deemed hazardous materials in aquatic environment. Alexandrium tamarense is a toxic HAB causing alga, which causes serious economic losses and health problems. In this study, the bacterium Deinococcus xianganensis Y35 produced a new algicide, showing a high algicidal effect on A. tamarense. The algicidal compound was identified as deinoxanthin, a red pigment, based on high resolution mass spectrometry and NMR after the active compound was isolated and purified. Deinoxanthin exhibited an obvious inhibitory effect on algal growth, and showed algicidal activity against A. tamarense with an EC50 of 5.636 µg/mL with 12h treatment time. Based on the unique structure and characteristics of deinoxanthin, the content of reactive oxygen species (ROS) increased after 0.5h exposure, the structure of organelles including chloroplasts and mitochondria were seriously damaged. All these results firstly confirmed that deinoxanthin as the efficient and eco-environmental algicidal compound has potential to be used for controlling harmful algal blooms through overproduction of ROS.

Comprehensive insights into the response of Alexandrium tamarense to algicidal component secreted by a marine bacterium.

Harmful algal blooms occur throughout the world, threatening human health, and destroying marine ecosystems. Alexandrium tamarense is a globally distributed and notoriously toxic dinoflagellate that is responsible for most paralytic shellfish poisoning incidents. The culture supernatant of the marine algicidal bacterium BS02 showed potent algicidal effects on A. tamarense ATGD98-006. In this study, we investigated the effects of this supernatant on A. tamarense at physiological and biochemical levels to elucidate the mechanism involved in the inhibition of algal growth by the supernatant of the strain BS02. Reactive oxygen species (ROS) levels increased following exposure to the BS02 supernatant, indicating that the algal cells had suffered from oxidative damage. The levels of cellular pigments, including chlorophyll a and carotenoids, were significantly decreased, which indicated that the accumulation of ROS destroyed pigment synthesis. The decline of the maximum photochemical quantum yield (Fv/Fm) and relative electron transport rate (rETR) suggested that the photosynthesis systems of algal cells were attacked by the BS02 supernatant. To eliminate the ROS, the activities of antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT), increased significantly within a short period of time. Real-time PCR revealed changes in the transcript abundances of two target photosynthesis-related genes (psbA and psbD) and two target respiration-related genes (cob and cox). The transcription of the respiration-related genes was significantly inhibited by the treatments, which indicated that the respiratory system was disturbed. Our results demonstrate that the BS02 supernatant can affect the photosynthesis process and might block the PS II electron transport chain, leading to the production of excessive ROS. The increased ROS can further destroy membrane integrity and pigments, ultimately inducing algal cell death.

Mameliella phaeodactyli sp. nov., a member of the family Rhodobacteraceae isolated from the marine algae Phaeodactylum tricornutum.

A novel Gram-staining-negative, aerobic, rod-shaped, non-motile, yellow bacterium designated strain KD53(T), was isolated from a culture of the alga Phaeodactylum tricornutum from Xiamen, Fujian Province, China. 16S rRNA gene sequence comparison showed that strain KD53(T) was a member of the Roseobacter clade within the family Rhodobacteraceae , forming a distinct lineage with species of the genus Mameliella . The 16S rRNA gene sequence similarities between strain KD53(T) and other strains examined were all less than 97.0%. Strain KD53(T) was found to grow optimally at 28 °C, at pH 7.5-8.0 and in the presence of 3% (w/v) NaCl. The dominant fatty acids of strain KD53(T) were C18 : 1ω6c and/or C18 : 1ω7c, C18 : 0 and C16 : 0. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content was 65 mol% and the major respiratory quinone was ubiquinone 10 (Q-10). On the basis of phenotypic data and phylogenetic inference, strain KD53(T) represents a novel member of the genus Mameliella , then the name Mameliella phaeodactyli sp. nov. is proposed. The type strain is KD53(T) ( =MCCC 1K00273(T) =KCTC 42178(T)).