Advances in using adaptive laboratory evolution technology for engineering of photosynthetic cyanobacteria / 生物工程学报

Cyanobacteria are the only prokaryotes capable of oxygenic photosynthesis, which have potential to serve as "autotrophic cell factories". However, the synthesis of biofuels and chemicals using cyanobacteria as chassis are suffered from poor stress tolerance and low yield, resulting in low economic feasibility for industrial production. Thus, it's urgent to construct new cyanobacterial chassis by means of synthetic biology. In recent years, adaptive laboratory evolution (ALE) has made great achievements in chassis engineering, including optimizing growth rate, increasing tolerance, enhancing substrate utilization and increasing product yield. ALE has also made some progress in improving the tolerance of cyanobacteria to high light intensity, heavy metal ions, high concentrations of salt and organic solvents. However, the engineering efficiency of ALE strategy in cyanobacteria is generally low, and the molecular mechanisms underpinning the tolerance to various stresses have not been fully elucidated. To this end, this review summarizes the ALE-associated technical strategies and their applications in cyanobacteria chassis engineering, following by discussing how to construct larger ALE mutation library, increase mutation frequency of strains and shorten evolution time. Moreover, exploration of the construction principles and strategies for constructing multi-stress tolerant cyanobacteria, and efficient analysis the mutant libraries of evolved strains as well as construction of strains with high yield and strong robustness are discussed, with the aim to facilitate the engineering of cyanobacteria chassis and the application of engineered cyanobacteria in the future.

Ontogenetic change in the diet of Semaprochilodus insignis (Characiformes: Prochilodontidae) during migration between two limnologically distinct environments in the Amazon Basin

Semaprochilodus insignis is an Amazonian migratory fish species, moving in large shoals between white- and black-water rivers. It has long been classified as a detritivorous fish. However, it is possible that the trophic plasticity of S. insignis could be higher than previously assumed. The objective of this study was to investigate the relative contributions of autotrophic energy sources to the diet of S. insignis in the Negro and Solimões rivers and to determine if the species undergoes an ontogenetic change in the diet. We found variations between the δ13C and δ15N values of S. insignis between the rivers. In the Negro River, periphyton (84%) was the principal energy source for adults, while juveniles in the Solimões River foraged predominantly on terrestrial plants/C3 macrophytes (50%) and phytoplankton (42%). These variations in isotopic signatures are likely associated with migratory movements of S. insignis at different life stages and hydrological periods. Instead of the previously assumed dietary classification, we suggest that S. insignis varies its diet ontogenetically, with adults acting as illiophagous in black-water while the young are detritivorous in white-water rivers. The results show that this species creates complex links between food chains, thus emphasizing the importance of conserving flooded areas.(AU)

Semaprochilodus insignis é uma espécie de peixe migratório da Amazônia, movendo-se em cardumes entre rios de água branca e preta. Tem sido classificado como um peixe detritívoro-iliófago. Porém, é possível que a plasticidade trófica de S. insignis seja maior do que se supunha. O objetivo deste estudo foi investigar as contribuições relativas de fontes de energia autotróficas para a dieta do S. insignis nos rios Negro e Solimões e se a espécie muda ontogenticamente a sua dieta. Encontramos variações entre os valores de δ13C e δ15N de S. insignis entre os rios: Negro, o perifíton (84%) foi a principal fonte de energia para adultos, enquanto para os juvenis do rio Solimões, foram predominantemente as plantas terrestres/macrófitas C3 (50%) e fitoplâncton (42%). Essas variações nas assinaturas isotópicas provavelmente estão associadas a movimentos migratórios do S. insignis em diferentes estágios da vida e períodos hidrológicos. Ao invés da classificação alimentar previamente assumida, sugerimos que o S. insignis varie sua dieta ontogeneticamente, os adultos são iliófagos em águas negras, enquanto os jovens são detritívoros em rios de águas brancas. Os resultados demonstram que essa espécie promove ligações complexas entre as cadeias alimentares enfatizando, dessa forma, a importância da conservação das áreas inundadas amazônicas.(AU)

Micropropagación de plantas de lechosa en recipientes de inmersión temporal a partir de brotes axilares / Micropropagation of papaya plants in temporary immersion recipients from axilary shoots

Se estandarizaron las condiciones de iniciación, multiplicación, enraizamiento y aclimatización de plantas hermafroditas de lechosa cv Maradol provenientes de brotes axilares, producidos en recipientes de inmersión temporal RITA®. En cada envase, contentivo de 200 ml de medio de cultivo líquido de Fitch, se colocaron cuatro brotes de 2 a 3 cm de longitud. Los biorreactores se conectaron a tres líneas de inmersión de 5, 2 y 1 min cada 4h y se colocaron 6 envases en promedio por línea, en condiciones de fotoperíodo de 16 h. Transcurridos 30 a 45 días, se cuantificaron los brotes y se clasificaron de acuerdo al tamaño: < 2 cm (pequeños), entre 2 a 3 cm (medianos), > 3 cm con y sin raíz (grandes). Los dos primeros tipos de brotes se continuaron multiplicando en los mismos medios; y los más elongados se aclimatizaron utilizando el Sistema Autotrófico Hidropónico (SAH). Se determinó la sanidad y la fidelidad de las plantas producidas mediante pruebas de ELISA y RAPD, respectivamente. Durante un periodo de 6 meses se reciclaron un total de 47 recipientes, los cuales produjeron 1.091 brotes: 377 pequeños; 482 medianos; 175 grandes sin raíz y 57 con raíz. Usando el SAH se obtuvo 89,5% de plantas aclimatizadas cuando se usaron brotes enraizados, y 41,6% a partir de brotes sin raíces. Con la combinación de las técnicas RITA y SAH se logró un sistema continuo y eficiente de producción de plantas sanas y fieles al tipo, en comparación con los métodos convencionales de micropropagación y aclimatización.

We standardized initiation, multiplication, rooting and acclimatization conditions of papaya cv Maradol hermaphrodite plants from axillary buds produced in temporary immersion reactor RITA®. Recipients contained 200 ml of Fitch liquid culture medium, and four shoots of 2 to 3 cm. in length were placed in each. The bioreactors were connected to three different immersion lines of 5, 2, and 1 min each 4h, with 6 containers per line on average, in 16 h photoperiod. After 30 to 45 days, the shoots produced were quantified and classified according to size: <2 cm (small), from 2 to 3 cm (medium), >3 cm with or without roots (large). The first two types of shoots were multiplied in the same culture media, and more elongated shoots were acclimatized using Autotrophic Hydroponic System (AHS). The sanity and fidelity of the produced plants were determined using ELISA and RAPD, respectively. For a period of six months 47 vessels were recycled and 1,091 shoots were produced: 377 small; 482 medium; 175 large without roots and 57 rooted shoots. Using AHS, 89.5% acclimatized plants were obtained when rooted shoots were used, and 41.6% from rootless shoots. With the combination of RITA and AHS techniques we achieved a continuous and efficient production of healthy and true to type papaya plants, in comparison to conventional micropropagation and acclimatization procedures.

The abundance and diversity of ammonia-oxidizing bacteria in activated sludge under autotrophic domestication.

Ammonia-oxidizing bacteria (AOB) play a key role in nitrogen-removal wastewater treatment plants (WWTPs) as they can transform ammonia into nitrite. AOB can be enriched in activated sludge through autotrophic domestication although they are difficult to be isolated. In this study, autotrophic domestication was carried out in a lab-scale sequencing-batch-reactor (SBR) system with two activated sludge samples. The ammonia removal capacity of the sludge samples increased during the domestication, and pH exhibited a negative correlation with the ammonia removal amount, which indicated that it was one important factor of microbial ammonia oxidation. The count of AOB, measured by the most probable number (MPN) method, increased significantly during autotrophic domestication as ammonia oxidation efficiency was enhanced. We investigated the changes in the community structure of AOB before and after domestication by amoA clone library and T-RFLP profile. It showed that AOB had been successfully enriched and the community structure significantly shifted during the domestication. Two groups of AOB were found in sludge samples: Nitrosomonas-like group remained predominant all the time and Nitrosospira-like group changed obviously. Simultaneously, the total heterotrophic bacteria were investigated by MPN and Biolog assay. The metabolic diversity of heterotrophs had changed minutely, although the count of them decreased significantly and lost superiority of microbial communities in the sludge.

Evaluación de un sistema para la micorrización in vitro en plantas demora de castilla (Rubus glaucus, Benth) / Evaluation of an in vitro mycorrhization system of blackberry plants (Rubus glaucus, Benth) / Avaliação de um sistema para micorrização in vitro em plantas de amora-preta (Rubus glaucus, Benth)

Objetivo. Obtener un sistema de micorrización in vitro en sistemas de cultivo autotrófico para plantas de mora de castilla (Rubusglaucus, Benth). Materiales y métodos. Se utilizaron esporas y fragmentos de raíces con vesículas del Hongo Formador de MicorrizaArbuscular (HFMA) Glomus sp. (GEV02). Se estableció un sistema de cultivo autotrófico para plántulas de mora, comparando dosmétodos de inoculación directa con el HFMA. Se cuantificó el número de esporas producidas, la longitud del micelio extraradical; asícomo el porcentaje de colonización del HFMA. Adicionalmente se midió la longitud aérea y radical, el peso fresco y seco de la partefoliar y radical para determinar el desarrollo de las plantas. Resultados. El sistema de cultivo autotrófico fue exitoso para plantas demora de castilla (Rubus glaucus, Benth); observándose un óptimo crecimiento de la parte aérea y radical de la planta. Adicionalmenteen este estudio se pudo obtener un sistema que permitió el desarrollo de Glomus sp (GEV02) bajo condiciones in vitro, con formaciónde estructuras típicas de la simbiosis como una buena colonización intraradical, con producción de arbúsculos y vesículas, así comoel desarrollo de micelio extraradical con hifas ramificadas y la formación de nuevas esporas. Conclusión. Las plantas de moramicropropagadas se asociaron con éxito, por primera vez, con un hongo formador de micorriza arbuscular bajo condiciones in vitro,permitiendo el desarrollo del sistema simbiótico HFMA Glomus sp., asociado a las raíces de plántulas de mora castilla micropropagadas...

Objective. Obtain an in vitromycorrhization system in autotrophic culture systems of blackberry plants (Rubus glaucus, Benth). Materials and methods. We usedspores and root fragments with vesicles of Arbuscular Mycorrhizal Fungus (AMF) Glomus sp (GEV02). We established an autotrophicculture system of blackberry plantlets comparing two methods of direct inoculation of the AMF. We measured the number of sporesproduced, the length of the extraradical mycelium as well as the percentage of colonization of the AMF. Additionally, we measuredthe shoot and root length, and the fresh and dry weight of the leaf and root parts to determine the plant development. Results. Theautotrophic culture system was successful for blackberry plants (Rubus glaucus, Benth; an optimal shoot and root growth was observed.Additionally, we obtained a system that allowed the development of Glomus sp. in in vitro conditions, with the formation of structurestypical of the symbiosis as well as a good intraradical colonization, with the production of arbuscules and vesicles, development ofextraradical mycelium with branched hyphae, and formation of new spores. Conclusion. For the first time, micropropagated blackberryplants associated successfully with an AMF under in vitro conditions, enabling the development of the symbiotic system AMF Glomussp. associated to roots of micropropagated blackberry plantlets...

Objetivo. Obter umsistema de micorrização in vitro em sistemas de cultura autotróficos para plantas de amora-preta (Rubus glaucus, Benth). Materiais emétodos. Foram usados esporos e fragmentos de raízes com vesículas do Fungo Formador Micorrízico Arbuscular (FFMA) Glomus sp.(GEV02). Foi estabelecido um sistema de cultivo autotrófico para mudas de amora-preta, comparando dois métodos de inoculação diretacom o FFMA. Foi quantificado o número de esporos produzidos, o comprimento do micélio extra radicular; bem como a porcentagemde colonização do FFMA. Além disso, foi medido o comprimento e o peso fresco e seco da parte folhar e radicular para determinar odesenvolvimento das plantas. Resultados. O sistema de cultivo autotrófico foi bem-sucedido para as plantas de amora-preta (Rubusglaucus, Benth), onde foi observado um crescimento ótimo da parte aérea e da raidicular da planta. Além disso, neste estudo foi obtido umsistema que permitiu o desenvolvimento de Glomus sp (GEV02) sob condições in vitro, com formação de estruturas típicas da simbiosecomo uma boa colonização intra radicular, com produção de arbúsculos e vesículas, assim como o desenvolvimento de micélio extraradicular com hifas ramificada e a formação de novos esporos. Conclusão. As plantas de amora-preta micropropagadas associaram-secom sucesso, pela primeira vez, com um fungo formador micorrízico arbuscular em condições in vitro, permitindo o desenvolvimentodo sistema simbiótico FFMA Glomus sp., associado às raízes das plântulas de amora-preta micropropagadas...

Analysis of Microbial Flora in Full Autotrophic Ammonium Removal System / 微生物学通报

The microbial flora of fungi,bacterial and actinomycetes in full autotrophic ammonium removal reactor and activated sludge was analyzed,and the amount of nitrite-oxidizing bacteria and ammonia-oxidizing bacteria was also compared.The result showed that the population,species,species number and dominates of microorganisms in full autotrophic ammonium removal reactor were different from that in activated sludge.In full autotrophic ammonium removal reactor,the amount of ammonia-oxidizing bacteria was increased remarkably which indicated that the accumulation of ammonia-oxidizing bacteria was a remarkable feature of full autotrophic ammonium removal system.

Nitrification and simultaneous denitrification by Azospirillum brasilense 12S.

Azospirillum brasilense, an associative diazotrophs from sorghum roots grows autotrophically on NH + 4 and CaCO3. NH + 4 a is also oxidized to NO – 2 and then denitrified. Addition of malate to the autotrophic medium enhances both NH + 4 oxidation as well as NO – 2 dissimilation. The incomplete nitrification linked denitrification results in a rapid loss of nitrogen from the growth medium. The bacterium also shows assimilatory NO3− and NO 2 reductases and fixes nitrogen at < 50 μg N/ml of NH + 4 , NO−3 or NO-2.

Correlation between inhibition of photosynthesis and growth of Chlorella treated with methyl parathion.

When methyl parathion (Ο,Ο dimethyl O p nitrophenyl phosphorothioate), an organophosphorous insecticide, was added to an exponentially growing culture of Chlorella protothecoides and the effects were followed for 12 days, the following observations were made: a) In autotrophic culture the cell number and the chlorophyll content decreased as compared to the control. These changes paralleled the inhibition of the rate of net photosynthesis, suggesting that the photosynthetic apparatus was the primary target of the insecticide action. b) The inhibition of cell growth (on cell number basis) also occurred in the case of heterotrophic culture at 100 μΜ insecticide concentration but the inhibition was less as compared to that of an autotrophic culture. c) The cell diameter in treated culture increased by 10 20% in both autotrophic and heterotrophic cultures. The observations, (b) and (c) suggest that apart from the photosynthetic apparatus, the insecticide has other sites of action, but the sensitivity of these sites to the insecticide is less than that of the photosynthetic apparatus.