Non-sterile Growth Research Articles

Indole-3-Acetic Acid Action in Outdoor and Indoor Cultures of Spirulina in Open Raceway Reactors

A significant research gap exists in investigating large-scale microalgae cultures exposed to outdoor conditions, with the addition of phytohormones using non-sterile growth media. Implementing these conditions is crucial for verifying the industrial viability of this strategy. This study aimed to evaluate the effect of indole-3-acetic acid (IAA) supplementation on Spirulina sp. LEB 18 cultures conducted indoors and outdoors in raceway bioreactors. The outdoor experiments were performed under uncontrolled environmental conditions. The indoor cultures were maintained within a thermostat-controlled chamber at a consistent temperature and lighting intensity. The outdoor experiments supplemented with IAA achieved a biomass concentration of 5.43 g L−1 and productivity of 173.9 mg L−1 d−1. These values increased 122.5% and 130.9% in biomass concentration and productivity, respectively, compared to the indoor experiments with the same supplementation. Moreover, roughly half of the biomass generated from outdoor cultivation with IAA consisted of carbohydrates (45%). Compared to indoor cultivation, this approach reduced production costs for biomass (55%) and lowered production costs for carbohydrates, proteins, and lipids by 86%, 44%, and 50%, respectively. The successful application of phytohormones in microalgae cultures, particularly under larger scale, nonsterile, and outdoor conditions, represents a significant advancement toward industrial implementation.

Dual-function oleaginous biocatalysts for non-sterile cultivation and solvent-free biolipid bioextraction to reduce biolipid-based biofuel production costs

Triacylglycerols (TAGs) are starting materials for the production of biolipid-based fuels such as biodiesel and biojet fuel. While various microorganisms can produce TAGs from renewable resources, the cultivation of TAG-producing microorganisms under sterilization conditions to avoid microbial contamination and application of solvent to extract TAGs from the TAG-filled microorganisms are costly. To overcome these challenges, this study reports the feasibility of a non-sterile cultivation of an oleaginous bacterium Rhodococcus opacus PD631SpAHB under saline conditions, followed by the use of a solvent-free, phage-lysis-protein-based bioextraction approach for TAGs release. The engineered strain PD631SpAHB was developed by introducing a recombinant plasmid carrying a phage lytic gene cassette (pAHB) into Rhodococcus opacus PD631 via transformation, followed by adaptive evolution under saline conditions. This newly developed strain is a salt-tolerant strain with the inducible plasmid pAHB to enable TAGs release into the supernatant upon induction. Cell lysis of PD631SpAHB was confirmed by the decrease of the optical density of cell suspension, by the loss of cell membrane integrity, and by the detection of TAGs in the culture medium. Up to 38% of the total TAGs accumulated in PD631SpAHB was released into supernatant after the expression of the lytic genes. PD631SpAHB strain is a promising candidate to produce TAGs from non-sterile growth medium and release of its TAGs without solvent extraction - a new approach to reduce the overall cost of biolipid-based biofuel production.

Engineering self-flocculating Halomonas campaniensis for wastewaterless open and continuous fermentation.

Halomonas has been developed as a platform for the next generation industrial biotechnology allowing open and nonsterile growth without microbial contamination under a high-salt concentration and alkali pH. To reduce downstream cost associated with continuous centrifugation and salt containing wastewater treatment, Halomonas campaniensis strain LS21 was engineered to become self-flocculating by knocking out an etf operon encoding two subunits of an electron transferring flavoprotein in the predicted electron transfer chain. Self-flocculation could be attributed to the decrease of the surface charge and increase of the cellular hydrophobicity resulted from deleted etf. A wastewaterless fermentation strategy based on the self-flocculating H. campaniensis was developed for growth and the production of poly-3-hydroxybutyrate (PHB) as an example. Most microbial cells flocculated and precipitated to the bottom of the bioreactor within 1 min after stopping the aeration and agitation. The supernatant can be used again without sterilization or inoculation for the growth of the next batch after collecting the precipitated cell mass. The wastewaterless process was conducted for four runs without generating wastewater. PHB accumulation by the self-flocculent strain was enhanced via promoter and ribosome binding site optimizations, the productivities of cell dry weight and PHB were increased from 0.45 and 0.18 g·L -1 ·hr -1 for the batch process compared to 0.82 and 0.33 g·L -1 ·hr -1 for the wastewaterless continuous process, respectively. This has clearly demonstrated the advantages of the wastewaterless process in that it not only reduces wastewater but also increases cell growth and product formation efficiency in a given period of time.

Tools to evaluate Herbaspirillum seropedicae abundance and nifH and rpoC expression in inoculated maize seedlings grown in vitro and in soil

The plant growth promoting bacteria Herbaspirillum seropedicae is an important model to study biological nitrogen fixation and it is proposed as crop inoculants for grasses. In this study we developed new tools to evaluate the abundance of H. seropedicae SmR1 and its expression in planta and investigated the association of the plant growth promoting bacteria H. seropedicae with maize grown in sterile and nonsterile conditions. Maize seedlings (P30F53) were inoculated with H. seropedicae SmR1 and grown in vitro and in soil. The plants were sampled at 4, 7 and 10 (in vitro) or 14, 21 and 28 days after inoculation (soil). Using qPCR we quantified H. seropedicae DNA and measured nifH, rpoC and hrcN levels of bacterial transcripts. In vitro assay inoculated plants presented highest amount of DNA and transcript contents, not detected in control plants. nifH and rpoC gene expression were detected on roots of inoculated maize cultivated in both growth conditions. However, it was not possible to detect hrcN gene expression in maize roots cultivated in soil. TaqMan assay is species-specific for H. seropedicae DNA and nifH and rpoC transcript levels could be used to monitor H. seropedicae gene expression in planta in sterile and nonsterile growth conditions. We developed specific, reliable and efficient tools to monitor H. seropedicae DNA abundance and H. seropedicae nifH and rpoC expression in planta.

Chromatin assembly factor CAF-1 represses priming of plant defence response genes.

Plants have evolved efficient defence systems against pathogens that often rely on specific transcriptional responses. Priming is part of the defence syndrome, by establishing a hypersensitive state of defence genes such as after a first encounter with a pathogen. Because activation of defence responses has a fitness cost, priming must be tightly controlled to prevent spurious activation of defence. However, mechanisms that repress defence gene priming are poorly understood. Here, we show that the histone chaperone CAF-1 is required to establish a repressed chromatin state at defence genes. Absence of CAF-1 results in spurious activation of a salicylic acid-dependent pathogen defence response in plants grown under non-sterile conditions. Chromatin at defence response genes in CAF-1 mutants under non-inductive (sterile) conditions is marked by low nucleosome occupancy and high H3K4me3 at transcription start sites, resembling chromatin in primed wild-type plants. We conclude that CAF-1-mediated chromatin assembly prevents the establishment of a primed state that may under standard non-sterile growth conditions result in spurious activation of SA-dependent defence responses and consequential reduction of plant vigour.

Colonization of lettuce rhizosphere and roots by tagged Streptomyces.

Beneficial microorganisms are increasingly used in agriculture, but their efficacy often fails due to limited knowledge of their interactions with plants and other microorganisms present in rhizosphere. We studied spatio-temporal colonization dynamics of lettuce roots and rhizosphere by genetically modified Streptomyces spp. Five Streptomyces strains, strongly inhibiting in vitro the major soil-borne pathogen of horticultural crops, Sclerotinia sclerotiorum, were transformed with pIJ8641 plasmid harboring an enhanced green fluorescent protein marker and resistance to apramycin. The fitness of transformants was compared to the wild-type strains and all of them grew and sporulated at similar rates and retained the production of enzymes and selected secondary metabolites as well as in vitro inhibition of S. sclerotiorum. The tagged ZEA17I strain was selected to study the dynamics of lettuce roots and rhizosphere colonization in non-sterile growth substrate. The transformed strain was able to colonize soil, developing roots, and rhizosphere. When the strain was inoculated directly on the growth substrate, significantly more t-ZEA17I was re-isolated both from the rhizosphere and the roots when compared to the amount obtained after seed coating. The re-isolation from the rhizosphere and the inner tissues of surface-sterilized lettuce roots demonstrated that t-ZEA17I is both rhizospheric and endophytic.

Vegetative Propagation of American Beech Resistant to Beech Bark Disease

The objective of this study was to develop vegetative propagation techniques—using tissue culture and grafting—for American beech (Fagus grandifolia) resistant to beech bark disease. Resterilizing the buds after excising bud scales reduced contamination of in vitro cultures derived from dormant buds. Application of a 7-day dark treatment before transferring shoots to the rooting medium improved rooting success. Plantlets gradually acclimatized to nonsterile growth conditions and set buds but failed to survive the dormant period. The application of 6-benzyladenine enhanced sprouting from roots collected from mature trees, but the excised shoots rooted poorly in vitro despite low contamination. Success of grafting scions from mature trees varied among genotypes and differed each year (30% in 2003, 12% in 2004, and 18% in 2005). Applying the growth hormone indole butyric acid to the scion before joining it to the rootstock did not increase grafting success. Survival of grafts was independent of rootstock age (1 or 2 years old). Grafting success increased when scion diameter was slightly larger than rootstock diameter. The rooting of sucker cuttings was successful for only one genotype. Critical steps, in which most failures in the propagation of American beech occur, are identified.

Deleterious properties of certain rhizosphere bacteria on field pea ( Pisum sativum) under gnotobiotic and non-sterile conditions

Three strains of Pseudomonas putida, one non-fluorescent and two fluorescent, were investigated in a series of complementary experiments to characterise their inhibitory effects on peas under different environmental conditions. Firstly, a gnotobiotic growth pouch system was developed to observe the deleterious effects of the strains on pea root development. A negative impact of the strains was observed on the development of the root morphology using these pouches which was accompanied with a subsequent reduction in root biomass. By using this method it was concluded that the deleterious effect of one of the strains was dependent on the inoculum density. Secondly, two complementary studies in non-sterile growth systems where pea seeds/seedlings were inoculated with the bacteria, showed deleterious effects on plant biomass by two of the strains. Thirdly, by using a sterile plant growth system allowing microscopic observations on root hair development, all strains were found to be able to induce root hair deformations on pea seedlings. The results showed that the mode of action for the deleterious effect differ between the strains. Based on our findings, we would like to emphasise the necessity to include a palette of different sterile and non-sterile growth systems to be able to identify characteristics of importance for deleterious rhizosphere bacteria.

Inoculation of conifer seed with plant growth promoting Bacillus strains causes increased seedling emergence and biomass

Seed inoculation with Bacillus strains LS and L6 significantly increased the rate of seedling emergence of white spruce but did not affect subsequent seedling growth. Lodgepole pine root growth was promoted by strain L5 in sterilized, but not non-sterile growth medium. Strain L6 promoted pine root growth in sterilized medium, but also caused significant increases in seedling emergence, shoot weight and height, root weight and surface area, and root collar diameter when tested in non-sterile pcat-vcrmiculite medium. The positive effects due to a single inoculation of pine with strain L6 at sowing were not detectable after I2 weeks growth. However, root growth was stimulated after 16 weeks growth if seedlings were re-inoculated with strain L6 mid-way through the experiment. Shoot-growth promotion was also detected when I yr old lodgepole pine seedlings were planted in pots and inoculated with strain L6. Douglas-fir seedlings grown from seed inoculated with strain L5 had increased root collar diameters, whereas those inoculated with strain L6 exhibited increased root surface area.