Axenic Algae Research Articles

Effect of acetochlor on the symbiotic relationship between microalgae and bacteria

In this study, two strains of symbiotic bacteria (SOB-1 and SOB-2) were isolated from Scenedesmus obliquus, and various algal-bacterial mutualistic systems were established under acetochlor (ACT) stress conditions. Following exposure to varying ACT concentrations from 2.0 to 25.0 μg/L, the capacity for co-cultured bacteria to degrade ACT was enhanced in 7 days by up to 226.9% (SOB-1) and 193.0% (SOB-2), compared with axenic algae, although bacteria exposed to higher ACT concentrations exacerbated algal metabolic stress, oxidative states, apoptosis and cellular lysis. ACT reduced carbohydrates in the phycosphere by up to 31.5%; compensatory nutrient plunder and structural damage by bacteria were the potential exploitation pathways determined based on the inhibition of bacterial infection using a glucanase inhibitor. The ACT-induced reduction in algal antimicrobial substances, including fatty acids and phenolics (by up to 58.1% and 56.6%, respectively), also facilitated bacterial exploitation of algae. ACT-dependent interspecific interaction coefficients between algae and bacteria generated from long-term symbiosis cultures implied that bacteria moved from mutualism (0 and 2.0 μg/L ACT) to exploitation (7.9 and 25.0 μg/L ACT). The population dynamic model under incremental ACT-concentration scenarios inferred that theoretical systematic extinction may occur in algal-bacterial systems earlier than in axenic algae. These outcomes provide interspecific insights into the distortion of algal-bacterial reciprocity due to the ecotoxicological effects of ACT.

How to Verify Non-Presence-The Challenge of Axenic Algae Cultivation.

Many phycological applications require the growth and maintenance of pure algae cultures. In some research areas, such as biochemistry and physiology, axenic growth is essential to avoid misinterpretations caused by contaminants. Nonetheless, axenicity—defined as the state of only a single strain being present, free of any other organism—needs to be verified. We compare the available methods to assess axenicity. We first purified unialgal Limnospira fusiformis cultures with an established series of axenicity treatments, and by including two additional treatment steps. The presumable axenic cultures were then tested for their axenic state by applying conventional tests on LB (lysogeny broth) agar-plates, 16S rRNA gene amplicon sequencing, flow-cytometry and epifluorescence microscopy. Only the plate tests indicated axenic conditions. We found a linear relationship between total cell counts of contaminants achieved by flow cytometry and epifluorescence microscopy, with flow cytometry counts being consistently higher. In addition, 16S rRNA gene amplicon sequencing demonstrated its superiority by not only being an efficient tool for axenicity testing, but also for identification of persistent contaminants. Although classic plate tests are still commonly used to verify axenicity, we found the LB-agar-plate technique to be inappropriate. Cultivation-independent methods are highly recommended to test for axenic conditions. A combination of flow-cytometry and 16S rRNA gene amplicon sequencing complement each other and will yield the most reliable result.

Development of a Microalgae-Based Continuous Starch-to-Hydrogen Conversion Approach

Eukaryotic algae represent a highly heterogeneous group in terms of organization, lifestyle, and metabolic capabilities. Unicellular green microalgae are capable of biohydrogen production through direct and indirect photolysis as well as dark fermentation. Most algae hydrogen studies focus on axenic algal cultures, although these are difficult and expensive to maintain for continuous operation. Moreover, the complex interplays and metabolic fluxes between algae and bacteria in natural ecosystems provide a number of clear biological and technological benefits to large-scale functional algae-based systems. Two green algae species from the Chlamydomonas and Chlorella genera were used to engineer stable synthetic communities by incorporating a starch-degrading bacterium from the Bacillus genus into the inter-kingdom consortium. Continuous photoheterotrophic biohydrogen production was achieved by elaborating an appropriate algal–bacterial ratio and fine-tuning the culture conditions for the synthetic consortia. Medium with starch as only carbon source served as a simple model of cheap substrate for algal hydrogen generation. The engineered pairwise algal–bacterial associations showed increased biomass and biohydrogen yield compared to the axenic control conditions. Chlorella sp. MACC-360 produced a significantly higher amount of hydrogen when both the bacterium partner and starch were added to the media compared to the axenic algae. Continuous, elevated algal hydrogen production was achieved in media supplemented with 8 g L−1 starch as sole carbon source when carefully selected initial cell number values were used for the Chlorella sp. MACC-360–B. amlyloliquefaciens co-cultures.

Identification of Volatiles of the Dinoflagellate Prorocentrum cordatum.

The dinoflagellate Prorocentrum cordatum, often called P. minimum, is a potentially toxic alga found in algal blooms. Volatile compounds released by the alga might carry important information, e.g., on its physiological state, and may act as chemical messengers. We report here the identification of volatile organic compounds emitted by two strains, xenic P. cordatum CCMP 1529 and axenic P. cordatum CCMP 1329. The volatiles released during culture were identified despite their low production rates, using sensitive methods such as open-system-stripping analysis (OSSA) on Tenax TA desorption tubes, thermodesorption, cryofocusing and GC/MS-analysis. The analyses revealed 16 compounds released from the xenic strain and 52 compounds from the axenic strain. The majority of compounds were apocarotenoids, aromatic compounds and small oxylipins, but new natural products such as 3,7-dimethyl-4-octanolide were also identified and synthesized. The large difference of compound composition between xenic and axenic algae will be discussed.

Cysteinolic Acid Is a Widely Distributed Compatible Solute of Marine Microalgae.

Phytoplankton rely on bioactive zwitterionic and highly polar small metabolites with osmoregulatory properties to compensate changes in the salinity of the surrounding seawater. Dimethylsulfoniopropionate (DMSP) is a main representative of this class of metabolites. Salinity-dependent DMSP biosynthesis and turnover contribute significantly to the global sulfur cycle. Using advanced chromatographic and mass spectrometric techniques that enable the detection of highly polar metabolites, we identified cysteinolic acid as an additional widely distributed polar metabolite in phytoplankton. Cysteinolic acid belongs to the class of marine sulfonates, metabolites that are commonly produced by algae and consumed by bacteria. It was detected in all dinoflagellates, haptophytes, diatoms and prymnesiophytes that were surveyed. We quantified the metabolite in different phytoplankton taxa and revealed that the cellular content can reach even higher concentrations than the ubiquitous DMSP. The cysteinolic acid concentration in the cells of the diatom Thalassiosira weissflogii increases significantly when grown in a medium with elevated salinity. In contrast to the compatible solute ectoine, cysteinolic acid is also found in high concentrations in axenic algae, indicating biosynthesis by the algae and not the associated bacteria. Therefore, we add this metabolite to the family of highly polar metabolites with osmoregulatory characteristics produced by phytoplankton.

A Newly Designed Automatically Controlled, Sterilizable Flat Panel Photobioreactor for Axenic Algae Culture.

In context of the global climate change, microalgae processes are gaining momentum as a biotechnological tool for direct fixation and valorization of greenhouse gases. Algae have the metabolic capacity to photosynthetically convert CO2 into high value products, such as food additives, under economic boundary conditions. High cost, commercial flat panel gas-lift bioreactors for microalgae cultivation at laboratory scale provide either small volumes or no sterile operation, which limits academic research. This brief report presents initial data for a new type of sterile operating flat panel gas-lift bioreactor with a unique asymmetrical U-shape. It utilizes automatable process control technologies that adhere to industrial standards to enhance data reproducibility and aid industrial scale up. The practicability was demonstrated using a Chlorella sorokiniana cultivation, which showed the typical growth behavior. Due to the sophisticated implemented control engineering technology, pivotal parameters as pH and temperature can be determined within a range of ±0.1 units, which was confirmed experimentally. The new flat panel gas-lift photobioreactor presented in this brief report fills the technology gap at laboratory scale with an autoclavable volume of 7.2 L. Moreover, it is easy to rebuild by means of the hereby provided blueprint, while exhibiting a six-fold cost reduction compared to commercially available flat panel photobioreactors.

Co-cultivation of siderophore-producing bacteria Idiomarina loihiensis RS14 with Chlorella variabilis ATCC 12198, evaluation of micro-algal growth, lipid, and protein content under iron starvation

Co-cultivation systems offer the potential to commercialize microalgae biomass. The key purpose of the study was to understand the relationship between siderophore-producing bacterium Idiomarina loihiensis RS14 and Chlorella variabilis ATCC 12198 strain for Chlorella growth enhancement. After observing growth enhancement in C. variabilis by adding metal chelator deferroxamine mesylate (siderophore standard) and purified siderophore from I. loihiensis (1 mg mL−1), a co-cultivation system was designed where axenic microalgae and co-cultured (microalgae + bacteria) aliquots were grown in (1:9, 9:1, 1:1) volumetric inoculum ratio (mL) under iron-sufficient and iron-deficient conditions. The co-culture volumetric ratio 1:1 (microalgae/bacteria) showed bleaching of microalgae and 1:9 showed less biomass (310 mg L−1) comparatively with 9:1 that increased 35% of biomass, i.e., 650 mg L−1 (axenic) to 1000 mg L−1 (co-cultured) in iron-deficient media. The inoculum ratios were optimized in 100 mL shake flask and 9:1 ratio was further scaled up with the similar conditions, and the co-culture showed 20% increase in biomass, i.e., 285.6 mg L−1 (axenic) to 356 mg L−1 (co-cultured). The co-cultured biomass contains 19.70% lipid content compared with axenic algae, i.e., 18.41% which shows 7% of increase in co-culture. Protein content increased to 30% in co-culture microalgae compared with axenic microalgae. Scanning electron microscope images show crumpled surface of Chlorella cells in co-cultured compared with its axenic cells. This finding is of interest for biofuel production from microalgae, often attained through nutrient-starvation processes leading to oil accumulation.

Cryopreservation studies of an artificial co-culture between the cobalamin-requiring green alga Lobomonas rostrata and the bacterium Mesorhizobium loti

Algal-bacterial co-cultures, rather than cultures of algae alone, are regarded as having the potential to enhance productivity and stability in industrial algal cultivation. As with other inocula in biotechnology, to avoid loss of production strains, it is important to develop preservation methods for the long-term storage of these cultures, and one of the most commonly used approaches is cryopreservation. However, whilst there are many reports of cryopreserved xenic algal cultures, little work has been reported on the intentional preservation of both algae and beneficial bacteria in xenic cultures. Instead, studies have focused on the development of methods to conserve the algal strain(s) present, or to avoid overgrowth of bacteria in xenic isolates during the post-thaw recovery phase. Here, we have established a co-cryopreservation method for the long-term storage of both partners in a unialgal-bacterial co-culture. This is an artificial model mutualism between the alga Lobomonas rostrata and the bacterium Mesorhizobium loti, which provides vitamin B12 (cobalamin) to the alga in return for photosynthate. Using a Planer Kryo 360 controlled-rate cooler, post-thaw viability (PTV) values of 72% were obtained for the co-culture, compared to 91% for the axenic alga. The cultures were successfully revived after 6 months storage in liquid nitrogen, and continued to exhibit mutualism. Furthermore, the alga could be cryopreserved with non-symbiotic bacteria, without bacterial overgrowth occurring. It was also possible to use less controllable passive freezer chambers to cryopreserve the co-cultures, although the PTV was lower. Finally, we demonstrated that an optimised cryopreservation method may be used to prevent the overgrowth potential of non-symbiotic, adventitious bacteria in both axenic and co-cultures of L. rostrata after thawing.

Strain-specific consumption and transformation of alga-derived dissolved organic matter by members of the Limnohabitans-C and Polynucleobacter-B clusters of Betaproteobacteria.

We investigated changes in quality and quantity of extracellular and biomass-derived organic matter (OM) from three axenic algae (genera Rhodomonas, Chlamydomonas, Coelastrum) during growth of Limnohabitans parvus, Limnohabitans planktonicus and Polynucleobacter acidiphobus representing important clusters of freshwater planktonic Betaproteobacteria. Total extracellular and biomass-derived OM concentrations from each alga were approximately 20 mg l-1 and 1 mg l-1 respectively, from which up to 9% could be identified as free carbohydrates, polyamines, or free and combined amino acids. Carbohydrates represented 54%-61% of identified compounds of the extracellular OM from each alga. In biomass-derived OM of Rhodomonas and Chlamydomonas 71%-77% were amino acids and polyamines, while in that of Coelastrum 85% were carbohydrates. All bacteria grew on alga-derived OM of Coelastrum, whereas only Limnohabitans strains grew on OM from Rhodomonas and Chlamydomonas. Bacteria consumed 24%-76% and 38%-82% of all identified extracellular and biomass-derived OM compounds respectively, and their consumption was proportional to the concentration of each OM compound in the different treatments. The bacterial biomass yield was higher than the total identifiable OM consumption indicating that bacteria also utilized other unidentified alga-derived OM compounds. Bacteria, however, also produced specific OM compounds suggesting enzymatic polymer degradation or de novo exudation.

Robust, high-productivity phototrophic carbon capture at high pH and alkalinity using natural microbial communities

BackgroundBioenergy with carbon capture and storage (BECCS) has come to be seen as one of the most viable technologies to provide the negative carbon dioxide emissions needed to constrain global temperatures. In practice, algal biotechnology is the only form of BECCS that could be realized at scale without compromising food production. Current axenic algae cultivation systems lack robustness, are expensive and generally have marginal energy returns.ResultsHere it is shown that microbial communities sampled from alkaline soda lakes, grown as biofilms at high pH (up to 10) and high alkalinity (up to 0.5 kmol m−3 NaHCO3 and NaCO3) display excellent (>1.0 kg m−3 day−1) and robust (>80 days) biomass productivity, at low projected overall costs. The most productive biofilms contained >100 different species and were dominated by a cyanobacterium closely related to Phormidium kuetzingianum (>60%).ConclusionFrequent harvesting and red light were the key factors that governed the assembly of a stable and productive microbial community.

Biomass production and nutrient assimilation by a novel microalga, Monoraphidium spp. SDEC-17, cultivated in a high-ammonia wastewater

To obtain suitable microalgae species for successful algal biomass production from low-cost wastewater, four axenic algae strains were isolated from a local lake. Through acclimation with the high-ammonia complex wastewater (CW) of a gourmet powder factory, one algae species showed good ability to yield biomass and endure high-ammonia conditions (>170mgL−1) in CW. This was verified as a Monoraphidium spp. by molecular identification, and named as SDEC-17. The algae were 27–60μm in length and 4–10μm in width, with relatively low specific surface area for withstanding ammonia ingress through the cell membrane. The final biomass densities of SDEC-17 in CW (1.29±0.09gL−1) and BG11 medium (1.31±0.08gL−1) did not show a statistically significant difference (p>0.05). Moreover, protein content was stimulated to 44% by CW, compared to 35% in BG11. Lipid accumulation of SDEC-17 was not significantly influenced by CW, and fatty acid profiles resembled those of palm oil. The algae would utilize ammonia first under conditions with various nitrogen sources present, and absorb large amounts of phosphorus from the wastewater. Thus, phosphorus and ammonia were removed with efficiencies of nearly 100%, satisfying the discharge standard of pollutants for municipal wastewater treatment plants. These results suggested that Monoraphidium spp. SDEC-17 is a promising candidate for algae biomass production and possibly chemical energy recovery from the complex wastewater.

파블로바 비리디스로부터 분리한 세균에 의한 미세조류의 생장 촉진

The marine microalga Pavlova viridis can grow fast and has the ability to accumulate essential nutrients for culturing marine animals, such as EPA and DHA, and it has been used as food for raring larval fish and prawn. The symbiotic relationship between the flagellate microalga Pavlova viridis and its associated bacteria was investigated. An axenic culture of P. viridis was obtained by repeated treatment of the microalga with an antibiotic cocktail. The axenic status was confirmed after sub-culturing three times in a sterile f/2 medium without an antibiotic. The axenic alga was then co-inoculated with five bacteria, arbitrarily designated as I1?I5, isolated from the alga to test the growth promotion of the algae. All bacterial strains promoted the growth of P. viridis, and bacterial isolate I3 was the most effective among the five bacteria tested. The cell number of P. viridis in the co-culture with I3 was significantly higher than that of the control culture. A sequence analysis of the 16S rRNA gene isolated from I3 revealed a 97% nucleotide sequence similarity to that of Citrobacter sp. The growth of strain I3 was also significantly enhanced by co-culturing with P. viridis, indicating a symbiotic relationship between the microalga and its associated bacterium. The association between the microalga and bacterium was confirmed by scanning electron microscopy.

Construction of an artificial microalgal-bacterial consortium that efficiently degrades crude oil

Four oil component-degrading bacteria and one oil-tolerant microalgae, Scenedesmus obliquus GH2, were used to construct an artificial microalgal-bacterial consortium for crude-oil degradation. The bacterial strains included Sphingomonas GY2B and Burkholderia cepacia GS3C, along with a mixed culture, named GP3, containing Pseudomonas GP3A and Pandoraea pnomenusa GP3B. GY2B could only degrade polycyclic aromatic hydrocarbons, GS3C was able to degrade aliphatic chain hydrocarbons, and GP3 could utilize both saturated and aromatic hydrocarbons. In combination with unialgal or axenic algae, the bacteria showed different effects on oil degradation. Unialgal GH2 was not suitable for the consortium construction, as it could not cooperate well with GS3C and GP3. The axenic GH2 exhibited no oil-degrading ability; however, it significantly promoted the degradation ability of the oil component-degrading bacteria, especially for degrading biorefractory polycyclic aromatic hydrocarbons. Axenic S. obliquus GH2, combined with the four bacteria mentioned above, formed an optimal algal-bacterial consortium. The artificial consortium demonstrated an elevated efficiency in degrading both aliphatic and aromatic hydrocarbons of crude oil.

CCAC Culture Collection of Algae at the University of Cologne: A new collection of axenic algae with emphasis on flagellates

CCAC Culture Collection of Algae at the University of Cologne: A new collection of axenic algae with emphasis on flagellates

Control of Vibrio spp. in Shrimp Hatcheries Using the Green Algae Tetraselmis suecica

The Vibrio spp. are the most common and harmful shrimp pathogenic bacteria causing serious diseases of larval and postlarval stages in the hatchery. The Vibrio inhibitory activity of green algae Tetraselmis suecica has been reported in vitro (Austin and Day 1990).In these experiments, the inhibitory activity was assessed in vivo in a commercial shrimp hatchery of the white prawn Fenneropenaeus indicus. The concentrate of the algae was used as a food supplement for the broodstock and also as partial live larval feed in the larval rearing tanks. Results showed that use of algae resulted in significant reduction of Vibrio numbers in maturation and spawning tank water, broodstock gut contents, egg and nauplii samples. The spawning tank water Vibrio count showed a negative correlation with egg hatching rate. The algae when used in larval tanks were found to reduce Vibrio count in both rearing water and larval samples, resulting in improved nauplii 5 to postlarvae 1 survival. Rearing tank experiments with axenic algae proved that the inhibitory effect was by Tetraselmis and not by associated bacteria. Usage of this algae in the hatcheries will be advantageous as it suppresses the dominance of Vibrio spp. may be including the potentially pathogenic ones.

Comparison of 14 CO 2 and 12 CO 2 Uptake and Release Rates in Laboratory Cultures of Phytoplankton

The accuracy of the 14 C method of estimating photosynthetic rates was evaluated by comparing inorganic 14 C and 12 C uptake and release rates in three species of microalgae. Net photosynthesis was defined as the net decrease in inorganic 12 C concentration during a given light period. The axenic algae cultures were incubated in a continuous flow-chamber system and the experiments conducted as time-course studies in light and dark. All experiments were characterized by higher 14 C uptake rates than 12 C during the first part of the light period, but in many of the experiments the 14 C uptake approached the 12 C net photosynthesis by time (...)

Mechanism of an early lysis by fatty acids from axenic Phormidium tenue (musty odor-producing cyanobacterium) and its growth prolongation by bacteria.

We have previously demonstrated that bacteria-containing Phormidium tenue, a cyanobacterium which produces musty odor 2-methylisoborneol, grew beyond 8 weeks, whereas axenic alga perished suddenly between the 3rd week and the 4th week while being cultured in the laboratory. This mechanism was investigated. It is assumed that when algal cells grow beyond a certain level, the supply of CO2 becomes inadequate and results in the rapid lysis of axenic alga. At that time, inhibitory substances liberated from algal cells kill the surviving alga. Since the process occurs continuously, this alga is finally annihilated. On the other hand, since inhibitory substances are metabolized or degraded by bacteria coexistent with alga, bacteria-containing P. tenue maintains growth for a long time. The growth-inhibitory substance was found to be unsaturated free fatty acids.

Effects of Marine Bacteria on the Culture of Axenic Oyster Crassostrea gigas (Thunberg) Larvae.

Bacteria-free oyster larvae (Crassostrea gigas) were cultured under aseptic conditions; they were fed axenic algae (Isochrysis galbana), and the medium was inoculated with isolated strains of marine bacteria. Twenty-one bacterial strains were tested, and most were detrimental to larval survival and growth. However, additions of strain CA2 consistently enhanced larval survival (21-22%) and growth (16-21%) in comparison with control cultures that were fed only algae. Size-frequency distributions of populations of larvae cultured for 10 days on axenic algae were skewed due to the poor growth of many individuals; whereas size-frequencies from populations of larvae fed axenic algae supplemented with CA2 bacteria were distributed normally. Strain CA2 may therefore make a nutritional contribution to the growth of oyster larvae. I. galbana did not grow under the light intensities used for larval culture; thus the improvement in larval growth cannot be attributed to bacterial enhancement of algal growth and, consequently, food availability. Naturally occurring microflora from Yaquina Bay, Oregon, depressed survival or growth of larvae-fed live algae.

ASCOPHYLLUM NODOSUM (PHAEOPHYTA) IN AXENIC CULTURE AND ITS RESPONSE TO THE ENDOPHYTIC FUNGUS MYCOSPHAERELLA ASCOPHYLLI AND EPIPHYTIC BACTERIA1

ABSTRACTAscophyllum nodosum (L.) Le Jol. was freed from bacteria and the endophytic fungus Mycosphaerella ascophylli Cotton by repeated treatment with chlorine solutions and grown in artificial seawater. Two types of axenic culture of different origin were obtained. Type 1 was developed from apices of A. nodosum collected in the sea. Type 2 was from plants which developed from adventitious embryos on rhizoids formed by type 1. This is the first time A. nodosum has been cultivated axenically. Growth of the axenic alga was increased by IAA, 21P and zeatin. Without external growth regulators some strains of the axenic alga deteriorated within a year; others developed a filamentous habit. Sulfur in a reduced state also stimulated growth. Addition of either glucose, mannose or mannitol to the medium caused the formation of calluslike layers of loosely packed colorless cells under the epidermis of the thalli and the epidermis was sloughed off. No increase in thallus length was noticed.Mycosphaerella ascophylli in axenic culture did not excude any substances stimulating growth of the alga, but that does not exclude an influence of the fungus on the alga in vivo. The fungus, when growing within the alga, seemed to have little influence on algal morphology. A bacterized but fungus‐free A. nodosum was cultivated in an artificial seawater for 8 years. In the bacteria‐free alga, the fungus protruded from the epidermis and evidently utilized the alga as a carbon source. The bacteria thus seem much more important than the fungus for normal growth of the Ascophyllum plant.