Fine Resolution Analysis of bacterial communities associated with Neochloris oleoabundans culture and insights into terpenes as contamination control agents

doi:10.21203/rs.3.rs-2634245/v1

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Fine Resolution Analysis of bacterial communities associated with Neochloris oleoabundans culture and insights into terpenes as contamination control agents

https://doi.org/10.21203/rs.3.rs-2634245/v1

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published 11 May, 2023

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Biological contamination is one of the main bottlenecks in microalgae production, reducing quality and productivity and sometimes leading to the complete loss of the cultures. Selecting terpenes can be a pathway toward eco-friendly contamination control in microalgae cultures. This work evaluated the presence of bacterial contaminants in N. oleoabundans cultures through HTS and 16S analysis and their susceptibility to six natural terpenes (α-pinene, β-pinene, limonene, trans-cinnamaldehyde, linalool, and eugenol). The principal phyla identified were Proteobacteria, Bacteroidetes, and Actinobacteria, and based on these data, 89 bacterial isolates of seven genera were obtained (36 Aureimonas sp., 27 Microbacterium sp., 5 Pseudomonas sp., 9 Bacillus sp., 14 Shinella sp., 1 Brevundimonas sp., and 1 Exiguobacterium sp.). The minimal inhibitory concentration of terpenes was 6.25 mg L^-1, with a reduction in bacterial counts of most isolates by 79% - 99%.

Bacteria

Microalgae

MIC

Terpene

Trans-cinnamaldehyde

Microalgae are photosynthetic microorganisms found mainly in freshwater and marine environments (Colling Klein et al. 2018; Fulbright et al. 2018). They are known for their easy adaption to extreme conditions, high photosynthetic efficiency, and biomass nutritional properties (Huang et al. 2014; Ma et al. 2016). Neochloris oleoabundans is a very interesting microalga for sustainable oil, biopolymers production, and wastewater treatment due to its high growth rate and culture conditions, such as temperatures above 25–30°C, CO₂ (without or supplementation: 0.04 and 5% (v/v)), irradiance level of 150 50µmol photons m^− 2 s^− 1 (Gouveia et al. 2009; Santos et al. 2012; Peng et al. 2015; Jaeger et al. 2018).

For large-scale production of N. oleoabundans, open systems are the most likely strategy due to the possibility of producing higher quantities of biomass that can be used in different industries with reduced production costs. However, open systems are susceptible to microbial contamination, which causes crashes and economic losses. There is little information on the influence of bacterial communities on algae cultures; therefore, it is vital to understand their behavior to control them. Competition for nutrients, parasitism, and predation are some of the interactions that can affect the development of microalgae cultures. Biomass production can be reduced up to 10 times because of nutrient competition, while direct predation can reduce cultures by up to 90% in 2–5 days (Wang et al. 2013; Ramanan et al. 2016; Molina et al. 2019). Different bacteria are associated with the natural phycosphere of microalgae, some of which are considered contaminants. Contaminant microorganisms can inhibit or reduce the development of a microalga of interest. Though maintaining axenic culture is complex and costly, for that reason, studying the relationships between microalgae bacteria is essential in production systems (Fulbright et al. 2018; Di Caprio 2020).

Several physical and chemical technologies have been developed to control the presence of different predatory organisms. Nevertheless, the efficiency of these methods depends on several factors, including detection stage, type of contaminant, and culture systems (Moreno-Garrido and Cañavate 2000; Wang et al. 2018; Deore et al. 2020). Currently, chemical treatment is the most used technique to inhibit or kill biological contaminants in microalgae cultures; for instance, pesticides were first used to reduce zooplankton. Other antimicrobial agents are antibiotics, sodium hypochlorite, sulfonamides potassium tellurite, and different detergents (e.g., tween, sodium lauryl sulfate) (Wang et al. 2013; Vaz et al. 2014; Lam et al. 2018; Vu et al. 2018). Even though the addition of chemical compounds can represent a possible solution, the use of these substances can be problematic due to the production of toxic by-products during the process and affect the growth of the target microalga (Scholz 2014; Van Haute et al. 2015; Day et al. 2017; Molina-Aulestia et al. 2021).

Essential oils such as clove or peppermint oils have known antimicrobial activity. These oils are rich in hydrophobic terpenes and terpenoids that affect the permeability of cells, showing activity against various microorganisms such as fungi and bacteria. Terpenoids such as eugenol, thymol, and carvacrol can cause coagulation of cell contents and, consequently, cell damage cox (Bakkali et al. 2008; Muhammad Asif Hanif et al. 2019). For this reason, using active compounds derived from natural sources is essential. Currently, techniques such as MIC (minimal inhibitory concentration) are used to evaluate the toxicity of natural substances, making it possible to establish the minimum concentration at which a substance inhibits the growth of microorganisms (Moreira et al. 2005; Parvekar et al. 2020).

This paper assesses the microbial diversity and identification of bacterial contaminants from cultures of the microalga N. oleoabundans UTEX 1185 and the antimicrobial activities of six essential oils. In previous studies, the toxicity of six terpenes (eugenol, linalool, limonene, α-pinene, β-pinene, and trans-cinnamaldehyde) on the microalga N. oleoabundans UTEX 1185 was evaluated. In this research, it was possible to show that beta-pinene and limonene were the most toxic terpenes to N. oleoabundans. At the same time, eugenol and linalool showed values above 250 mg L-1, turning them into a green alternative for managing microalgae culture (Molina-Aulestia et al. 2022).

Algal strain and culture conditions

The microalga Neochloris oleoabundans UTEX 1185 was cultivated in BG₁₁ medium (Stanier et al. 1971). The inoculum was produced using 1000-mL Erlenmeyer flasks with 400 mL of culture. The cultures were maintained with a photoperiod of 12:12 h with an illumination of 60 µmol photons m^− 2 s^− 1 at 25°C and manually stirred twice daily. To maintain the cultures' viability, dilutions were performed using BG₁₁ every two months, maintaining a final cell concentration of 1.5E + 8 cell mL^− 1.

Determination of the total number of bacterial in the microalgae culture

To determine the total bacteria, 100 µL of N. oleoabundans UTEX 1185 culture was added to 900 µL of sterile peptone water 0.1%, followed by serial dilutions and pour plate method. The plates were incubated at 25°C for 48 hours using Nutrient Agar (Kasvi, Brazil) (Vu et al. 2018).

Bacterial Metagenome

Using the Illumina platform, the bacteria in the culture of N. oleoabundans UTEX 1185 was determined by the 16S ribosomal DNA (rDNA) gene. The culture was evaluated initially and 12 months after control and maintenance. The cultures were maintained in the conditions described in the section Algal strain and culture conditions.

One milliliter of each sample was centrifuged at 12000 × g for 1 min. The cell pellet was resuspended in 500 µL of Tris-EDTA, and the extraction of total DNA was performed according to (de Carvalho Neto et al., 2018). The total DNA was quantified with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). The hypervariable V3-V4 region was amplified from the total DNA extracted using primers degenerated 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’), containing complementary adaptors for Illumina platform sing KlenTAQ polymerase (Sigma-Aldrich) PCR generated bar-coded amplicons under the following conditions: 95°C for 3 min, and 18 cycles at 95°C for 0.5 min, 50 cycles for 0.5 min, and 68°C for 1 min, followed by a final extension at 68°C for 10 min. Samples were sequenced in the MiSeq platform using the 500 V2 kit, following standard Illumina protocols (Illumina, San Diego, CA, USA). Chimeric sequence detection, removal of noises from pre-cluster, and taxonomic attribution were also performed using standard parameters of the QIIME (Quantitative Insight Into Microbial Ecology) software package, version 1.9.0 (r). Applying the UCLUST method (Edgar 2010), sequences presenting identity above 97% were considered the same operational taxonomic units (OTUs) according to the SILVA database (Quast et al. 2013). Sequences derived from Illumina sequencing were deposited in the NCBI database.

Isolation and characterization of cultivable bacteria Neochloris oleoabundans UTEX 1185

Based on the bacterial metagenome results, different agar and culture conditions were chosen to isolate the most abundant cultivable bacteria. One hundred µL of the N. oleoabundans UTEX 1185 culture was spread on the surface of the Nutrient Agar (Kasvi, Brazil-NA), BG₁₁ Agar (BG₁₁) (Stanier et al. 1971), BG₁₁ filtered out agar (FL), Luria-Bertani (LB) Agar (Kasvi, Brazil), Glucose- Yeast Extract- Malt Extract Agar (GYM), RH Agar (Himedia – Brazil), BG₁₁ filtered out (10%) and BG₁₁ (90%) (BF) agar, and Tryptic Soy Agar (TSA) (Kasvi, Brazil). Each agar plate was incubated at following conditions: C1 = 25°C, light; C2 = 25°C, dark; C3 = 30°C, light; C4 = 30°C, dark; C5 = 37°C, light; C6 = 37°C, dark. To mimic the growth conditions of the microalgae, the plates of conditions C1, C3, and C5 were maintained for seven days using the same illumination conditions (60 µmol photons m^− 2 s^− 1 and with 12:12 photoperiod). Based on their morphological characteristics (color and coarse appearance of colonies on the agar plate), eighty-nine colonies were isolated and purified by repetitive streaking (Benson 2001; Choi et al. 2008; Fernández Olmos et al. 2010).

Molecular identification

Eighty-nine isolates were selected for 16S ribosomal DNA (rDNA) analysis for molecular identification. The extraction protocol was performed according to (Maske 2021) with modifications. The cell pellets were obtained after centrifugation at 1200 ×g for 10 min. The supernatant was resuspended in 2 mL saline solution (NaCl 0.9%) and transferred to a new sterile tube to centrifuged at 10000 ×g for 5 min. Then, the supernatant was discarded, and 500 µL of NaCl 0.9% solution and 27.5 µL of sodium dodecyl sulfate (20% w/v in distilled, deionized water) were added by incubation at room temperature. 250 µL of phenol-chloroform-isoamyl alcohol (25:24:1; Sigma Aldrich) was added, homogenized by inversion, and centrifuged at 12000 ×g for 10 min. The supernatant was collected, and 250 µL of chloroform-isoamyl alcohol (24:1; Sigma Aldrich) was added and homogenized, followed by centrifugation at 12000 ×g for 10 min. The supernatant was collected, and the DNA was precipitated with 3x (v/v) absolute ethanol and centrifugated at 12000 ×g for 20 min. Pellets were washed with 80% ethanol, dried, and resuspended in ultrapure water. The concentration of isolated DNA was determined using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, EUA).

The 16S rRNA region was amplified by polymerase chain reaction (PCR) using 27F and 1492R primer (Lane et al. 1985). The amplification reaction was performed in a thermal cycler Veriti (Applied Biosystems, Paisley, UK). Amplifications were performed in a final volume of 25 µL containing 2 µL DNA (50 ng µL^− 1), 0.55 µL dNTPs (10 mM), 0.75 µL MgCl₂ (50 mM), 2.5 µL buffer (10x), 0.5 µL of primers 27F and 1492R (10 µM), 0.2 µL Ludwing DNA polymerase (5U µL^− 1) and 18 µL of water. PCR generated amplicons under the following conditions: initial denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 0.25 min, annealing at 55°C for 0.75 min, extension at 72°C for 1.5 min, and final extension at 72°C for 6 min.

The PCR products were sequenced by automatic capillary electrophoresis on ABI 3730xl DNA analyzer (Applied Biosystems, Paisley, UK). The obtained chromatograms were inspected using BioEdit 7.0.5.3 (Hall 1999), and the sequences were compared against GenBank 16S rRNA sequences using standard BLAST. The genetic identification of the studied strains and reconstruction of phylogenetic relationships was performed using sequences deposited in the GenBank database of available type strains of each genus identified, besides the sequences generated in this study. The 16S rRNA gene sequences obtained have been deposited in the GenBank database. DNA sequence alignments were performed using Mafft version 7 (Katoh and Toh 2008) (https://mafft.cbrc.jp/alignment/server/) and manually corrected, when necessary, in MEGA version 7. A neighbor-joining phylogenetic tree was constructed using the MEGA X version 10.1 program (Kumar et al. 2018). The evolutionary distances were computed by Maximum Composite Likelihood Method (Cavalli-sforza and Edwards 2008) and maximum parsimony. The robustness of individual branches was estimated by bootstrapping with 1000 replicates (Felsenstein 1985). The accession numbers are ON197225 - ON197312.

Antimicrobial activity

Chemicals

Six terpenes previously studied and with recognized antimicrobial activity were used: Eugenol, Linalool, (R)- (+)-Limonene, trans- Cinnamaldehyde, α-pinene and (-)-β-pinene (Sigma-Aldrich, Brazil). The six solutions were prepared using pure Tween 20 (8.4 mL), the terpene under evaluation (1.6 g), and the BG₁₁ medium (990 mL). The stock concentration was 1600 mg L^− 1 (Molina-Aulestia et al. 2022).

Culture medium and inoculums

The stock cultures of microorganisms identified in Isolation and characterization of cultivable bacteria Neochloris oleoabundans UTEX 1185 were maintained on nutrient agar plates at 4°C. The inoculum was prepared by suspending a loopful of bacterial cultures into 10 mL of Nutrient Broth (Kasvi, Brazil) and were incubated at 25°C, with a 12:12 photoperiod and an illumination of 60 µmol photons m^− 2 s^− 1 for 48 h. The cultures were transferred to Erlenmeyer flasks with a final volume of 50 mL of Nutrient Broth (Mohamed et al. 2014). Finally, the cultures were diluted in 0.1% saline solution until the turbidity corresponded to 0.5 McFarland (NCCLS 1999; Mendes et al. 2011).

Preliminary assessment of antimicrobial activity

Disc Diffusion Method

The toxicity of essential oil was identified by employing the agar disc diffusion method 100 mgL^− 1 solution was prepared for each oil. The suspension of microorganisms was inoculated using a disposable swab on the surface of Nutrient Agar medium plates. Then, 6 mm diameter nitrocellulose discs were added, previously impregnated with 10 µl of the essential oil solutions. The plates were placed in an oven at a controlled temperature (25°C) for 24 hours with a 12:12 photoperiod and a light intensity of 60 µmol photons m^− 2 s^− 1. The antibiotic chloramphenicol dissolved in ethanol at 100 mg L^− 1 was used as a positive control, and sterile Tween 20 was used as a negative control. The inhibition zones were measured by diameter. The extract was classified as active when it presented an inhibition halo (NCCLS 1999; Pfaller et al. 2002; Alim et al. 2009; Mendes et al. 2011; Mohamed et al. 2014).

Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) was analyzed using a 96-well microplate assay based on the screening. First, the cultures were adjusted to the 0.5 McFarland scale, and 100 µl of a solution at 200 mg L^− 1 was added to each essential oil in the first row. Next, 50 µl of nutrient broth was pipetted into each well from the second to the last row of the microplate. Subsequently, serial dilutions of the antimicrobial agent were performed by pipetting 50 µl from the first row to the second row and repeating this process for the third and so on. The seventh and eighth rows do not contain antimicrobial agents. Finally, 50 µl of bacteria were inoculated into each well from the second to the eighth row. The final concentrations of the antimicrobial agents were 100, 50, 25, 12.5, and 6.25 mg L^− 1. In the last column added, chloramphenicol (100 mg L^− 1) was a positive antimicrobial agent. The samples were incubated at 25°C for 24 h with a 12:12 photoperiod and a light intensity of 60 µmol photons m^− 2 s^− 1. Subsequently, an aliquot of 100 µL was removed from all assay wells and inoculated onto nutrient agar plates under similar conditions. Finally, 30 µL of resazurin (150 µg mL^− 1) was added, and the original microplate was incubated for 1 h to evaluate the cell viability (CLSI 2012; Veiga et al. 2019).

Total viable count of bacteria and bacterial metagenome

The total viable cells and the community bacterial concentration were evaluated in the N. oleoabundans. The initial bacterial concentration was 4.85x10⁵ CFU mL^− 1. In contrast, after 12 months of maintenance of cultures (section 2; material and methods), the final concentration was 5.8x10⁴ CFU mL^− 1, representing a reduction of more than 50% of viable bacteria. The bacterial community was also analyzed at two intervals. A total of 11 phyla were identified in the original culture. The dominant groups were Proteobacteria and Bacteroidetes, with 81% and 10%, respectively. In the culture analyzed 12 months later, only four phyla were identified, showing a reduction of 63%. The phyla with the highest prevalence were Proteobacteria, 92%, and Actinobacteria, 4%. Bacteroidetes and Proteobacteria. And at the family level, Hyphomonadaceae (35.5%), Xanthomonadaceae (18.2%), and Pseudomonadaceae (12.7%) showed a significant presence in the initial culture, while for the culture analyzed 12 months later, the families Xanthomonadaceae (56.5%), Phyllobacteriaceae (18.9%) and Aurantimonadaceae (6.4%) had the highest taxonomic frequency (Fig. 1). The total diversity identified is detailed in the Supplementary material, Table S1-S4. Further analysis showed that the genera Parvibaculum (59.5%) and Pseudomonas (23%) were abundant in the initial culture, while 12 months later, the genera Anaplasma (2.3%) and Sphingopyxis (1.6%) were more prevalent.

Alpha diversity analysis

5.700 and 43.4000 sequences were obtained from the 16S gene sequences from the initial culture and 12 months (MC) after, respectively. Each analysis's alpha rarefaction (Supplementary material S1, S2) curves suggest that it did not reach the plateau. Therefore, the instability shows that not all subdominant species were identified.

Isolation, Determination, and Molecular identification of Bacterial Community in Neochloris oleoabundans UTEX 1185

The highest number of isolated strains was obtained in conditions C1 (n = 39; 25°C; light), C3 (n = 24; 30°C; light), followed by C2 (n = 16; 25°C; dark) and, finally, C4 (n = 10; 30°C; dark). No growth was verified in conditions C5 (37°C; light) and C6 (37°C; dark) (Table 1). Of 89 isolates were identified by 16S rRNA gene sequencing, corresponding to Aureimonas sp. (n = 34), Microbacterium sp. (n = 26), Bacillus sp. (n = 8), Pseudomonas sp. (n = 5), Exiguobacterium sp. (n = 1), Brevundimonas sp. (n = 1) and Shinella sp. (n = 14). For taxonomic characterization, the isolates sequences were compared with the available at NCBI. Based on phylogenetic analyses, the strains Shinella (strain 77) and Microbacterium (strain 89) are suggested as new species, as they formed a new clade, as shown in the figure (Supplementary material, Fig. S6, S8). Finally, the strains that showed the most considerable phylogenetic distance in the clades of each genus analyzed were selected (Supplementary material, Fig. S2-S8).

Antimicrobial activity

Inhibition Zone Determination (Disc Diffusion Method)

Based on the results of phylogenetic analyses, it was possible to identify 32 microorganisms in which the phylogenetic distance in the clade was more significant. These strains were selected to evaluate the toxicity of the six essential oils by the disk diffusion method. It could detect activity against ten bacteria (see Table 2). The terpene solutions showed different degrees of antimicrobial activity, except for eugenol, which failed to inhibit the growth of any of the microorganisms evaluated. In the case of linalool, it was possible to observe inhibition only on the genus Bacillus at 0.9 mm, as shown in Table 2. Similar results were observed for the case of limonene, the inhibition zone was 0.7 mm, and the strains in which the effect was observed were the genus Bacillus. α -pinene and β- pinene showed more significant inhibition against all the bacteria tested; the range obtained was 0.7-1 mm and 0.6-3 mm, respectively. Finally, the oil that exhibited the best activity against most bacteria was Trans-cinnamaldehyde, measured between 0.8-2 mm (see Table 2). In the case of the Brevundimonas strain, it was observed that none of the essential oils inhibited growth.

Minimum Inhibitory Concentration (MIC)

Based on the results described in section 3.3 (Inhibition Zone Determination), six strains and five essential oils were selected to determine the minimum inhibitory concentration. The MIC and percentage inhibition values for each strain are described in Table 3. Pseudomonas was the most resistant strain to all the essential oils since it was impossible to observe inhibition at any of the concentrations analyzed. For Microbacterium, Aureimonas, and Exiguobacterium, the MIC was 6.25 mg L^− 1 for all oils. Microbacterium was more sensitive to limonene, while Aureimonas and Exiguobacterium had a higher percentage of inhibition to Beta-pinene (see Table 3). Bacillus genus presented resistance for Alpha-pinene and Linalool oils; the MIC value ranges between 12.5–50 mg L^− 1, and the highest percentage of inhibition was observed using limonene. Finally, Beta- pinene and Limonene oils did not affect Shinella cultures. The MIC value is between 6.25-50 mg L^− 1 for the genera evaluated (Microbacterium, Shinella, Bacillus, Aureimonas, and Exiguobacterium). While the inhibition was up to 99% (Shinella) using linalool.

Total viable count of bacteria and bacterial metagenome

Currently, studies on bacterial communities associated with microalgae refer to natural environments; however, there needs to be more information on the different interactions under laboratory conditions. Determining these interactions is essential to establish control processes for reducing possible bacterial contaminants in microalgae cultures. This work evaluated the culturable bacteria in two periods (initial and 12 months after maintenance). It was possible to observe the reduction of more than 50% of the total culturable bacteria. The removal of the cultivable bacteria may be due to the maintenance and conservation processes of the culture. In controlled culture conditions, all microalgae are associated with bacterial strains that may have some considered contaminants or inhibitors. Therefore, it is essential to maintain the culture at different intervals. These intervals will depend on the growth rate of the species, as well as environmental factors such as light, nutrients, and temperature, among others (Wang et al. 2013; Fulbright et al. 2016; Vu et al. 2018).

There are reports on cultivating and quantifying total bacteria in some microalgae cultures. For instance, Tetraselmis suecica was produced using an F medium in two conditions. In the laboratory, the microalgae were maintained in a sterile medium, and the total bacterial concentration was 102.4x10⁶ CFU mL^− 1. On the other hand, T. suecica outdoor culture was cultivated under a semi-continuous regime from February to November; the total concentration was reached in the winter and was 26.5x10⁶ CFU mL^− 1 (Biondi et al. 2017). Another case is the production of Arthrospira platensis using a SOT medium under continuous illumination at 30°C and 150 rpm. In both cases, the bacterial population was 3.7x10⁶ CFU mL^− 1, higher than those obtained in the present work (Choi et al. 2008).

In non-axenic microalgae cultures, a zone known as phycosphere is associated with different bacterial strains, some present in the natural habitat and others that entered the culture as contaminants, even in closed systems. In this zone, bacterial growth is stimulated by different extracellular products of the algae, so bacteria-microalgae interactions can be precise, resulting from a balance between stimulatory and inhibitory activities. Due to these interactions, variations have been observed in a microspatial context or on a larger time scale during different culture stages (Ramanan et al. 2016; Biondi et al. 2017). Studying these interactions could facilitate algal production; for instance, in the aquaculture industry, it is possible to use non-pathogenic bacteria as biocontrol agents (Riquelme 2003).

The bacterial community associated with N. oleoabundans culture was also analyzed at two intervals (initial and 12 months after maintenance). Reports show that the phyla level, Bacteroidetes, and Proteobacteria were previously identified in the Nannochloropsis salina and T. suecica (Goecke et al. 2013; Kimbrel et al. 2019; Piampiano et al. 2019). Some bacterial groups can stimulate microalgal growth. However, these heterotrophic bacterial groups developed in an algal culture that was initially devoid of organic and used sources generated by the microalgae to grow (le Chevanton et al. 2013). Nevertheless, communities restrict algal development due to nutrient competition (Wang et al. 2013) or by releasing anti-algal substances (Banerjee et al. 2002; Sambles et al. 2017).

It is known that the highest concentrations of organic carbon are found in the phycosphere, which may favor the presence of chemotactic bacteria, leading to potentially favorable physical attachment of bacteria to microalgal cells (Smriga et al. 2016; Kimbrel et al. 2019). Though it is not possible to limit this region to an exact part of a cell, it is probable that some bacterial taxa do not adhere to algal cells and probably could live close to microalgal cells. In addition, the attachment process (bacteria - microalgae) may be transient (attachment followed by detachment or loose attachment). Therefore, some groups that show attachment are likely to have an unattached contingent at any given time (Seymour et al. 2017).

Isolation, Determination, and Molecular identification of Bacterial Contaminants in Neochloris oleoabundans UTEX 1185

Environmental factors can influence the development of bacterial communities associated with microalgae cultures. For instance, the temperature directly impacts bacterial growth, which was possible to observe during the isolation process (Fulbright et al. 2018b). The highest number of isolated strains was obtained using temperatures between 25°C − 30°C (C1 and C3), while higher temperatures inhibited growth, making it impossible to isolate bacteria in conditions C5 and C6 (Table 1). N. oleoabundans UTEX 1185 exhibits optimal growth at 25°C; it is possible that the isolated microorganisms were adapted to these conditions (Singh and Singh 2015).

There are reports on the presence of Microbacterium and Shinella genera in microalgae cultures such as Botryococcus braunii, T. suecica, and Chlorella sp. (Biondi et al. 2017; Sambles et al. 2017). The genera Pseudomonas, Bacillus, Brevundimonas, and Exiguobacterium were also reported as bacteria associated with the culture of Microchloropsis salina, Chlorella vulgaris, Scenedesmus obliquus (Krohn-Molt et al. 2013; Fulbright et al. 2016; Fisher et al. 2019; Liu et al. 2020a). Some of these bacteria showed inhibitory effects on microalgae growth. For instance, some Bacillus species can be lethal to Microcystis aeruginosa, Chlorella, and Scenedesmus (Mu et al. 2007). There are reports that bacteria are more effective than algae in phosphate removal, reducing algal growth under phosphate-limiting conditions (Guerrini et al. 1998).

Antimicrobial activity

Inhibition Zone Determination (Disc Diffusion Method) and Minimum Inhibitory Concentration (MIC)

Currently, some studies report on the antimicrobial activity and the use of essential oils on bacterial groups of clinical and food interest. However, based on these characteristics, the present study investigated the effect of six terpenes on bacteria isolated from the culture of N. oleoabundans UTEX 1185.

In the disc diffusion method, it was observed that eugenol did not inhibit the growth of any of the bacterial genera tested (Table 2). There are reports on the effect of this terpene on various pathogenic bacteria and fungi; for instance, Staphylococcus aureus, Escherichia coli, Candida spp., and Aspergillus niger, among others. Nevertheless, the concentrations evaluated (MIC and MBC) were higher than those used in this work (100 mg L^− 1) (Marchese et al. 2017). Although the genera Pseudomonas and Brevundimonas presented inhibition halos (disk diffusion method, Table 2) using the concentration of 100 mg L^− 1, it was impossible to determine the MIC value (Table 3), suggesting the need to evaluate higher concentrations.

There are reports on the effect of different terpenes on the genus Pseudomonas (Dias et al. 2022). For instance, in the work by Liu et al. (2020b), the antibacterial activity of linalool was evaluated on Pseudomonas fluorescens. On the other hand, the genus Brevundimonas, despite having few species, is considered to have pathogenic potential. Currently, there are no reports of using terpenes to control this genus of bacteria. MIC and MBC values have been determined for different antibiotics, such as piperacillin-tazobactam, amikacin, and doripenem (Lee et al. 2011). For the genera Bacillus, Microbacterium, Aureimonas, Exiguobacterium, and Shinella, it was possible to determine MIC values for some of the terpenes evaluated (Table 3), and none of the oils evaluated showed 100% inhibition; therefore, these substances are bacteriostatic, i.e., they manage to inhibit the growth of microorganisms without causing immediate death, so their effect could be reversible.

Several factors can influence the effect of essential oil; for instance, oxygenates (terpenoids) show better antimicrobial activity than hydrocarbons such as (+)-α-pinene. Additionally, another point to be considered is the mechanism of action.

The terpenes effect is structural and functional at the membrane level (Bakkali et al. 2008). However, as terpenes are lipophilic compounds, their interaction with the hydrophobic part of the membrane may induce effects on anisotropy and dipolar organization. Permeabilization, a possible mechanism, is associated with the loss of ions, reduction of membrane potential, collapse of the proton pump, and depletion of the ATP pool. Another possibility to explain cell damage is the coagulation of the cytoplasm, damage to lipids and proteins, and may also produce leakage of macromolecules and cell lysis, as well as inhibit enzymes such as protease, histidine carboxylase, amylase, and ATPase (Lambert et al. 2001; Turina et al. 2006).

Prevention and control are essential tasks that help to avoid significant losses in production systems. Currently, contamination of microalgae crops is one of the main problems to be controlled. There are several reports on the adverse effects of contaminants. Therefore, it is essential to develop new technologies. In the present work, the tested terpenes showed activity on most bacteria isolated from N. oleoabundans UTEX 1185. This indicates that these substances could be used as control agents for biological contaminants in microalgae cultures. Therefore, the use of naturally occurring antimicrobial substances is of great interest due to the possibility of being used as management alternatives in different crops. However, further studies are needed to determine optimal concentrations, interactions, and efficacy in large-scale systems.

The phylum Proteobacteria, Bacteroidetes, and Actinobacteria were identified in the cultures of N. oleoabundans UTEX 1185, while some of the genera identified were Parvibaculum, Pseudomonas, Anaplasma, and Sphingopyxis. Eighty-nine microorganisms were isolated, including the following genera: Aureimonas sp., Microbacterium sp., Pseudomonas sp., Bacillus sp., Shinella sp., Brevundimonas sp., Exiguobacterium sp. The highest number of isolates was obtained at 25ºC in the presence of light. It was possible to observe that Beta-pinene 50 mg L^− 1 inhibited bacteria Bacillus sp. In contrast, Alpha-pinene, Linalool, and Trans-cinnamaldehyde, a concentration of 6.25 mg L^− 1, efficiently inhibited most isolates. The inhibition percentages found were 79%-99%.

CRediT authorship contribution statement

Denisse Tatiana Molina-Aulestia: Methodology, Investigation, Formal analysis, Writing – original draft. Júlio César de Carvalho: Methodology, Conceptualization, Supervision, Visualization, Writing – review & editing. Gilberto Vinícius de Melo Pereira: Formal analysis, Writing – review & editing. Alexander da Silva Vale: Methodology, Investigation, Formal analysis, Writing – review. Dão Pedro de Carvalho: Investigation, Writing – review. Vanete Thomaz Soccol: Formal analysis, Writing – review & editing. Carlos Ricardo Soccol: Formal analysis, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no know competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability Statements

The datasets generated during and analyzed during the current study are available from the corresponding author upon reasonable request.

Funding Information

This research was funded by CNPq, the Brazilian National Council for Scientific and Technological Development, grant number 407543/2013-0, and CAPES, the Coordination of Improvement of Higher Education Personnel - PROEX program.

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Table 1. Conditions and a total of microorganisms isolated

	C1 – Light, 25°C						C2 – Dark, 25°C						C3 – Light, 30°C						C4 – Dark, 30°C
Conditions	NA	TSA	LB	BG₁₁	BF	FL	NA	TSA	LB	BG₁₁	BF	FL	NA	TSA	LB	BG₁₁	BF	FL	NA	TSA	LB	BG₁₁	BF	FL
Aureimonas	0	3	1	1	1	0	0	0	2	0	3	2	0	2	2	3	3	2	0	0	0	6	0	3
Microbacterium	3	4	0	0	0	0	0	1	2	0	2	2	0	2	1	0	4	4	0	1	0	0	0	0
Bacillus	4	3	0	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0
Pseudomonas	5	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Exiguobacterium	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0
Brevundimonas	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0
Shinella	14	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

C5 - C6 do not show growth

Table 2. Inhibition zones (mm) of growth of tested microorganisms by essential oils Inhibition

	Essential oil (mm)
Strain	Linalool	α-pinene	β- pinene	Trans cinnamaldehyde	Limonene
Microbacterium	0	0	2.345	2.454	2.291
Microbacterium	0	1.116	1.248	0.504	0
Microbacterium	0	0	0	7.020	0
Microbacterium	0	0	0	1.003	0
Microbacterium	0	0	0	1.024	0
Microbacterium	0	0.814	0.535	1.257	0
Exiguobacterium	0	0.801	0.696	1.262	0
Bacillus	0.925	0.816	0.761	1.208	0.734
Bacillus	0.911	0.714	0.916	0.880	0.800
Aureimonas	0	0.781	3.135	1.854	0
Shinella	0.905	0.705	0	1.105	0

Table 3. Minimal Inhibitory Concentration (MIC) and Inhibition percent of bacterial isolated of N. oleoabundans culture (mg L^-1)

Strains	Alpha-pinene		Beta-pinene		Linalool		Limonene		Trans-cinnamaldehyde
Strains	MIC	%	MIC	%	MIC	%	MIC	%	MIC	%
Bacillus sp	>100^a	0	50	89	>100^a	0	12.5	92	12.5	89
Microbacterium sp	6.25	97	6.25	93	6.25	97	6.25	99	6.25	93
Shinella sp	6.25	98	>100^a	0	12.5	99	>100^a	0	6.25	93
Aureimonas sp	6.25	82	6.25	83	6.25	82	25	81	6.25	79
Pseudomonas sp	>100^a	0	>100^a	0	>100^a	0	>100^a	0	>100^a	0
Exiguobacterium sp	6.25	84	6.25	89	6.25	79	6.25	57	6.25	80

^aMIC value above 100 mg L^-1

No competing interests reported.

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Fine Resolution Analysis of bacterial communities associated with Neochloris oleoabundans culture and insights into terpenes as contamination control agents

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