There is an urgent need for sustainable protein supply routes with low environmental footprint. Recently, the use of hydrogen-oxidizing bacteria (HOB) as a platform for high-quality microbial protein (MP) production has regained interest. This study aims to investigate the added value of using conditions such as salt and temperature to steer HOB communities to lower diversities, while maintaining a high protein content and a high-quality amino acid profile. Pressure drop and hydrogen consumption were measured for 56 days to evaluate autotrophy of a total of six communities in serum flasks. Of the six communities, four were enriched under saline (0.0, 0.25, 0.5, and 1.0 mol NaCl liter−1) and two under thermophilic conditions (65°C). Five communities enriched for HOB were subsequently cultivated in continuously stirred reactors under the same conditions to evaluate their potential as microbial protein producers. The protein percentages ranged from 41 to 80%. The highest protein content was obtained for the thermophilic enrichments. Amino acid profiles were comparable to protein sources commonly used for feed purposes. Members of the genus Achromobacter were found to dominate the saline enrichments, while members of the genus Hydrogenibacillus were found to dominate the thermophilic enrichments. Here, we show that enriching for HOB while steering the community toward low diversity and maintaining a high-quality protein content can be successfully achieved, in both saline and thermophilic conditions.
IMPORTANCE Alternative feed and food supply chains are required to decrease water and land use. HOB offer a promising substitute for traditional agricultural practice to produce microbial protein (MP) from residual materials and renewable energy. To safeguard product stability, the composition of the HOB community should be controlled. Defining strategies to maintain the stability of the communities is therefore key for optimization purposes. In this study, we use salt and temperature as independent conditions to stabilize the composition of the HOB communities. Based on the results presented, we conclude that HOB communities can be steered to have low diversity using the presented conditions while producing a desirable protein content with a valuable amino acid profile.


Mankind is predicted to grow to almost 10 billion people in the coming 30 years (1). As a consequence, the world’s demand for protein is expected to double (2). Assuming the current feeding patterns, satisfying human requirements for protein with traditional farming methods will not only lead to more intense competition for natural resources but also to an increase in waste production. This will ultimately result in a rise in greenhouse gas emissions and further deforestation, land degradation, and biodiversity loss (3). An alternative solution is the production of microbial protein (MP) by bacteria, fungi, yeast, or algae. The key difference of MP production, when compared with conventional animal/plant protein production, lies in the fact that nutrient and water loss can be avoided, therefore decreasing its ecological impact (3, 4). Moreover, bacteria have the advantage of using a wide variety of substrates and producing a large array of products, which can be considered as added value (5, 6).
Chemolithotrophs such as hydrogen-oxidizing bacteria (HOB), also known as Knallgas bacteria, are of particular interest in this framework due to their ability to capture carbon dioxide. Using hydrogen as an electron donor and oxygen as an electron acceptor opens the possibility of detaching protein production from agricultural practice (7). When implementing such a process, predefined communities of several types of bacteria, contrary to cultures of single species, can be valuable due to their increased ability to adjust their activity to changes in environmental conditions (8). The use of a dedicated group of species, i.e., community of which the composition is restricted and that can be maintained within defined boundaries, implies a higher metabolic diversity and thus a higher competitive advantage when faced with the intrusion of undesired microorganisms or changes in the environment (9). This phenomenon is known as the competitive exclusion principle. While communities cultivated in open systems have been reported to show an increase in adaptability and activity (8, 10), the inexistence of growth constraints can also result in continuously changing growth conditions (11), which ultimately can lead to a turnover in species composition. Variation in the amount and abundance of species could imply a lack of control over the MP over time.
One strategy to assure a stable and safe microbial community, without relying on traditional sterilization processes, comprises the use of conditions, such as salinity, temperature, pressure, or pH, that favor specific organisms and thus allow for a more defined combination of organisms (12). In addition to offering a potentially safer path to MP production (13, 14), the use of such dedicated HOB communities may also result in the opportunity to steer the product, i.e., to push the community toward producing certain desired compounds.
Halophilic bacteria, organisms that require salt (NaCl) for growth, thrive in high-salinity environments mainly through the production of compatible solutes (water-soluble organic compounds) (15). Other organisms, such as halophilic archaea, follow a different strategy to cope with high-salinity environments and accumulate potassium ions intracellularly (16).
Such compounds are commercially relevant due to their potential use in the cosmetics and food industries, both as moisturizers and as protein stabilizers. Furthermore, halophiles can be used as feed in aquaculture and are also potential sources of unique enzymes, antioxidant pigments, mycosporine-like amino acids (17), and exopolysaccharides (EPS). Thermophiles are generally defined as organisms able to thrive at temperatures over 55°C and have for the last several decades received increasing attention, as they express a broad variety of heat-resistant enzymes (thermozymes) (18, 19) and represent an important source for novel biocatalysts (20). Thus, organisms able to cope with high salinity and high temperature represent a potential source for food industrial purposes.
The present research aims at exploring protein production by HOB enriched from saline and thermophile environments. We show that enriching for HOB while steering the community toward low diversity and maintaining a high-quality protein content can be successfully achieved in both saline and thermophilic conditions. The ability to steer microbial composition while maintaining the product’s quality may lead to valuable biotechnological applications.


Enrichment with HOB at high-salt and high-temperature conditions.

Salt (0, 0.25, 0.50, and 1.0 mol · liter−1 NaCl) and temperature (65°C) were used to investigate the possibility of steering HOB communities toward low diversity. Salt and temperature were chosen for their accessibility and applicability. Three different inocula were selected for this purpose, and soil from a salt production site was used to enrich for salt-tolerant HOB communities (see Fig. S1 in the supplemental material). The highest tested salt concentration was selected based on the concentration of NaCl in sediment used as inoculum (1.0 mol · liter−1 NaCl). Decreasing concentrations were chosen to predict the minimum level of salt required to steer the communities toward lower diversity. Sediment from a geyser and compost were used to enrich for thermophile HOB communities. The thermophile enrichment processes were performed at 65°C, as that was the average temperature of both samples. A total of six enrichments were obtained (Table 1); four were inoculated using saline soil (CM 1, CM 2, CM 3, and CM 4), one using compost (TC), and one using sediment retrieved nearby the geyser (TG). Hydrogen consumption showed that, after 56 days, the enrichment process resulted in active HOB communities in each of the serum flasks (Fig. 1).
FIG 1 Enrichment of HOB communities under saline (CM 1 to CM 4) (a) and thermophile conditions (TC and TG) (b). Hydrogen consumption was used for all enrichments as a measure for HOB activity for an experimental period of 56 days. Error bars represent standard deviation (n = 3).
TABLE 1 Enrichment conditions for three inocula in saline and thermophile conditions
Enrichment codeInoculumNaCl (mol · liter−1)Temp (°C)
CM 1Sediment from a salt production site0.030
CM 2Sediment from a salt production site0.2530
CM 3Sediment from a salt production site0.5030
CM 4Sediment from a salt production site1.030
TGSediment from a geyser0.065

Structure and composition of the enriched communities.

As the enrichments were performed in autotrophic conditions, i.e., using inorganic carbon and energy sources, bacteria and/or archaea were expected to dominate the enriched communities. To determine the content of archaea and bacteria in each community, a quantitative real-time PCR (qPCR) analysis was initially performed, which showed that bacteria were dominating the microbial communities (see Table S1 in the supplemental material). For this reason, further structure and composition analyses were performed using a bacterium-targeting strategy only, i.e., 16S rRNA amplicon sequencing using primers targeting specifically this group of organisms. The rarefaction curves of 16S rRNA amplicons suggested that the sequencing depth was sufficient to describe the communities in all samples (see Fig. S2 in the supplemental material).

(i) HOB-enriched communities at high salt levels.

To evaluate the differences between the bacterial communities enriched with HOB at different NaCl concentrations, a principal coordinate analysis (PCoA) based on the Bray-Curtis dissimilarity matrix was used (Fig. 2a). The results indicated high similarity between the communities enriched at high concentrations of NaCl (0.5 and 1.0 mol · liter−1, CM 3 and CM 4, respectively) compared with the communities enriched with 0.25 NaCl mol · liter−1 (CM 2) and without the addition of NaCl (CM 1). The latter two are also shown to be different from each other. Alpha diversity by means of amplicon sequence variant (ASV) richness and Shannon diversity (Fig. 2b and c) was higher for the community enriched without salt (CM 1) than the community enriched with 1.0 mol · liter−1 NaCl (CM 4). Figure 2d depicts the phylogenetic classification of the communities enriched with HOB at different NaCl concentrations at the genus level. The results showed a diversity shift from the community enriched with no addition of salt (CM 1) to the communities enriched with increasing amounts of NaCl (CM 2, CM 3, and CM 4). While the saline communities were dominated by members of the Achromobacter genus (95.6%, 97.3%, and 97.01%, respectively), the nonsaline community contained members belonging to both Alcaligenaceae and Burkholderiaceae families (54.2 and 36.7%, respectively). In total, seven known bacterial families were detected, including Alcaligenaceae, Burkholderiaceae, Oxalobacteraceae, Xanthomonadaceae, Microbacteriaceae, Enterobacteriaceae, and Bacillaceae.
FIG 2 Diversity of HOB communities enriched under saline conditions. (a) Principal coordinates based on Bray-Curtis dissimilarities calculated from the rarefied table. Note that nearly all variance is explained along the first principal coordinate axis. (b, c) Alpha diversity of bacterial communities under saline conditions. (d) A phylogeny (maximum likelihood 16S rRNA marker-based) comprising all amplicon sequence variants (ASV) depicts associated genus-level taxonomy and class-level grouping. The tree is combined with a heatmap presenting relative read abundances per ASV. Node support is based on 100 bootstrap trees, and tree scale denotes estimated evolutionary distances among taxa (GTR + G + I nucleotide substitution model). Sample CM 1 represents the bacterial diversity before enrichment is applied using saline conditions at incremental concentrations (CM 2, CM 3, and CM 4). ASV richness (a) and Shannon diversity H′ estimates (b) for each sample are calculated from a rarefied feature table with 14,650 sequences per sample. The rarefied data capture the full diversity of the samples; rarefaction curves are presented in Fig. S2a in the supplemental material.

(ii) HOB communities enriched at high temperature.

HOB communities enriched at 65°C (TC and TG) showed comparable ASV richness (Fig. 3a) but were less even, as the Shannon diversity index was 3 times lower (1.8 and 0.58) for the community retrieved from the geyser sediment than the community retrieved from compost (Fig. 3b). The top genera for the thermophilic communities included five members from the Bacilli class, Caldibacillus, Geobacillus, Kyrpidia, and Hydrogenibacillus as well as members from the Clostridia, Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, and Coriobacteriia classes. Interestingly, seven different ASVs were assigned to Hydrogenibacillus, the dominant genus in both communities.
FIG 3 Diversity of HOB communities enriched under increased temperature. (a, b) Alpha diversity of bacterial communities experiencing elevated temperature. (c) A phylogeny (maximum likelihood 16S rRNA marker-based) comprising all amplicon sequence variants (ASV) depicts associated genus-level taxonomy and class-level grouping. The tree is combined with a heatmap presenting relative read abundances per ASV. Node support is based on 100 bootstrap trees, and tree scale denotes estimated evolutionary distances among taxa (GTR + G + I nucleotide substitution model). ASV richness (a) and Shannon diversity H′ estimates (b) for each sample are calculated from a rarefied feature table with 16,080 sequences per sample. The rarefied data capture the full diversity of the samples; rarefaction curves are presented in Fig. S2b in the supplemental material.

Protein content and amino acid composition.

Once autotrophic growth was confirmed, five out of the six HOB communities were cultivated in a 2-liter continuous stirred-tank reactor (CSTR) to evaluate their potential as microbial protein producers. The thermophilic communities displayed a similar bacterial structure, and thus only one (TG) was selected for further analysis.
Protein content (Fig. 4a) and amino acid composition (Fig. 4b) were evaluated in biomass grown under sequential batch configuration and harvested at the end of the cultivation period. With respect to the protein content, a variation of up to 23% was observed among the saline communities, with the highest content corresponding to the community enriched with 0.5 mol · liter−1 NaCl (63%) and the lowest content corresponding to the community enriched without the addition of salt (41%). The former is considerably higher than the commonly used soybean meal (45%) and slightly lower than reference protein feed additives, such as fishmeal (66%) or bacterial meal (68%). Protein content of the thermophilic community (TG) was 80%. In addition to the protein content, variation in the essential amino acid profile was also observed among the saline communities. The profile of the communities enriched with 0.25 and 1 mol · liter−1 NaCl is similar (except for arginine and lysine) and comparable to that of bacterial meal, fishmeal, and single-cell protein produced by a Sulfuricurvum species bacterial culture described by Matassa et al. (11). The community enriched with 0.5 mol · liter−1 NaCl follows the same pattern showing, however, increased levels of certain amino acids, such as arginine, leucine, and valine. The community enriched with no addition of salt, conversely, had a profile closer to the one of soybean meal.
FIG 4 Protein content per cell dried weight (a) and essential amino acids profile (b) of the microbial communities grown at increasing salinity levels (CM1, CM2, CM3, and CM4) and high temperature (TG) (this study) compared with the HOB-enriched community grown by Matassa et al. (11) as well as bacterial meal, fishmeal, and soybean meal as reported by Overland et al. (58). Arg, arginine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Cys, cysteine; Phe, phenylalanine; Tyr, tyrosine; Val, valine. Error bars represent standard deviation (n = 3).
This shows that salt and temperature are promising conditions to drive the enriched HOB communities toward lower diversities.

Growth performance.

The main parameters analyzed under batch conditions included growth rate (h−1), volumetric productivities (g cell dry weight [CDW] · liter−1 · day−1), and nitrogen upgrade efficiency (%). As reported in Table 2, volumetric productivities varied between 0.082 and 0.161 g CDW · liter−1 · day−1 for the saline communities. The highest value was obtained for 0.25 mol · liter−1 of NaCl. Accordingly, higher growth rate (0.0211 h−1) and nitrogen upgrade efficiency (51%) were observed for the same community. TG presented a volumetric productivity of 0.098 g CDW · liter−1 · day−1 and a nitrogen upgrade efficiency of 23%.
TABLE 2 Parameters of HOB cultivation obtained under batch configuration, averaged over three different sequential batch tests
Enrichment codeTemp (°C)NaCl (mol · liter−1)Growth rate (h−1)Volumetric productivity (g CDW · liter−1 · day−1)N upgrade efficiency (%)
CM 13000.0116 ± 0.0400.115 ± 0.04339.6 ± 0.7
CM 2300.250.0211 ± 0.0110.161 ± 0.01050.5 ± 1
CM 3300.500.0117 ± 0.0120.106 ± 0.01440.9 ± 0.6
CM 4301.00.0089 ± 0.070.082 ± 0.00719.9 ± 0.3
TG65 0.0109 ± 0.0100.098 ± 0.01223.42 ± 0.2


The environmental impact of the current farming strategies together with the prospect of increasing protein demands has led to the revival of MP as a suitable protein source for feed and food (21). Guaranteeing a stable and constant product composition is a key feature for establishing successful reactor-based MP production technologies. So far, such need has been met by the use of axenic conditions (22, 23), which require a strict sterile production process. Natural communities have recently come forward as an innovative strategy to overcome sterility requirements (11). Controlling the composition of communities, however, is crucial, as changes might lead to a product of unknown nutritional value. Here, we show that both salt and temperature can be used as independent conditions to drive the enriched HOB communities toward lower diversities.

Characteristics of the enriched bacterial communities.

The enrichment strategies successfully resulted in six enriched HOB communities, four enriched in the presence of different NaCl concentrations and two enriched at 65°C.
Communities CM 1, CM 2, CM 3, and CM 4 were enriched at 0.0, 0.25, 0.5, and 1.0 NaCl mol · liter−1, respectively, and were found to be dominated by members of the Achromobacter genus at relative abundances over 95%. Representatives of the Alcaligenaceae family, to which the Achromobacter genus belongs, occupy diverse ecological niches ranging from water and soil to animals (24) and have been previously recognized as facultatively lithoautotrophic hydrogen oxidizers (25). Achromobacter representatives have been associated with saline environments and have also been reported as abundant members of HOB enrichments from soil samples (26). The Achromobacter genus has recently been given attention due to the ability of some members to infect immunocompromised humans (27).
Communities TG and TC were enriched at 65°C and were dominated by members of the genus Hydrogenibacillus at relative abundances over 98%. The Hydrogenibacillus genus belongs to the family Bacillaceae and contains one species, the thermophilic and facultatively chemolithoautotrophic Hydrogenibacillus schlegelii (previously Bacillus schlegelii) (28, 29).
In other studies that report on similar enrichment strategies, i.e., HOB grown in a nonaxenic sequential batch configuration that were inoculated from environmental samples, the spontaneous communities were shown to be highly diverse and to evolve to a different composition over time (11, 26). Both Matassa et al. (11) and Ehsani et al. (26) observed the segmentation of the HOB-enriched communities into the following three distinct groups: autotrophic HOB, heterotrophic bacteria, and predatory bacteria.
Similar to any other food or feed product, microbial protein is required to be safe for consumption (30). This means, in a broad sense, a low RNA content, absence of toxins (produced by either the host bacteria or the contaminant bacteria), and no allergenic potential (31). In axenic conditions, the risk of toxins can be overcome by carefully selecting the production organism (host bacteria) and the process conditions (avoiding contamination). When a community under nonrestrictive conditions is used, the control of its composition becomes more challenging. The use of temperature as a selection factor offers a dual advantage. As shown in this study, temperature led to the enrichment of HOB, which can be considered as safe, with a high-quality protein profile while maintaining diversity at low levels. The use of a temperature higher than 50°C also impairs the growth of the most common foodborne pathogens (3234), often responsible for toxin production. The endospores of spore-forming pathogens, however, may remain viable even when subjected to temperatures higher than 50°C.

Potential as MP producers.

The protein content of the HOB biomass produced at 65°C was about 10% higher than that of HOB biomass grown at room temperature, whereas the protein content of the HOB biomass grown at 0, 0.25, 0.50, and 1.0 NaCl mol · liter−1 was 30, 13, 8, and 16% lower, respectively. The former shows a protein content more similar to fishmeal. Although these findings lack the complementary animal studies on allergenicity and digestibility, they clearly illustrate that HOB grown either at elevated salt (0.50 NaCl mol · liter−1) or at higher temperatures (65°C) remain comparable to high-quality protein sources.
While the protein content and the amino acid profile remain within the FAO’s recommendations (35), all of the HOB-enriched communities were characterized by yield performances much lower than other reported HOB cultures (11, 36, 37) grown at 30°C and without the addition of NaCl. The volumetric productivity of communities CM 2, CM 3, CM 4, and TG was 12, 18, 23, and 19 times lower than the average productivity, respectively, presented for instance by Matassa et al. (11), 0.078 ± 0.012 g CDW · liter−1 · h−1. Hydrogen is well known to have a very low solubility in water (38). When temperature or salinity increases, hydrogen solubility further decreases. The low solubility of hydrogen gas with the resulting low transfer rate is thus likely to have hampered bacterial growth. The resistance of the gaseous substrate diffusion at the gas-liquid interface was recognized as the limiting step for other microbial processes, such as syngas fermentation (39, 40), methanotrophic MP production (41), or hydrogenotrophic denitrification (42). In this study, a conventional CSTR was used to cultivate the HOB-enriched communities. Despite being simple and easy to operate, the gas delivery scheme of gas sparging does not allow for an efficient hydrogen transfer. To overcome the challenge of delivering hydrogen in an efficient manner, Epsztein et al. (42) designed a novel pressurized high-rate hydrogenotrophic reactor without gas purging. Briefly, the novel reactor allows for the maintenance of a gas-liquid equilibrium without pressure build-up and without any hydrogen gas loss from the gas phase. The reactor is operated under an unsaturated flow regime as a trickling filter where water is recirculated over the biofilm carriers (42). In another study, a U-loop fermenter for methanotrophic MP production has been proposed. In this case, liquid or gas-liquid mixtures are injected through one or more jets into one large tank where the bioreaction takes place. By doing so, high superficial gas velocities were achieved, outperforming the traditional CSTR (43). Both designs could be employed as a strategy to improve HOB productivities in saline and thermophile conditions.

Concluding remarks.

The use of salt and temperature, as proposed in this study, allows for the enrichment of restricted HOB communities, providing an alternative to stabilize them and to achieve a more defined composition. The protein content of the HOB biomass produced at 65°C was about 10% higher than that of HOB biomass grown at room temperature and bacterial meal. In both conditions (saline and thermophile), the amino acid profile was comparable to traditionally used protein sources. Further research is required to optimize the performance of the process in terms of biomass yield.


Sampling and enrichment.

For the saline enrichments, 5 g of sediment retrieved from a salt pond (Salinas de Castro Marim, Portugal) was resuspended in 100 ml of distilled water and incubated in a rotary shaker at room temperature for 1 h. Serial dilutions were performed in mineral medium supplemented with 0, 0.25, 0.5 and, and 1 mol liter−1 NaCl. The mineral medium (1 liter) contained 2.3 g KH2PO4, 4.0 g Na2HPO4·7H2O, 1 g NH4Cl, 0.5 g MgSO4·7H2O, 0.5 g NaHCO3, 0.01 g CaCl2·2H2O, 0.05 g ferric ammonium citrate, and 1 ml trace element solution. The trace element solution (1 liter) contained 0.6 g H3BO3, 0.4 g CoCl2·6H2O, 0.2 g ZnSO4·7H2O, 0.06 g MnCl2·4H2O, 0.06 g NaMoO4·2H2O, 0.04 g NiCl2·6H2O, and 0.02 g CuSO4·5H2O. A total volume of 10 ml of serial dilutions 10−5 to 10−10 was transferred to gas-tight 100-ml serum bottles. The remaining volume of the serum flasks was used as headspace. For the thermophile enrichments, 5 g of sediment retrieved nearby a geyser (Iceland) was resuspended in 100 ml of distilled water and incubated in a rotary shaker at room temperature for 1 h. Serial dilutions were also performed in mineral medium but without salt supplementation. A total volume of 10 ml of serial dilutions 10−5 to 10−10 was transferred to gas-tight 100-ml serum bottles. The previous process was repeated for the compost sample. The flasks were continuously flushed for a period of 48 h with a hydrogen (30%), carbon dioxide (15%), and air (45%) mixture using long syringes and incubated at 30°C and 65°C for the saline and thermophile enrichments, respectively. Gas consumption was detected by pressure drop measurements and further confirmed by gas chromatography (GC) analysis (Varian CP-4900 gas chromatograph, thermal conductivity detector (TCD), helium carrier gas, and a Mol Sieve 5A Plot [10 m by 0.53 mm] column). After hydrogen consumption was observed, weekly refreshments were performed until reproducible growth was attained. Enrichment conditions are summarized in Table 1. The experimental design is also described in Fig. S1 in the supplemental material.

Bioreactor design and operation.

A 2-liter glass vessel equipped with a metallic propeller was used to assess growth performance. Temperature and pH were automatically controlled and set to 30°C and 6.8, respectively. For the thermophile cultures, the temperature was adjusted to 65°C. Stirring was set to 250 rpm. The gas mixture was fed through a lab-scale gas disperser placed at the bottom of the vessel and designed to cover the entire surface. Hydrogen was locally produced through a lab-scale electrolyser, and compressed air was used to provide the oxygen. Gas flows were monitored using mass flow controllers (Bronkhorst High-Tech BV, The Netherlands) and kept at 65 ml of H2/min, 32 ml of CO2/min, and 120 ml of air/min.

Analytical methods.

Growth was monitored over time by cell dry weight (CDW), measured in duplicate after water was evaporated at 105°C for 24 h and organics were burned at 550°C for 2 h. Before the analysis, pellets were collected by centrifugation at 13,000 × g for 15 min three times, each time resuspending the biomass pellet in demineralized water. Total nitrogen was determined using cuvette tests (Hach Lange) in the collected pellet. The final protein content of CDW was obtained by applying a conversion factor of 6.25 to the obtained total nitrogen value (44). NH4+-N concentrations were determined by liquid chromatography-organic carbon detection (LC-OCD). Sample solution was first passed through a 0.45-μm polyethersulfone filter before dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) analysis using a liquid chromatography-organic carbon detection-organic nitrogen detection (LC-OCD-OND; model 8, DOC-Labor) with built-in Siemens Ultramat 6E nondispersive infrared detector (NDIR) coupled with a proprietary organic nitrogen detector (UV 220 nm) and UV detector (254 nm). The column used was a Toyopearl HW-50S, 30 µm, 250 mm (250 mm by 20 mm each), and it separated each sample into the following five fractions: biopolymers, humic substances, building blocks, low-molecular-weight (LMW) acids, and LMW neutrals. The mobile phase was a phosphate buffer (28 mmol, pH 6.58).

Amino acid composition.

Protein hydrolysis was performed in lyophilized biomass using methanesulfonic acid (4 M) at 160°C in a nitrogen atmosphere to prevent oxidation. Tryptamine (0.2%) was added to minimize tryptophan degradation. The obtained hydrolysate was partly neutralized with sodium hydroxide (4 M) and further diluted. Free amino acids were analyzed with an Agilent 6410 liquid chromatograph-tandem mass spectrometer (LC-MS/MS) using a Merck SeQuant ZIC-cHILIC column for separation and selective electrospray triple quad LC-MS/MS MRM transitions for detection and quantification.


Calculations were as follows:
Nitrogen upgrade efficiency(%)=NiNoNi×100
Volumetric productivity(gliter × day)=CDWV×T
Protein content(%)=(ProteinCDW)×100
where Ni is the initial nitrogen concentration (mg/liter), No is the final nitrogen concentration (mg/liter), CDW is the cell dry weight (g/liter), V is the volume of the reactor (liters), and T is the operational time (days).

Bacterial community analysis preparation sequencing.

Pellets were obtained by centrifuging liquid samples for 15 min at 13,000 × g. Subsequently, total DNA was extracted by using the PowerBiofilm DNA isolation kit (Qiagen, USA) according to the manufacturer’s protocol. The extracted DNA was quantified using the QuantiFluor double-stranded DNA (dsDNA) kit and a Quantus 2.0 fluorometer (Promega, USA), and DNA purity and quality were confirmed by measuring the absorbance at 260 and 280 nm (Nanodrop 2000; Thermo Scientific, Waltham, MA, USA) and via agarose gel electrophoresis, respectively. A control PCR was carried out using the primers P338 and P518r (45) following the protocol of reference 46. The PCR product quality was verified via agarose gel electrophoresis to confirm that no PCR inhibition took place.

Real-time PCR.

The StepOnePlus real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) was used for real-time PCR (qPCR) analysis of technical triplicates of a 100-fold dilution of the DNA samples (diluted with DNase-free water) for the total bacteria and total archaea. The primer sets used for total archaea (ARC787F and ARC1059R) were previously described (47). Total bacteria were quantified using the P338f and P518r primers (48). The reaction mixture of 20 µl was prepared using the GoTaq qPCR master mix (Promega, Madison, WI, USA) and consisted of 10 µl of GoTaq qPCR master mix, 3.5 µl of nuclease-free water, 0.75 µl of each primer (final concentration of 375 nM), and 5 µl of template DNA. A two-step thermal cycling procedure, which consisted of a predenaturation step of 10 min at 94°C, followed by 40 cycles of 10 s at 94°C and 1 min at 60°C, was used. Linearized plasmids were used to create a 10-fold dilution series of 101 to 107 copies μl−1, and these were also analyzed in technical triplicates (see Table S1 in the supplemental material).

Amplicon sequencing and data processing.

The DNA extracts were sent to LGC Genomics GmbH (Berlin, Germany), where 300-bp paired-end sequencing was carried out on an Illumina MiSeq using V3 chemistry. Amplicon sequencing was performed by targeting the V3-V4 hypervariable region of the 16S rRNA gene using bacterial primers 341F and 785R, with an additional wobble position in the reverse primer to make it more universal (49). The PCR protocol was carried out as described by De Vrieze et al. (49). Sequence data processing comprised quality control and amplicon sequence variant (ASV) calling using QIIME2 (50) and DADA2 (51), respectively (see Table S2 in the supplemental material). Representative sequences for each ASV were used to assign taxonomy using a naïve Bayes classifier trained on full 16S sequences of the curated SILVA database v.132 (50, 5254).

Bacterial diversity.

To construct a maximum likelihood tree, a Crenarchaeota sequence (accession number AY861903) was first added to the representative sequences as an outgroup. Sequences were then imported in R (R Core Team, 2018) as a DNAStringSet for subsequent profile-to-profile multiple alignment with the DECIPHER package (54). A phylogeny was constructed with the phangorn package (55) assuming nucleotide substitution rates following the general time reversible (GTR + G + I) model (56), which was visualized using the ggtree package. The sample-by-feature table, taxonomic information, and phylogeny were imported into a phyloseq object (57) to estimate ASV richness, Shannon diversity H′, relative ASV abundances, and beta diversity using principal coordinate analysis (PCoA) of the Bray-Curtis dissimilarity matrix. Alpha diversity was estimated based on rarefied feature tables (halophiles, 14,650 reads per sample; thermophiles, 16,080 reads per sample). Rarefaction curves were generated to confirm that the full bacterial diversity could be estimated at the normalized depths (see Fig. S2 in the supplemental material). Moreover, the feature tables contained no ASVs assigned to eukarya, chloroplast, or mitochondria.

Data availability.

The sequences reported in this paper have been deposited in the NCBI Sequence Read Archive (SRA) under project number PRJEB39592.


This work was performed in the cooperation framework of Wetsus, European Centre of Excellence for Sustainable Water Technology (https://www.wetsus.nl). Wetsus is cofunded by the Dutch Ministry of Economic Affairs and Ministry of Infrastructure and Environment, the European Union Regional Development Fund, the Province of Fryslân, and the Northern Netherlands Provinces. This work has also received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 665874 and by the Geconcentreerde Onderzoeksactie, Ghent University (BOF15/GOA/006), as well as by the companies Avecom and DCWater.
We thank the research theme “Protein from Water” for the fruitful discussions.
We declare no conflict of interests.
Raquel G. Barbosa contributed conceptualization, investigation, formal analysis, and writing (original draft; review and editing); H. Pieter J. van Veelen contributed formal analysis and writing (NGS methodology); Vanessa Pinheiro contributed investigation; Tom Sleutels contributed supervision and writing (review); Willy Verstraete contributed supervision and writing (review); and Nico Boon contributed conceptualization, supervision, and writing (review).

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Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 87Number 429 January 2021
eLocator: e02439-20
Editor: Shuang-Jiang Liu, Chinese Academy of Sciences


Received: 4 October 2020
Accepted: 19 November 2020
Published online: 29 January 2021


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  1. hydrogen oxidizing bacteria
  2. HOB
  3. enrichment
  4. high salinity
  5. high temperature
  6. microbial protein



Center for Microbial Ecology and Technology (CMET), Ghent University, Ghent, Belgium
Wetsus, European Centre of Excellence for Sustainable Water Technology, Leeuwarden, The Netherlands
Wetsus, European Centre of Excellence for Sustainable Water Technology, Leeuwarden, The Netherlands
Vanessa Pinheiro
Wetsus, European Centre of Excellence for Sustainable Water Technology, Leeuwarden, The Netherlands
Tom Sleutels
Wetsus, European Centre of Excellence for Sustainable Water Technology, Leeuwarden, The Netherlands
Willy Verstraete
Center for Microbial Ecology and Technology (CMET), Ghent University, Ghent, Belgium
Avecom NV, Wondelgem, Belgium
Center for Microbial Ecology and Technology (CMET), Ghent University, Ghent, Belgium


Shuang-Jiang Liu
Chinese Academy of Sciences


Address correspondence to Nico Boon, [email protected].

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