Light and oxygen facilitating the directly treatment food wastewater and poly-β-hydroxybutyrate, 5-aminolevulinic acid, pigment productions by Rubrivivax gelatinosus Open Access

Purple nonsulfur bacteria (PNSB) are photosynthetic bacteria (Choorit et al. 2002; Asao et al. 2011; Chen et al. 2020). They can realize wastewater purification and the recycling of biomass resources simultaneously. So far, scientists have used PNSB to treat multiple types of wastewaters (Kobayashi & Kurata 1978; Zilles et al. 2002; Dong et al. 2003). Meanwhile, PNSB biomass is a valuable bio-resource to generate bio-polymers, poly-β-hydroxybutyrate, pigments, and 5-aminolevulinic acid (Jensen et al. 1961; Greene & Mascarenhas 1964; Kobayashi & Kurata 1978). Therefore, PNSB wastewater processing technology averts the surplus activated sludge problems as PNSB themselves can be utilized as resources. In a variety of wastewaters, the nontoxic and harmless class wastewater is optimal for PNSB biomass resource recycling.

In spite of the PNSB wastewater processing technique having many merits, it still cannot finish industrial application due to the limitation of a traditional theory. This is because the nontoxic and harmless wastewater usually includes quite a lot of macromolecular organic materials. According to microbial ecological succession law (Kobayashi & Tchan 1973, 1978; Siefert et al. 1978), the large population of heterotrophic bacteria was observed first in natural organic wastewater. At the same time, the macromolecules (macromolecular carbohydrates, protein) were degraded into small molecules (monosaccharide, lower fatty acid, amino acid). Afterward, the heterotrophic bacteria slowly reduced and PNSB increased rapidly. These indicated that PNSB could not decompose macromolecular organic and only directly utilized small molecules. Thus, the traditional theory stated that PNSB could not directly treat wastewater containing macromolecular organics. They included most types of nontoxic wastewaters suitable for PNSB treatment, such as protein and polysaccharide wastewaters (Kobayashi & Tchan 1973, 1978; Siefert et al. 1978).

The traditional theory restricts the direct application of PNSB wastewater treatment technology. As a result, the pretreatment (i.e., solubilization treatment) is often applied to degrade macromolecules into small ones in PNSB wastewater treatment. Then, PNSB is utilized for the following treatment in wastewaters containing macromolecular organics (Kobayashi & Tchan 1973, 1978; Siefert et al. 1978). However, this extra process will increase the energy and cost consumption and treatment process. It also uses up the growth substratum that otherwise will be absorbed by PNSB to generate biomass. Furthermore, PNSB may also be contaminated by infectious bacteria from the pretreatment process, making that PNSB biomass cannot be recycled and reused as a resource. So, achieving PNSB direct treatment of macromolecular organic wastewater is very key to the wide use of PNSB wastewater processing technology.

Rubrivivax gelatinosus belongs to the PNSB (Choorit et al. 2002; Zhi et al. 2020). Before the experiment in the medium (Hyun et al. 1989; Buranakarl et al. 1998; Tanskul et al. 2003; Oda et al. 2004), it was found that R. gelatinosus could secrete extracellular enzymes (protease and amylase). This suggested that R. gelatinosus could degrade macromolecular organics (protein and starch) and thus had the potential to directly treat wastewaters containing macromolecular organics.

However, in previous studies for R. gelatinosus, most efforts were committed to wastewater treatment after pretreatment (Choorit et al. 2002), the photosynthetic structure (Ranck et al. 2005), and hydrogen production (Maness & Weaver 2002). Little information is reported concerning the R. gelatinosus direct treatment of wastewaters containing macromolecular organic. So far, it is yet unknown whether R. gelatinosus can directly treat the macromolecules in organic wastewater. In addition, the light and oxygen conditions are the most important for R. gelatinosus. They influence the multiple metabolic activities of R. gelatinosus. Under certain light and oxygen conditions, R. gelatinosus could secrete protein-degrading enzymes (Pemberton et al. 1998; Bauer et al. 2003; Gomelsky et al. 2008) and then may impact R. gelatinosus direct treatment of the wastewaters containing macromolecules organic.

Therefore, the objectives of the current research are as follows: (i) to clarify whether R. gelatinosus can directly treat wastewaters containing macromolecules organic; (ii) to investigate the effect of light–oxygen conditions on R. gelatinosus direct treatment of wastewaters containing macromolecules organic and biomass, poly-β-hydroxybutyrate (PHB), 5-aminolevulinic acid (5-ALA), and pigment productions. The typical wastewaters containing macromolecules organic, namely, food wastewater (soybean protein and starch wastewaters), were chosen for this current study.

In the current study, R. gelatinosus was used. It was isolated from the fish pond (Xu et al. 2004). They grew to the logarithmic stage in the medium. After that, they were placed at 4 °C to spare.

The improved RCVBN medium contains the following elements: DL-malic acid (4.0 g/L), magnesium sulfate (0.12 g/L), ammonium sulfate (1 g/L), calcium chloride (0.075 g/L), potassium dihydrogen phosphate (0.5 g/L), dipotassium hydrogen phosphate (0.3 g/L), Na₂EDTA (0.020 g/L), yeast extract (100 mg/L); microelements: Fe³⁺ (0.0025 mol/L), Mn²⁺ (0.009 mol/L), Zn²⁺ (0.0033 mol/L), Co²⁺ (0.0024 mol/L), Cu²⁺ (0.0024 mol/L), and pH: 6.8.

Typically, polysaccharides and proteins were macromolecular substances (natural organic matter). In this work, the corresponding macromolecular organic wastewater was the starch wastewater and soybean protein wastewater, respectively. All two macromolecular organic wastewaters were high-consistency organic outlet water, including a great quantity of macromolecular organic contaminants. The abundant blow-down of three wastewaters would give rise to acute environmental contaminant questions. Besides, they were also classic nonhazardous and had a large content of growth media to R. gelatinosus. Thus, they were selected for this study.

The soybean protein wastewater and starch wastewater were obtained, respectively, from the Guangzhou Soybean Products Machining Factory and Guangzhou Starch Processing Plant. They were filtered first by 100 hole net. Afterward, the wastewaters were thinned by water. Ultimately, the features for soybean protein wastewater were as follows: chemical oxygen demand (COD) was 6,000 mg/L and the protein was 3,200 mg/L; the features for starch wastewater were as follows: COD was 6,700 mg/L and starch was 6,100 mg/L.

The photosynthetic reactor was presented in Figure S1. For batch culturing, 600 mL of two different wastewaters were added to the two different bioreactors. After bottling, two bioreactors containing different wastewaters were disinfected by sterilizing the pot. After sterilization, these bioreactors containing wastewater were placed in the sterilized ultra-clean cabinet until cooling. Afterward, R. gelatinosus (191.8 mg/L according to our screening experiment) was put in the wastewater in the sterilized ultra-clean cabinet. The initial pH value of each reactor was 7.0. The photosynthetic reactor with wastewater and R. gelatinosus was placed on a thermostat (25–29 °C). R. gelatinosus was grown in wastewater under sterile conditions. They were also provided with 120 rpm mixing revolutions by a shaker.

In the current work, illumination condition (3,000 lux) was achieved by regulating the spacing from 100 W bulbs to the reactor. The oxygen condition was achieved by injecting pure oxygen (98%). Dissolved oxygen (DO) content was kept using a flow valve. Micro-oxygen and aerobic conditions were maintained within 0.09 and around 2.0 mg/L DO, respectively. The black textile was covered on the reactor surface in order to achieve darkness. The anaerobic condition was achieved by injecting pure nitrogen (98%).

Wastewater samples (5 mL) were got from different bioreactors. They were separated by a high speed (9,000 rpm, 10 min). After the COD, protein, and starch contents, the activity of protease and amylase in the supernatant fluid was determined. The poly-β-hydroxybutyrate (PHB), 5-ALA, pigment, and biomass productions in collected R. gelatinosus were determined. American Public Health Association (APHA) methods were applied to determine biomass (dry cell weight) and COD. The COD was measured by the potassium dichromate method. Biomass was measured by the drying method at 100 °C.

The collected R. gelatinosus cell was broken by ultrasound at 40 kHz under 4 °C. The process lasted for 20 min each time and was carried out three times. After, PHB and 5-ALA productions were tested using a spectrophotometer (ThermoSpectronic, Rochester, NY, USA) at 235 and 480 nm, respectively. Pigment (chloromycetin and carotenoids) productions were tested using a spectrophotometer at 663, 646, and 470 nm. The detailed process was completed according to Wellburn (1994); Rahman et al. (2015); Dhaliwal & Chandra (2020); and Li et al. (2021).

Ultraviolet spectrophotometer (ThermoSpectronic, Rochester, NY, USA) was used to determine protease and amylase activities at 440 and 540 nm, respectively, based on Gessesse et al. (2003) and Zhang et al. (2007).

The protein and starch contents in two wastewaters were measured, respectively, through the Kjeldahl instrument and enzymatic hydrolysis (China's national food safety standards GB/T 5009.9-2008 (2008), GB 5009.5-2010 (2010)).

Three parallel groups were set in each experimental process. The data are expressed as mean ± error.

After 96 h of treatment, under light–anaerobic conditions, the COD removals for soybean protein wastewater and starch wastewater were around 80 ± 4% and 70 ± 3%, respectively (Figure 1). Under light–micro-oxygen conditions, the COD removals for the aforementioned two wastewaters reached around 70 ± 2.5 and 60 ± 3%, respectively. The COD removals of two wastewaters were higher under light–anaerobic conditions than light–micro-oxygen conditions.

Moreover, it is shown in Figure 1 that under dark–aerobic, dark–anaerobic, dark–micro-oxygen, and light–aerobic conditions, the COD removals for both two wastewaters reached around 23 ± 1.1 and 5 ± 0.2%, respectively, after 96 h. Moreover, their COD removals had almost not changed after 24 h. This was because the soybean protein wastewater and starch wastewater contained around 23 ± 1.1 and 5 ± 0.2% of small molecules, respectively. After 24 h, the small molecules were exhausted. Thus, COD removal almost did not change after 24 h. This proposed that R. gelatinosus could not immediately treat soybean protein wastewater and starch wastewater under dark–aerobic, dark–anaerobic, dark–micro-oxygen, and light–aerobic conditions.

Furthermore, as shown in Figure S2, the domesticated R. gelatinosus also had the same result as shown in Figure 1 for COD removals in two wastewaters.

Simultaneously, as shown in Table 1, after 96 h of treatment, R. gelatinosus biomass, poly-β-hydroxybutyrate (PHB), 5-ALA, and pigment productions in two wastewaters increased significantly under light–micro-oxygen and light–anaerobic conditions. The biomass, PHB, pigment, and 5-ALA contents reached at least 2000, 0.6, 198, and 0.085 mg/L in two wastewaters, respectively. Under light–anaerobic conditions, the biomass, PHB, pigment, and 5-ALA contents were at least 10 times the initial content in two wastewaters. Under the other four light and oxygen conditions, the biomass, PHB, 5-ALA, and pigment production increased slightly or decreased after 96 h, which was consistent with the result shown in Figure 1.

These results indicated that R. gelatinosus could directly treat the wastewaters containing macromolecules from organic and biomass resource recycling, which was different from the understanding of traditional theory. This was because the traditional theoretical perspective studied the ecological succession of mixed bacteria in natural organic wastewater. After ecological succession, R. gelatinosus only uses the small molecules in wastewater. In this study, the pure R. gelatinosus was used directly to treat the wastewaters containing macromolecules organic from the point of wastewater treatment. There was no microbial ecological succession in the wastewater in this work. Therefore, under appropriate light–oxygen conditions, R. gelatinosus demonstrates the characteristic of directly treating the wastewaters containing macromolecules organic. In short, the results of this article expanded the understanding of the traditional theory of PNSB (R. gelatinosus) directly purifying the macromolecules of organic wastewaters.

Generally, the macromolecules organic wastewaters contain abundant macromolecular organic substances. Starch and soybean protein wastewaters are constituted mainly by protein and starch, respectively, which are the main sources of COD in relevant wastewater. Figure 1 shows that R. gelatinosus could directly treat the two wastewaters and removed COD greatly under light–micro-oxygen and light–anaerobic conditions. These indicate that R. gelatinosus may remove the macromolecular organic substances (protein and starch) in soybean protein and starch wastewaters.

Figure 2 indicates that under light–micro-oxygen and light–anaerobic conditions, R. gelatinosus could remove the protein and starch in two wastewaters. This might be relevant to extracellular enzyme synthesis and secretion because both protein and starch removal requires the involvement of corresponding extracellular enzymes (protease and amylase).

Therefore, in order to clarify the effect of light–oxygen conditions on protease and amylase synthesis and secretion, the protease activity in soybean protein wastewater and amylase activity in starch wastewater were measured.

Figures 1–3 show R. gelatinosus could directly treat the soybean protein and starch wastewaters under light–micro-oxygen and light–anaerobic conditions, but could not under other light–oxygen conditions. According to Yang et al. (2018), light and oxygen were the most important factors for R. gelatinosus. For light, it was necessary to condition photosynthesis (PS) and determined the PS and expressions of PS genes in R. gelatinosus. As an external stimulus, light also regulated a variety of metabolic activities and signal transduction pathways for R. gelatinosus. According to Ranchou-Peyruse et al. (2006), a two-component signal transduction system (TCSTS) was a kind of signal transduction system existing in bacteria. TCSTS participated in a variety of physiological and biochemical processes and was the regulatory mechanism of its metabolic activities (the synthesis of protease and amylase) (Ranchou-Peyruse et al. 2006). As shown in Figures 1 and 2, COD, protein, and starch were degraded and removed.

However, two enzymes were still detected under light aerobic conditions (Figure 3). The main reason was the oxygen. For oxygen, it had an inhibitory effect on the expression of related genes on two enzymes (Pemberton et al. 1998; Bauer et al. 2003; Gomelsky et al. 2008). Moreover, oxygen concentration decided the level of gene expression. Then the synthesis and activity of the enzyme were also affected. Based on the previous literature (Pemberton et al. 1998; Bauer et al. 2003; Gomelsky et al. 2003), oxygen tension in the environment determined the transcription of oxygen-dependent PS genes. It was concluded that for R. gelatinosus, light stimulated enzyme synthesis. However, oxygen concentration decided the amount of enzymes.

The natural water body environment belonged to light aerobic or dark anaerobic conditions. Under two conditions, two enzymes were not synthesized. Sometimes, the natural water body also might be low light–micro-oxygen. Even if the enzyme was synthesized under this light-oxygen, the enzyme activity was very low during 24 h, as shown in Figure 3. At this time, the amount of organic matter absorbed by R. gelatinosus was very small. So, they could not grow and reproduce rapidly. Meanwhile, heterotrophic microorganisms secreted many degrading enzymes. Therefore, they occupied the majority of organic matter and reproduced in large quantities. Therefore, the traditional theory thought they could not degrade macromolecular organic matter. However, R. gelatinosus wastewater treatment technology had advantages that could not be ignored, namely, no secondary pollution to the environment because of biomass recycling.

As shown in Table 1, the growth and the content of PHB, 5-ALA, and pigment were significantly increased with the removal of organic compounds in the two wastewaters by R. gelatinosus. This was due to the light and oxygen conditions controlling the synthesis of the degrading enzyme of R. gelatinosus. These enabled R. gelatinosus to utilize the organics in wastewater to produce cell, PHB, pigment, and 5-ALA contents. Previous studies had used wastewater as a medium to produce PHB and pigments for other microorganisms. Rahman et al. (2015) studied that microalgae produced PHB using wastewater as media. Hashemi et al. (2021) used yeast wastewater to produce pigments by filamentous fungi. In this work, it was found that R. gelatinosus directly used food processing wastewater to produce the PHB, 5-ALA, and pigment, which reduced the secondary pollution of excess sludge to the environment.

This study expanded the viewpoint of traditional theory (R. gelatinosus untreated directly wastewater). Under light–anaerobic and light–micro-oxygen, R. gelatinosus directly treated the macromolecules’ organic wastewaters and biomass, PHB, 5-ALA, and pigment productions were enhanced. Light activated the synthesis of protease and amylase. However, oxygen has inhibited the synthesis of enzymes. Only the amount of oxygen is at micro-oxygen or anaerobic, and the above synthesis is conducted. It was realized that R. gelatinosus directly treated wastewater and accumulated nutrients for recycling, which reduced the secondary pollution of excess sludge into the environment.

This work was supported by the startup research fund.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.