Large-scale non-destructive plastic disposal is a major global issue not only for the environment and ecosystem, but also for human health 1. One way to overcome these issues, is to generate plastics from biological resources with biodegradable properties 2. Microbial biopolymers are found in nature as intracellular compounds, and often produced when bacteria are placed in adverse environmental conditions. These material act as convenient energy sources in their natural state 3. Ploy-hydroxy-alkanoates (PHAs) are classified as a group of biodegradable and biocompatible polymers produced by microorganisms and can be a potential replacement for synthetic polymers such as polypropylene 4–8.
Poly-hydroxy-butyrate (PHB) is a type of PHAs, with the property of biodegradability 7–11. It is stored within the cytoplasm of a bacteria and can be extracted, and purified by centrifugation and multi-stage washing operations. It is used as an effective thermoplastic polymer and has many properties similar to standard commercial polymers like polypropylene 3. It can be completely degraded by microorganisms, converted to water and carbon dioxide, and returned to nature over a year 12. Morris Lemoigne, a French microbiologist, was the first who obtained PHB in 1925, and its general formula was described as (C4H6O2)n13.
The use of PHB has increased over past years. One of its simplest but most important application, is for packaging of food containers, beverage bottles, plastic films and bags, cosmetics packaging, shampoo bottles, cardboard and paper covers, sanitary towels, etc. Utilization of these polymers has reduced garbage accumulation in the environment 14. Recently, medical applications of PHB such as in cardiovascular products, nerve tissue repair, nutrition of humans and animals, orthopedic applications, urology, and wound healing have widely been considered 15–17 18,19.
Isolated microorganisms from poor natural environments such as lake water and soil have the capability to survive more than those living in more advanced diets 20,21. Streptomyces species are good examples of such microorganisms. About 10000 metabolites are produced by Streptomyces species which are classified as Actinomycetes. They are recognizable in culture media with high pigmentation and diversity of colony 10. Streptomyces species grow in pH range of 6.5-8.0 22, most types are mesophilic and grow in temperature range of 10–37°C 23. There are more than 500 Streptomyces species and the appearance of colony, natural compression, and color on agar medium, makes it easier to detect their colonies 11.
Many researchers have reported production of PHB from different bacterial species such as Bacillus sp., Pseudomonas sp., Methylobacterium sp., Ralstonia sp., and Alcaligenes sp., via submerged fermentation 24–27. Chandani et al. 28 estimated PHB content in Acinetobacter sp. K3 isolate to be 79.4% w/w, which was obtained at pH of 8, and temperature of 40°C, while mannitol and urea were used as carbon and nitrogen sources, respectively. Khanafari et al. 29, used a culture of Azetobacter bacteria in a whey medium, temperature of 35°C and pH of 7 for production of PHB. Adding different nitrogen sources except ammonium nitrate had positive effect on PHB production and its maximum value was achieved in presence of Meat extract where 4.43 g/L PHB was produced, while increasing O2 content by shaking at 122 rpm decreased PHB production from 4.43 to 0.04 g/L.
Jeyasree et al. 30 incubated Bacillus Subtilis MTCC1790 within nutrient broth culture medium with 2%(v/v) and at different temperatures of 37, 47, 57°C with shaking speed of 225–250 rpm. PHB accumulation within the strain was reached its maximum at 24 h while maximum growth rate was obtained after 48 h.
Hong et al. 31 incubated Vibrio Proteolyticus bacteria in M9 medium including 1% (w/v) glucose, 0.1% (w/v) yeast extract, and 5% sodium chloride and tested various carbon and nitrogen sources. The produced PHB was analyzed by Gas Chromatography (GC). Results showed that the highest amount of biomass and PHB were obtained after 48 h, while they decreased after 72 h which was attributed to the decomposition of PHB after running out of fructose.
Mostafa et al. 32 optimized production of PHB using Erythrobacter aquimaris bacteria isolated from marine environment. Maximum PHB accumulation within bacteria was achieved after 5 days of incubation at pH of 8.0, temperature of 35°C, where peptone and glucose were used as nitrogen and carbon sources, respectively. In another work, Mostafa et al. 33 isolated marine bacterium Pseudodonghicola xiamenensis and used it for production of PHB in date syrup culture medium. They reported the highest production rate of PHB after 96 h of incubation at pH of 7.5 and temperature of 35°C in the presence of 4% NaCl and peptone as nitrogen source, where using date syrup resulted in PHB concentration of 15.54g/L and a PHB yield of 38.85%.
Sanhueza et al.34 investigated influence of carbon source on properties of PHB produced by Paraburkholderia xenovorans LB400. They used glucose, mannitol, and xylose as carbon sources and found that PHB produced by this strain was less crystalline that commercial one. Produced PHB using xylose as carbon source showed characteristics like human skin, while PHB produced using mannitol had larger molecular weight than others.
Amadu 35 reviewed synthesis of PHB in microbes cultivated in wastewaters. TO our knowledge no relevant work found that used Streptomyces species isolated from soil for production of PHB.
In this work, firstly different isolates of Streptomyces coded as G2, 6, G8, E17, and N5 for investigation of their performance in production of PHB. Then, values of effective parameters including carbon and nitrogen sources, pH, temperature, incubation time, and shaking speed were obtained from the literature and used as base case in first phase of PHB production, called extraction phase. After incubation, samples were taken from culture mediums containing different species and presence of PHB were examined by Sudan Black staining method. Also, different analysis methods including, Gas Chromatography-Mass Spectrometry (GC-MS), spectrophotometry, and weighing methods. Finally, optimization of effective parameters was examined with the method of one-factor-at-a-time 36 and their optimum values were found.