Valorization of cashew industry wastewater as a carbon and nutrient source for the microbial growth and production of the polyhydroxyalkanoates: A potential biopolymer by Bacillus species

Abstract Wide applications of conventional plastics across the world have made its production inevitable. To avoid the ecocide occurring due to conventional plastics, studies on the production and extraction of renowned biopolymers like polyhydroxyalkanoates (PHAs) are explored. PHA is a family of polyesters naturally produced by bacterial fermentation with the potential to replace conventional hydrocarbon-based polymers. The efficient utilization of industrial discard as renewable feedstocks in the production of eco-friendly products such as bioplastics has been studied. This study focuses on the production of PHA using cashew industry wastewater (CIW). Since cashew industry wastewater (CIW) is rich in phenolic compounds and other sources, this study explores the possibility of eliminating the toxic phenol compounds from the waste by the production of PHA using cashew industry wastewater as a secondary source of carbon by paving the way to eco-friendly process. In this context, PHA-producing bacterium was isolated from wastewater samples collected from cashew industries. The morphological, microscopic, and biochemical characterization revealed that the isolated strain CFM1 is Bacillus sp. The strain CFM1 that gave maximum (30%) PHA in control medium was taken up for further studies with wastewater. The concentration of sucrose in the control production media was replaced with cashew industry wastewater. Wastewater was introduced to the medium at different concentrations (10–100%). As CIW concentration was increased, the sucrose concentration was decreased in the medium accordingly. CFM1 isolates produced 34% PHA with 20% wastewater. The biopolymer produced was characterized by FTIR and NMR.


Introduction
Polyhydroxyalkanoates are biopolymers produced by various types of bacteria and fungi.These biopolymers are biodegradable and possess properties similar to petroleum-derived polymers (Koller et al., 2005).Petroleum-based polymers are non-biodegradable and have become a major environmental concern.PHAs are valuable alternative to these plastics.
PHAs are energy-rich compounds which are stored by bacteria inside their cells under a nutrientlimiting environment.Poly(3-hydroxybutyrate) and poly(4-hydroxybutyrate) are the most commonly used polymers.Polyhydroxy-valerate, polyhydroxy-hexanoate, and polyhydroxyoctanoate are copolymers and belong to the types of medium chain length PHA (mcl PHA).PHA displays superficial physicochemical properties such as serving as a better oxygen barrier, water vapor barrier, and better odour barrier compared to polypropylene and polyethylene terephthalate (Tsang et al., 2019).
These unique properties have promoted PHA usage in a number of fields such as packaging and medical industries.The physicochemical properties of PHA are known to be affected by the species of the microorganism, carbon source provided, and the operating parameters of the reactor (Weber et al., 2002).PHA has been commercially produced by many manufacturers such as Tianjin Green Biosciences Co. Ltd in China, Metabolix in the USA, and Biocycle PHB industrial in Brazil (Tsang et al., 2019).
The main problem related to the production of PHA is the high cost of production.The high cost arises due to expensive carbon sources such as refined sucrose or glucose, fermentation conditions, and effective downstream process (Koller et al., 2017).To tackle the carbon cost many other alternative waste products from agricultural, industrial sector waste streams are identified and tested for microbial polyesters production.Several waste products such as hydrolyzed rice and wheat straw (Ahn et al., 2016;Cesário et al., 2014), pea-shell slurry (Patel et al., 2012), fruit pomaces of cherries, grapes & apricots (Follonier et al., 2014), residual glycerol from biodiesel production, castor oil, palm oil, waste frying oil, jatropa oil and whey (Gomez Cardozo et. al, 2016), spent coffee bean oil (Cruz et al., 2014), refined lactose and whey (Berwig et al., 2016), refined sucrose and unrefined sugarcane molasses (Geethu et al., 2019), wastewater from various sources such as Mars chocolate factory (Tamis et al., 2014), municipality, starch and cheese industry, paper and pulp industry (Yan et al., 2006), brewery (Ben et al., 2016), cardboard industry (Bhuwal et al., 2013), and oil mill (Dionisi et al., 2005) have been tested as a carbon source for PHA production.
The present work was studied with the objective of understanding whether cashew nut wastewater is a suitable substrate for polymer production.Additionally, as the cashew wastewater contains phenol, an environmental pollutant, it is harmful to release into the environment.Cashew tree (Anacardium Occidentale L.) is a well-known plant from Anacardiaceae family and grown in different parts of the globe specially in Brazil, India, and Nigeria.The cashew nut processing industries are one of the major sources of income in the coastal states of India like Karnataka, Kerala, Goa, Tamilnadu, and Maharashtra.India is one of the largest exporters of cashew nuts in the world, and the Kollam district in Kerala is known as the cashew capital of India.Earnings from cashew nut export for FY20 were US$566.67 million, and India accounts for nearly 65% of global cashew kernel export.Raw cashew nuts need to be processed before delivery to consumers.After cleaning and drying, two different types of methodologies are used for processing of raw cashews, i.e, roasting in drums and cooking in steam boilers.
A mixture of phenolic compounds such as 70% anacardic acid, 18% cardol, and 5% cardanol makes up the natural cashew nut shell liquid (Mgaya et al., 2019).During processing, because of high temperature, anacardic acid undergoes decarboxylation to produce cardanol; these components leach out of the shell and contribute to the toxicity of the wastewater (Rwahwire et al., 2019).Since wastewater contains harmful phenolic compounds, it is dangerous to the environment if discharged without efficient treatment (Leite et al., 2015;Martins et al., 2009).However, industries consider wastewater treatment as an economic burden owing to the heavy investments with only long-term returns (Raji & Packialakshmi, 2022).
Conversion of this waste into valuable products before planning treatment is hence a durable option.Zhang et al. (2018) demonstrated the use of the toxic phenolic compound as a carbon source for the production of polyhydroxyalkanoates (PHA).Since cashew industry wastewater (CIW) is rich in phenolic compounds and other sources, this study explores the possibility of the production of PHA by using cashew industry wastewater as a secondary source of carbon.
This research contributes to the development of an eco-friendly process by utilizing CIW as a valuable resource for the production of biopolymers, which would otherwise be considered a waste, By exploring the potential of PHAs in this context, the study provides insights into sustainable waste management practices and the creation of environmentally friendly materials.

Sample collection
Samples were collected from a Cashew nut processing factory located in Brahmavar taluk, Udupi District, Karnataka, India.The sample was collected from the steam boiler used for the processing of cashew nuts, cooled, and stored in a 5-liter container.The container was sealed tightly, and the samples were taken to the lab and stored at 4°C for further use.

Wastewater characterization
Wastewater analysis is an important step because it helps to determine whether it is suitable to be used as a medium for PHA production.Wastewater was analyzed according to American Public Health Association (APHA) standards to determine the amount of organic matter-biological oxygen demand (BOD), chemical oxygen demand (COD), oil and grease, total suspended solids, nutrients like phosphates, nitrates, and sulphates, pH, microbial load (CFU/ml), and total phenolic content.

Isolation and screening of PHA-producing bacteria
Bacterial isolation was carried out using the serial dilution technique, where wastewater sample is sequentially diluted and from that diluted sample 100ul was put on the Nutrient Agar Petri plate and incubated for 24 h at 30°C.After the incubation, the bacterial colonies were taken and transferred to 5 ml Nutrient broth and incubated for 16-18 h at 30°C; then the 5 ml broth culture broth was transferred to standard PHA production medium and on incubated on a shaker 170 rpm for 72 h at 30°C.After incubation cells were harvested by centrifugation and intracellular PHA was extracted using hypochlorite solution.The bacterial colony that gave maximum PHA yield was taken for further studies.The pure culture of CFM1 on nutrient agar was preserved at 4ºC and subcultured regularly once in 3-4 weeks.

Production of PHA using wastewater
The isolate which showed maximum production is labelled as CFM1.Morphological tests such as gram staining, endospore staining, and other biochemical tests were carried out to identify the isolated bacteria (Talaiekhozani, 2013).Control experiment of PHA production was performed with the shake flask method using standard e production media containing (g/L): Na 2 HPO 4 .2H 2 O, 2.2; (NH 4 ) 2 SO 4 , 1.5; KH 2 PO 4 , 1.5; MgSO 4 , 0.2 and 2 % sucrose as a carbon source, pH 7 and incubated at 32°C, 170 rpm for a duration of 72 h.
For wastewater experiment, the concentration of sucrose in the standard PHA production media sucrose was gradually replaced from 2% to 0% with the increase in cashew wastewater concentration from 10% to 100%.The production was carried out in a 250-ml Erlenmeyer conical flask with 50 ml production media at 32°C, 170 rpm for a duration of 72 h.All the experiments were done in duplicate.

Estimation of biomass
The cells were collected by centrifugation (Plastocraft SSR-V/FM) at 7000 rpm for 10 min and washed with distilled water and dried at 80°C in airflow drier to a constant weight.Biomass was measured by gravimetric method.

Extraction of PHA
The dried biomass was suspended in 4 % sodium hypochlorite solution (1 ml for 20 mg of biomass) and hydrolyzed at 37°C for 1 h.The hydrolysate was centrifuged, and the sediments were washed with distilled water and acetone.The PHA obtained was then dissolved using chloroform and was air-dried in a petri plate of known weight.Extracted PHA was obtained as a thin film which was further analysed (Geethu et al., 2019).

FTIR
FTIR is typically used as a tool to analyze the structural information of polymers.The PHA film obtained after each experimental trial was dissolved in chloroform and thoroughly mixed with potassium bromide (KBr) and pelletized.This pellet was placed in the sample chamber in FTIR spectrophotometer and exposed to infrared radiation to obtain a spectrum using Shimadzu 8400 S with spectral range of 4000-400 cm −1 .

1 H NMR
NMR is a very powerful technique in PHA characterization that accommodates double bonds.The two-dimensional nuclear technique helps in the analysis of the specific double bond location in the monomer. 1 H NMR spectrum was obtained by dissolving 5 mg of PHA film in deuterated chloroform and analyzed in Bruker Ascend 400 NMR spectrometer at 22°C.

Results and discussion
Characterization of wastewater is an important step in both wastewater treatment and resource recovery as it determines the fate of wastewater.It also helps us in determining the type of treatment to be employed and whether the wastewater can be used for resource recovery or energy generation.Various parameters for wastewater characterization are listed in Table 1.
Wastewater analysis indicated the presence of high amount of organic matter (BOD) and phenolic compounds (294 mg/L).The microbial load value indicates that, even though a high number of phenolic compounds are present in the wastewater, microbes are still able to survive in it.Phosphate and nitrates were present in minute quantities (mg/L), and sulphate concentration was zero.Nutrients, such as sulphate, nitrate, phosphate, and magnesium, are essential that are required for the growth of bacteria.PHA production occurs under nutrient-limiting conditions and excess carbon.But the bioavailability of these nutrients in the wastewater was insufficient for the growth, so these extra nutrients were externally added, as given in methodology (production medium).

Isolation and screening of PHA-producing bacteria
Bacterial isolates were screened and the strain that accumulated PHA with more than 20% of cell biomass (12 isolates) was selected for further PHA production.Amongst 12 isolates, the CFM1 strain gave the maximum yield with 2 g/L of biomass and PHA 0.61 g/l with 30% product production, in control medium containing 2% sucrose as carbon source.The isolated strain is Gram Positive bacteria and has the ability to form endospore.The bacterium was identified as Bacillus sp.Based on microscopic, colony morphology and biochemical analysis.The Bacillus sp is a aerobic soil bacteria and a well-known PHA producer.The PHA granule was first identified and isolated from Bacillus megaterium species (Geethu et al., 2019).

PHA production using wastewater
The PHA production from Bacillus sp (CFM1) was done further using cashew industry wastewater.The concentration of sucrose in the standard medium was gradually replaced with an increasing concentration of wastewater in the range of 10% to 100%.PHA yield is calculated as % wt of PHA (g/L) in the dry cell weight (g/L); CFM1 isolate was able to accumulate a maximum of 34.04% PHA at a concentration of 20% wastewater in the media as shown in Figure 1.As the concentration of wastewater was gradually increased, the concentration of sucrose in the media decreased gradually.For every 10% increase in wastewater concentration, sucrose concentration decreased by 0.2%.From Figure 1, it is evident that CFM1 at 20% waste water concentration was able to yield the same amount of PHA as control samples.
From Figure 1, it is clear that after 20% of wastewater in the media, both biomass and PHA yield was maximum but it is gradually decreased as the concentration of CIW increased and CFM1 isolate did not yield an appreciable amount of biomass and PHA when only wastewater was used as a carbon source.Hence, a reduction in bacterial cell mass with an increase in wastewater concentration could be attributed to the increased toxicity of phenolic compounds.
Cashew wastewater contains a high amount of suspended solids and phenolic compounds (Table 1), which seem to support bacterial growth by providing more surface area for growth and shielding them against shear force created by constant agitation (Obruca et al., 2018).The presence of phenolic compounds might be acting as a stress-inducing factor leading to the increased accumulation of PHA (Chahal et al., 2016).Cashew wastewater containing phenols acts as a carbon source till a permissible limit; later, it inhibits the growth of the organism.Phenol disturbs the function of the cell membrane at a higher level, and it can stop cellular function leading to cell death.The trend in decrease in the biomass and PHA accumulation can be observed from 30% wastewater.Biomass drastically decreased from 0.82 g/L to 0.34 g/L and PHA from 0.22 g/L to 0.04 g/L.This signifies that there is an optimal level of phenol which acts as a carbon source, and the biomass can also utilize the phenol for growth.

FTIR analysis
Fourier transform infrared spectroscopic analysis is one of the powerful tools to obtain data on the polymer structure as every chemical entity in the sample has its own transmittance values.
The functional groups of PHA were confirmed by the FTIR study.Figures 2 and Figure 3 show the FTIR spectra of PHA with control and 20% wastewater, respectively.A sharp peak near wavelength 1720 cm −1 corresponds to the C=O stretching vibration of the carbonyl ester group (RC=O) of PHA (Vishnuvardhan Reddy et al., 2009).Spectral range from 1050 cm −1 to 1300 cm −1 represents the valence vibration of the carbonyl group (Biradar et al., 2015;Shamala et al., 2009).Absorption at 1378 cm −1 and 1455 cm −1 corresponds to CH3 and -CH2 groups, respectively (Vishnuvardhan Reddy et al., 2009).Peaks around 2930 cm −1 and 2975 cm −1 belong to the stretching vibration of methylene and methyl groups.By comparing obtained IR spectra result with previous studies (Biradar et al., 2015;Shamala et al., 2009), we can conclude that in the present work, irrespective of the carbon source, FTIR spectra obtained for all samples are similar.A little variation in the peak values could be attributed to the degree of crystallinity of the PHA (Xiao & Jiao, 2011).

1 H NMR analysis
According to Figures 4 and 5, peaks from 0.86 ppm-0.95ppm and 1.26-1.28ppm represent the -CH 3 group of hydroxyvalerate and hydroxybutyrate respectively.A peak at 1.586 ppm represents the -CH 2 side of hydroxyvalerate.Peaks from 2.430 ppm to 2.645 ppm depict -the CH 2 group of hydroxybutyrate and hydroxyvalerate bulk structure.Peaks from 5.22 ppm to 5.28 ppm represent -the CH group of HB & HV bulk structure.From Figures 4a,b and 5, it is clear that NMR peaks observed in all the samples indicate the presence of hydroxybutyrate and hydroxyhexanoate (PHHx) when compared with the standard NMR spectra.Similar peaks have been reported and identified by Bhattacharyya et al. (2012).So, the obtained PHA is a co-polymer of PHB and PHHx.However, study of 13 C NMR and other characterizations is essential for complete explanation and confirmation of the copolymer structure.
Phenol is a six carbon aromatic compound, catalyzed to catechol by the membrane-associated enzyme phenol hydroxylase.It is further postulated catechol is oxidized by catechol 1, 2-dioxygenase or catechol 2, 3 -dioxygenase resulting in the final product pyruvate or adipic acid metabolized for PHA biosynthesis resulting in the copolymer of PHB and PHHx.Catechol can be cleaved, and its products can inhibit PHA synthesis or hinder other metabolic activities of the cell due to the higher concentration of phenol or its products (Reddy et al., 2015).This study provides evidence for the production of PHA from industrial wastewater that contains toxic phenolic substances like anacardic acid, cardol, and cardanol.When sucrose concentration in the production media was gradually decreased with the addition of wastewater, the percentage yield of the PHA also increased till a certain concentration of the wastewater with CFM1 isolates.When compared with the PHA production using standard media, CFM1 isolates were able to give a higher yield with 20% wastewater in the media.This result indicates that the CFM1 isolates can utilize the wastewater as a source of carbon and the presence of other nutrients such as phosphate and nitrate boosts their growth.A high amount of suspended solid supports the growth of the bacteria by providing more surface area for multiplication and by protecting them against the shear force created by continuous agitation.The PHA accumulation inside the bacterial cell increases when they are subjected to nutrient stress or stress created by some external factors such as growth inhibitors, dissolved oxygen level, and agitation rate.

Conclusions
• The cashew industry wastewater contains a high number of phenolic compounds and these compounds seem to induce stress on bacterial growth, which results in increased accumulation of PHA inside the bacterial cell.
• Partial replacement of sucrose with wastewater in the media results in decreased cost of production.
• It provides an opportunity to investigate into the biochemical mechanisms for PHA biosynthesis by using carbon sources especially phenolic contents in the cashew industry wastewater.
• The renewable carbon source holds a potential for production of PHA by employing statistical modelling to optimize and enhance the yield which also can be taken to large-scale studies.

Figure
Figure 1.PHA and biomass quantity (g/l) produced by CFM1 strain.