Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive
Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Utilization of Handmade Paper Industries Effluent Fortified with Growth-promoting Microbes for Enhancing Vigna radiata Growth

S
Sunita Chauhan1,*
P
Poonam C. Singh2
R
Rahul Mishra1,3
1Kumarappa National Handmade Paper Institute, Sanganer, Jaipur-302 029, Rajasthan, India.
2CSIR-National Botanical Research Institute, Lucknow-226 001, Uttar Pradesh, India.
3Khadi and Village Industries Commission, Jaipur-302 004, Rajasthan, India.

ABSTRACT

Background: Biofertilizers play an essential role in bringing sustainability to agriculture. They are being focused all over the world to develop and improve technologies for biofertilizers to make them cost-effective and enhance productivity. Handmade papermaking from cow dung results in the generation of effluents, raw liquor (RL) and alkaline black liquor (BL), which are rich in organic carbon and nutrients. The present study was carried out to study the compatibility of RL and BL in agriculture along with microbial biofertilizers.

Methods: One of the waste effluents (RL) was recently reported to have plant growth-promoting characteristics with the promising results of seed germination and pot studies using green gram seeds at KNHPI. In the present study, the RL and BL efficacy were tested with microbial cultures procured from different sources and isolated at KNHPI for enhancing their biofertilizer potential. The cultures, namely mung Rhizobium (MR), Phosphate Solubilizing Bacteria (PSB-NB), Enterobacter kobei (ECM), Bacillus species isolated from soil B.St (S), Bacillus species isolated from textile industry waste B.St (W) were selected for detailed study in different combinations.

Result: The combination of RL and BL with different PGPR strains of a compatible nature was observed to show enhanced seed germination and plant growth. The present study provides an insight into the utilization of waste by-products of the handmade paper industry that will have socio-economic implications. This may contribute to bring sustainability to the agriculture sector and help provide employment, income generation and reduce environmental pollution.

INTRODUCTION

The pulp and paper sector is recognized as the sixth largest contributor to industrial pollution, following the oil, cement, leather, textile and steel industries, by releasing diverse contaminants into the environment (Savant et al., 2006; Mehmood et al., 2019). Papermaking is highly energy-intensive and water-consuming in terms of freshwater utilization (Pokhrel and Viraraghavan, 2004). Water consumption varies with production methods and may reach up to 60 m³ per ton of paper, generating ~50 m3 of wastewater (Thompson et al., 2001; Ashrafi et al., 2015; Molina-Sanchez et al., 2018). Freshwater is used at every stage, from raw material cultivation to pulping, bleaching and sheet formation, resulting in substantial wastewater generation (Chauhan and Meena, 2021).
       
Major pollutants of pulp and paper effluents include lignin, fatty acids, dark colour compounds, biochemical oxygen demand (BOD), chemical oxygen demand (COD), adsorbable organic halides (AOX) and volatile organic compounds (VOCs) (Sharma and Singh, 2021). According to the Ministry of Environment and Forest (MOEF), the pulp and paper industry is categorized under the red category due to its high pollution potential (Patel et al., 2021). Wastewater generation ranges from 220-380 m3 per ton of paper produced and is often highly coloured and toxic (Badar and Farooqi, 2012). In India, total water consumption may reach 250-300 m3 per ton of paper (Chaudhary and Paliwal, 2018; Patel et al., 2021). Various treatment approaches, including aerobic, anaerobic, electrochemical, photocatalytic, ozonation, coagulation-flocculation and adsorption techniques have been investigated (Singh and Tripathi, 2020; Touhari et al., 2023). Biological treatment methods rely on microbes to convert organic pollutants into biomass (Sharma and Singh, 2021). However, efficient treatment depends on wastewater characteristics, quantity and reuse requirements, emphasizing the need for well-defined management strategies (Singh and Tripathi, 2020).
       
Handmade paper refers to sheets produced manually through traditional sheet-forming processes. The Khadi and Village Industries Commission (KVIC) includes paper made using Cylinder Mould Machine/Vat (CMM or CMV) with a deckle width of 102 cm in handmade paper category. Handmade paper is considered socially responsible and sustainable as it is tree-free, eco-friendly and resource-efficient (Chauhan et al., 2022).
       
It is justified as a cost-effective and energy-saving alternative with strong economic and environmental benefits (Reddy, 2015). Although handmade and mill-made paper share similar principle of production, differences exist in processing, energy use and environmental impact (Chauhan and Hussain, 2009; Chauhan et al., 2022). Both sectors generate coloured effluents that possess environmental and aesthetic concerns, necessitating proper treatment and disposal.
       
Kulshreshtha et al., (2011) have evaluated the health perspectives of the effluents of the handmade paper industry of the Sanganer region and reported them to be mutagenic with either one strain of Salmonella typhimurium or with both. The presence of dyes and chemicals in the effluents of handmade paper and pulp industries is revealed by its colour, low pH and high COD (Kulshreshtha et al., 2010a). The handmade paper and cardboard industries also produce sludge, at the rate of approximately 10 kgd-1 per industry, in the form of shortened pulp residues which cannot be recycled again. These residues are accumulated in the drain or street and produce enormous odour. Kulshreshtha et al. (2013) have reported the cultivation of Pleurotus citrinopileatus on the sludge of handmade paper and cardboard industrial waste. Agro-industrial and industrial wastes have been exploited for centuries in Asia for the production of oyster mushrooms (Zervakis et al., 1995; Sivrikaya et al., 2002; Kuforiji and Fasidi, 2009; Kulshreshtha et al., 2010b).
       
Recycled water is defined as wastewater that is treated and reused to supplement water supply (US EPA, 1992). The beneficial utilization of treated wastewater for agriculture is the major water reuse application worldwide (US EPA, 2004). Research shows that wastewater irrigation can result in significant changes to soil’s physical, chemical and biological properties. Kunhikrishnan et al. (2012) have reported the influence of wastewater irrigation on the transformation and bioavailability of heavy metal (loid)s in soil. González et al. (1992) have explored the possibility of utilizing kraft black liquors generated in a paper mill, using Eucalyptus globulus as raw material, for the production of a Nitro-Humic soil conditioner. Liu et al. (2021) have reported a pulping black liquor-based polymer hydrogel as a water retention material and slow-release fertilizer. Similarly, Haile et al., (2021) have reviewed the prospects of making different high-value-added biomaterials from pulp and paper mill wastes.
       
Therefore, in view of all the above background, the present study was taken up to explore the possibility of improving the use of effluents in the handmade paper industry using cow dung as raw materials. The effluents were augmented with different PGPR strains and evaluated in green gram (Vigna radiata i.e. mung). While the utilization of industrial effluents (Baskar et al., 2026) and PGPRs (Kajić et al., 2025) (in agriculture has been explored independently, this study contributes by systematic evaluation of their combined application, particularly using cow dung-based handmade paper effluents, for improving mung bean growth. Thus the study offers a novel integration of handmade paper industry effluents with plant growth-promoting microbial consortia, providing new insights into their combined potential as eco-friendly biofertilizer formulations for enhancing Vigna radiata growth. 

MATERIALS AND METHODS

The present study was carried out in the Biotechnology laboratory of Kumarappa National Handmade Paper Institute (KNHPI), Jaipur, Rajasthan during the Financial year’ 2021-22.
 
Procurement of standard microbial strains of PGPR
 
Pseudomonas sps (PSB-NB) and the bacterial strains of rhizobia, namely mung Rhizobium (MR), chickpea Rhizobium (CPR) and pea Rhizobium (PR), were procured from the National Botanical Research Institute (NBRI), Lucknow. Azotobacter sp. (Azo-R) was kindly received from Krishi Vigyan Kendra, Rajsamand. Bacillus megaterium (BM), Bacillus safensis (B.sf) and Enterobacter kobei (ECM) were procured from National Agriculturally Important Microbial Culture Collection (NAIMCC), ICAR-National Bureau of Agriculturally Important Microorganisms (ICAR-NBAIM), Mau. Two Bacillus species were isolated from garden soil (Bst.S) and textile industry effluents (Bst.W) at KNHPI, Jaipur, Rajasthan.
 
Collection of waste effluents of handmade papermaking
 
Two types of waste streams/effluents of cow dung based handmade paper industry were collected. Raw liquor (RL) obtained before pulping of the cow dung squeezed in the dewatering machine and after pulping by NaOH (BL) were used in the study (Narayana et al., 2024).
 
Examining the capability of microbial cultures to utilize RL/BL as the sole source of nutrient
 
RL and BL plates were prepared separately by adding agar powder (Bacteriological grade, HiMedia, GRM026) at the rate of 3% of the effluents used (RL and BL). The cultures were streaked on RL and BL agar plates. The inoculated plates were incubated at 28°C for 3 to 5 days and the growth of bacterial culture indicated the utilization of RL and BL by the microbes used. Sterilized water agar media supplemented with un-autoclaved RL and BL separately were also included in the study. These plates were inoculated with the available PGPR strains. The plates were then incubated at 28°C and observed for growth, if any.

Biocompatibility test between microbial strains
 
Nutrient agar medium was used for checking the compatibility of Rhizobium with different combinations of the available microbial strains. Three standard methods of checking biocompatibility viz. Well Diffusion method, Cross Streaking, Disc and Swab method were used.
 
Preparation of modified RL and BL solutions
 
The microbial cultures were grown in their specific media/broth. Rhizobium was grown in Yeast Mannitol Broth (YEMA, Hi Media, M721), AzoR in Jensen’s Broth (Hi Media, GM973) and ECM in King’s B broth (glycerol-10 gpl, K2HPO4-1.5 gpl, MgSO4-1.5 gpl, Peptone-20 gpl). Nutrient broth (HiMedia) was used for growing Bst. (S), Bst.(W), PSB-NB and B.sf All the inoculated plates were incubated for 48 H at 28°C. The 48 H growth cultures were used as primary inoculum (1% of broth) to inoculate the autoclaved and un-autoclaved broth of RL and BL. The biocompatible cultures were inoculated at the rate of 1% each. The inoculated RL and BL solutions were then incubated at 28°C for a period of 48 H. Another set was maintained without incubation- “instant sets”. The treatments of different PGPR strains and RL or BL used are given in Table 1 and 2.

Table 1: Un-autoclaved and autoclaved set of RL used for in vitro seed germination assay of mung bean.



Table 2: Un-autoclaved and autoclaved sets of RL and BL with the given consortiums of PGPR strains used.


 
Surface sterilization of mung seeds       
                
Seeds were surface sterilized using 0.01% mercuric chloride solution. For this, Mung seeds were dipped in mercuric chloride solution for 3 minutes and then washed 10 times with sterilized distilled water. The seeds were then allowed to dry inside the Laminar Air Flow chamber.
 
In vitro seed germination study
 
In the in vitro seed germination assay, the study aimed to investigate the impact of modified RL/BL solutions on the germination potential of the certified mung bean seeds (Vigna radiata) (Variety bullet) procured from M/S Rajasthan agriculture research institute (RARI), Durgapura, Jaipur. Petri dishes were autoclaved with the circles of Whatman filter paper number 1. The seeds were soaked for four hours in the RL and BL solutions, along with a control set of soaking seeds in tap water.
 
Estimation of the vigour index
 
In each of the experiments conducted, the vigour index for each treatment was determined according to the formula from Abdul-Baki and Anderson (1973).
 
Vigour index = [Mean of root length (cm) + Mean of shoot length (cm)] × Percentage of seed germination

RESULTS AND DISCUSSION

Examining the capability of microbial cultures to utilize RL and BL as the sole source of nutrients
 
All the microbial cultures procured were evaluated for their ability to utilize autoclaved and un-autoclaved RL and BL. All the cultures could grow very well in the autoclaved RL plates. In the case of un-autoclaved RL plates, apart from the inoculated cultures, the indigenous bacteria of the RL also grew in the plates. However, the inoculated bacteria showed growth along the streaks. The culture growth on solidified RL plates is shown in Fig 1A (autoclaved) and 1B (un-autoclaved). Similarly, the cultures grown on solidified BL plates are shown in Fig 2A (autoclaved) and 2B (un-autoclaved).

Fig 1: Growth of microbial cultures on autoclaved (A) and un-autoclaved (B) RL plates.



Fig 2: Growth of microbial cultures on autoclaved (A) and un-autoclaved (B) BL plates.


 
Characterization of Rhizobium strains
 
The three Rhizobium strains were characterized morphologically and biochemically. The colony characteristics on YMA plates and microscopy showed a typical Rhizobium growth pattern.
 
Biochemical characterization of Rhizobial strains
 
All the Rhizobium strains showed typical growth on YMA. The colonies were white to cream with exopolysaccharides secretion. The colonies didn’t take the colour of congo red dye when grown on YMA plates. All the strains were gram-negative and confirmed by Methyl Red (MR test), Catalase and Bromothymol Blue tests. The negative response was recorded for the Voges Proskauer (VP) test, Starch Hydrolysis and Citrate Utilization tests, which showed the absence of Enterobacteriaceae, common contaminants. All the strains endured temperatures up to 45°C. Chowdhury (2015) and Tyagi et al. (2017) reported a similar trend in the biochemical tests.
 
Biocompatibility studies
 
In natural conditions, bacteria live in communities and show various kinds of interactions like mutualism, antagonism, synergism, etc. Therefore, their biocompatibility is essential for the use of microbial consortiums. The natural ability or capability of microbes living or existing together in harmony reflects the biocompatibility among them. So, it is always recommended that the biocompatibility of the proposed microbial strains be checked before developing a biofertilizer sample. Kumar and Chandra (2008) have also suggested that the compatibility of Rhizobium sps. should be evaluated before using different consortiums in the field while studying Lentil plants using PSB and PGPRs as biofertilizers. Therefore, the biocompatibility check was performed in the study.  The MR was found to be compatible with bacterial strains viz. BM, ECM, PSB, Bst.(W) and Bst (S) (Table 3). On the other hand, B.sf was found to dominate over Rhizobium sps. Whereas, Rhizobium sps. dominated over all the other Bacillus sps. Overall, MR was found to dominate all the bacterial strains tested except for B.sf Based on phosphate solubilization, IAA production and ARDRA profile, Rhizobium and Bacillus strains have been reported to be compatible with each other (Dhole et al., 2022). 

Table 3: Compatibility among the microbial cultures.


 
In vitro seed germination assay
 
The germination assay was carried out to screen the microbes+ RL or BL combination, which showed effective PGP activity. As per the results shown in Table 4, RL+MR was the best in shoot length for both the autoclaved (39.62% over control) and un-autoclaved (9.83% over control) sets. The un-autoclaved RL+MR was the best combination for root length, resulting in an increase of 32.35% in root length than the control. However, in the autoclaved version, maximum root length could be achieved by using a combination of RL+BM+MR+Gypsum. This showed an increase of 30.31% over control. Maximum fresh weight was achieved in the case of un-autoclaved RL+MR (90.72% over control). In the autoclaved set, RL gave the maximum fresh weight (i.e. 7.58% higher than the control). Dry weight was obtained with the combination of RL+BM+MR+Gypsum for both the autoclaved and un-autoclaved sets, which was 120% and 141.46% higher than the respective control values (Table 4).

Table 4: In vitro seed germination assay using Un-autoclaved and autoclaved RL added with different microbial cultures.


       
For the autoclaved sets of instant RL, maximum shoot length and fresh weight could be achieved in the RL+MR+PSB treatment and maximum root length (6.66% over control) was obtained in the RL+MR+BM treatment. Maximum dry weight was obtained in the RL+MR+ECM, which was 28.16% higher than the controls (Table 5).

Table 5: In vitro seed germination assay (Un-autoclaved and autoclaved set of RL-instant).


       
For the un-autoclaved set of instant RL, maximum shoot length was obtained in tap water, whereas maximum root length (530%) and fresh weight (55.55%) were obtained in only RL. Dry weight was maximum (18.18% over control) in the case of only RL and RL+MR+BM.
       
For the autoclaved set of instant BL, maximum shoot length was obtained in the case of BL+MR+ECM (6.35% more than the control) and maximum root length was found equal in the case of water and BL+MR+ECM. Fresh weight was found to be maximum in the case of water, while dry weight was maximum (1.64% increase over control) in the case of BL+MR (Table 6).

Table 6: In vitro seed germination assay (Un-autoclaved and autoclaved set of BL-Instant).


       
For the un-autoclaved set of instant BL, shoot length (14.35 % more than the control), fresh weight (57.44% higher than the control) and dry weight (which was 12.08% more than the control) were found to be the best in BL+MR+B.st(W) while maximum root length was achieved in the case of BL+MR+BM which showed an increase of 24.76% over control.
       
For the autoclaved set of RL-48 hrs, maximum shoot length (i.e. 81.12% more than the control) was achieved in the case of RL+MR+PSB while maximum root length (83.25% over control) was found in RL+MR+B.sf Both the maximum fresh weight (146.26% higher than the control) and dry weight (98% more than the control) were obtained in only RL (Table 7).

Table 7: In vitro seed germination assay (Un-autoclaved and autoclaved set of RL-48H).


       
For the un-autoclaved set of RL-48 Hrs, maximum shoot length (8.9% higher than the control) was obtained in the case of RL+MR+BM whereas maximum root length (i.e. 51.81% higher than the control) and fresh weight (108% more than the control) were found in only RL. Dry weight (12.67% more than the control) was maximum in the case of RL+MR.
       
For the autoclaved set of BL-48 hrs, maximum shoot length (67.72% higher) and fresh weight (6% more than the control) were obtained in the case of BL+MR+ECM, whereas maximum root length and dry weight were found in the case of tap water (Table 8).

Table 8: In vitro seed germination assay (Un-autoclaved and autoclaved set of BL-48 hours).


       
For the un-autoclaved set of BL-48 hrs, maximum shoot length, root length and fresh water were obtained in the case of tap water. However, if we compare it with the autoclaved water, then the BL+MR+BM showed the maximum result for root length. Similarly, dry weight (29.19% more than the control) was also the maximum for BL+MR+BM.
       
Ahmad et al. (2012) have reported that the combined application of Rhizobium and Plant Growth Promoting Rhizobacteria (PGPR) improves the growth and productivity of Mung bean (Vigna radiata L.) under salt-stressed conditions compared with the un-inoculated control or with the individual inoculation of either the Rhizobium or the Phosphate Solubilizing Bacteria (Pseudomonas sps.). In similar lines, mung Rhizobium has shown better effects in combination with the PGPR strains used.
       
Similarly, Korir et al., (2017) have also shown the synergistic effect of Rhizobium sps. and PGPR, especially the BM, in the bean growth of another leguminous plant, namely Phaseolus vulgaris L., i.e. common bean. This is in concurrence with our results of getting a better effect of mung Rhizobium when combined with the BM.
       
As per Kaur and Sharma (2016), out of the various bio-fertilizer treatments on Vigna radiata L (Mung bean), the net returns of combined inoculation of Rhizobium and PGPR were found to be maximum. Although they reported that seed yield increased with the inoculation of different biofertilizers, the combined inoculation of Rhizobium and PGPR produced significantly higher seed yield over all the treatments. So, they also reported the synergistic effect of Rhizobium and PGPR sps. on mung bean plants.
       
In one of the studies reported by Yadav et al. (2024), the plants grown with individual biofertilizers of Rhizobium sps. did not show significant results in the morphological and physical parameters. Still, the consortium of biofertilizers, i.e., Rhizobium sps., Pseudomonas putida and Frateuria aurantia, showed observable yield improvements in mung bean plants.
       
The PGPR strain named Enterobacter kobei has shown good response while using the BL. Hayat et al., (2010) have also reported that the bacteria belonging to the species Azospirillum, Enterobacter, Klebsiella and  Pseudomonas, have been shown to attach to the root and efficiently colonize root surfaces in which Rhizobium bacteria is already present. Therefore, such PGPRs have the potential to contribute to sustainable plant growth promotion in a synchronized manner if they are compatible with each other.
       
In the case of microbial consortia, even though they are compatible, their combinations may not always result in higher yields. Many other ecological interactions are going on in the rhizosphere upon consortium application. These interactions may affect the performance of bioinoculants, be it positively or negatively. Inoculants and their consortiums do have an effect on plant growth and yield, but many combinations have to be tried to achieve higher ecological success (Renu et al., 2016).
       
Khan et al., (2026) reported the role of Plant Growth Promoting Rhizobacteria (PGPR) and plant growth regulators in enhancing the growth of chickpea (Cicer arietinum L.) and minimizing pesticide induced stress in plants.
       
Neha et al. (2024) demonstrated that co-inoculation of PGPR and Rhizobium with Vigna radiata significantly enhanced root nodulation, biomass accumulation, nutrient uptake and yield attributes. The mung bean was compared with un-inoculated control and single inoculation treatments. Improved soil health and higher N and P uptake indicated the synergistic role of both microbial inoculants in promoting plant growth and soil fertility.
       
Thus the findings of the present study are in concurrence of the previous reports indicating a positive effect of using Rhizobium in combination with selected PGPR strains. Therefore, an integrated application of potent PGPR strains and Rhizobium with handmade paper industrial effluents may serve as an effective and sustainable approach for improving mung bean productivity. Fig 3 shows the overall effect of biofertilizers tested on vigour index of mung seeds.

Fig 3: Effect of biofertilizers tested on vigour index of mung seeds.

CONCLUSION

From the systematic detailed study, it can be concluded that the effluents of cow dung based handmade papermaking are capable of supporting the growth of bacterial cultures tested. Out of the two types of effluents (RL and BL), RL was found to be a better option for promoting the plant growth parameters. Nevertheless, the quality of these effluents may be improved by inoculating them with the consortium of biocompatible PGPR strains. Both the types of  effluents showed a higher biofertilizer potential in un-autoclaved form than the respective autoclaved forms. Overall, it can be concluded from the germination assay results that RL+ MR is the best combination for both autoclaved and un-autoclaved versions when it was used after 48hours incubation. However, in the case of instant utilization of the modified RL, only RL or water was found to be the best. This might be because the inoculated bacteria could not grow to the sufficient number capable enough to bring out the positive changes. Thus for both RL as well as BL, addition of MR was found to be very effective. Further, the addition of BM with MR showed positive results in both RL as well as BL. This combination was also found best when natural additives like Gypsum was added to modify the effluents.

ACKNOWLEDGEMENT

The financial support provided by the Directorate of Science and Technology, Khadi and Village Industries Commission (KVIC), Mumbai is thankfully acknowledged.
       
The authors are sincerely thankful to the Director, CSIR-National Botanical Research Institute (NBRI), Lucknow and Dr. Puneet Singh Chauhan, Principal Scientist, Division of Plant Microbe Interactions, NBRI, Lucknow for kindly providing cultures and their guidance for the study. 
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

REFERENCES


  1. Abdul-Baki, A.A. and Anderson, J.D. (1973). Vigour determination in soyabean seed by multiple criteria. Crop Science. 13: 630-633. http://dx.doi.org/10.2135/cropsci1973.00 11183X001300060013x.

  2. Ahmad, M., Zahir, Z.A., Asghar, H.N. and Arshad, M. (2012). The combined application of rhizobial strains and PGPR improves growth and productivity of mung bean (Vigna radiata L.) under salt stressed conditions. Ann. Microbial. 62: 1321-1330. https://doi.10.1007/s13213-011-0380-9.

  3. Ashrafi, O., Yerushalmi, L. and Haghighat, F. (2015). Wastewater treatment in the pulp-and-paper industry: A review of treatment processes and the associated greenhouse gas emission. Journal of Environmental Management. 158: 146-157. https://doi.org/10.1016/j.jenvman.2015.05.010.

  4. Badar, S. and  Farooqi, I. (2012). Pulp and Paper Industry-Manufacturing Process, Waste Water Generation and Treatment. In: Environmental Protection Strategies for Sustainable Development-Strategies for Sustainability. [Malik, A., Grohmann, E. (eds)], Springer, Dordrecht. pp 397-430. https://www.researchgate.net/publication/226218540.

  5. Baskar, M., Kayalvizhi, C. and Bose, C.S.M. (2026). Co-friendly utilisation of distillery effluent in agriculture-A review. Agricultural Reviews. 24(1): 16-30. 

  6. Chaudhary, S. and Paliwal, R. (2018). Techniques for Remediation of Paper and Pulp Mill Effluents: Processes and Constraints. In: Handbook of Environmental Materials Management, 12. Springer International Publishing, Cham. https:// doi.org/10.1016/j.biortech.2011.03.048.

  7. Chauhan, S. and Prasad, S., Meena, S. and Bhatnagar, P. (2022). Exploring the less travelled path of ecofriendly handmade paper production. Innovations in Environmental Biotechnology. (pp.387-414). 10.1007/978-981-16-4445-0_17. https:// www.researchgate.net/publication/360640232.

  8. Chauhan, S. and Meena, B.L. (2021). Introduction to pulp and paper industry: Global scenario. Physical Sciences Reviews. 6(5): 81-109. https://doi.org/10.1515/b_psr- 2020-0014.

  9. Chauhan, S. and Hussain, G. (2009). “Machine made Vs. Handmade Paper” Souvenir of Handmade Paper Industry Published by KVIC During the International Conference on Pulp and Paper Industry (PAPEREX-2009): 75-80.

  10. Chowdhury, D. (2015). Isolation, identification and characterization of bacteria (Rhizobium) from chick pea (Cicer arietinum) and production of biofertilizer. Jr. of Biotechnology and Bioscience. 3(12): 26-29. https://www.researchgate. net/publication/301799385.

  11. Dhole, A.M., Shelat, H.N., Patel, H. and Jhala, Y. (2022). Appraisal of synergistic (co-inoculation) effect of rhizobium and non-rhizobial bacterial endophytes of Vigna radiata. Research Square. https://doi.org/10.21203/rs.3.rs- 1375242/v1.

  12. González, C., Alvarez, R. and Coca, J. (1992). Use of kraft black liquors from a pulp mill for the production of soil conditioners. Waste Management and Research. 10(2): 195-201. https://doi.org/10.1016/0734-242X(92)90072-S.

  13. Haile, A., Galebo, G.G., Tesfaye, T., Mengie, W., Mebrate, M.A., Abuhay, A. and Limeneh, D.Y. (2021). Pulp and paper mill waste-utilization and prospects for high value added biomaterials. Bioresources and Bioprocessing. 8: 35. https://doi.org/10.1186/s40643-021-00385-3.

  14. Hayat, R., Ali, S., Amara, U., Khalid, R. and Ahmed, I. (2010). Soil beneficial bacteria and their role in plant growth promotion:  A review. Annals of Microbiology. 60(4):  579-598. https:// doi.org/10.1007/s13213-010-0117-1.

  15. Kajić, S., Gradinščak, A., Königshofer, D., Fadljević, I., Sikora, S. and Rajnović, I. (2025). Characterization of plant growth promoting rhizobacteria and their benefits on soybean growth. Legume Research. 48(1): 131-136. doi: 10.18805/LRF-819.

  16. Kaur, J. and Sharma, P. (2016). Response of summer mung bean (Vigna radia L.) to rhizobium and PGPR inoculation under different plating methods. Ann. Agric. Res. New series. 37(4): 87-90. https://www.researchgate.net/publication/ 375640816.

  17. Khan, R., Singh, R. and Kumar, R. (2026). Effect of plant growth promoting rhizobacteria (pgpr) and plant growth regulators (PGR) on antioxidant properties of chickpea (Cicer arietinium L.) in presence of toxic effect of thiamethoxam.  Indian Journal of Agricultural Research. 60(1): 115- 122. doi: 10.18805/IJARe.D-6377.

  18. Korir, H., Mungal, N.W., Thuita, M., Hamba, Y. and Masso, C. (2017). Co-inoculation effect of rhizobia and plant growth promoting rhizobacteria on common bean growth in a low phosphorus soil. Frontiers in Plant Science. 8: 141. https://doi: 10.3389/fpls.2017.00141.

  19. Kuforiji, O.O. and Fasidi, I.O. (2009). Biodegradation of agro- industrial wastes by a edible mushroom Pleurotus tuber- regium (Fr.). J. Environ. Biol. 30(3): 355-358. PMID: 20120458. https://pubmed.ncbi.nlm.nih.gov/20120458/.

  20. Kulshreshtha, S., Mathur, N. and Bhatnagar, P. (2011). Handmade paper and cardboard industries: In health perspectives. Toxicology and Industrial Health. 27(6): 515-521. doi: 10.1177/0748233710391992.

  21. Kulshreshtha, S., Mathur, N. and Bhatnagar, P. (2010a). Genotoxic evaluation of handmade paper industrial effluent-A case study. International Journal of Chemical Sciences. 8: 2519-2528.

  22. Kulshreshtha, S., Mathur, N., Bhatnagar, P. and Jain, B. (2010b). Bioremediation of industrial waste through mushroom cultivation. Journal of Environmental Biology/Academy of Environmental Biology, India. 31: 441-444.

  23. Kulshreshtha, S., Mathur, N., Bhatnagar, P. and Kulshreshtha, S. (2013). Cultivation of Pleurotus citrinopileatus on handmade paper and cardboard industrial wastes. Industrial Crops and Products. 41: 340-346. 10.1016/ j.indcrop.2012.04.053.

  24. Kumar, R. and Chandra, R. (2008). Influence of PGPR and PSB on Rhizobium leguminosarum bv. viciae strain competition and symbiotic performance in lentil l. World Journal of Agricultural Sciences. 4(3): 297-301. https://www. researchgate.net/publication/242235746.

  25. Kunhikrishnan, A., Nanthi, S., Bolan, K.M., Seth, L., Ravi, N. and Kim, W. (2012). Chapter Five-the Influence of Wastewater Irrigation on the Transformation and Bioavailability of Heavy Metal (Loid)s in Soil, [Editor(s): Donald, L.] Sparks, Advances in Agronomy, Academic Press. 115: 215- 297. https://doi.org/10.1016/B978-0-12-394276-0.000056.

  26. Liu, X., Li, Y., Meng, Y., Lu, J., Cheng, Y., Tao, Y. and Wang, H. (2021). Pulping black liquor-based polymer hydrogel as water retention material and slow-release fertilizer. Industrial Crops and Products. 165: 113445. https:// doi.org/10.1016/j.indcrop.2021.113445.

  27. Mehmood, K., Rehman, S.K., Wang, F.F., Mahmood, Q., Jadoon, A., Javed, M.F. and Ahmed (2019). Treatment of pulp and paper industrial effluent using physiochemical process for recycling. Water. 11(11): 2393. https://doi:10.3390/ w11112393.

  28. Molina-Sanchez, E., Leyva-Díaz, J.C., Cortes-Garcia, F.J. and Molina-Moreno, V. (2018). Proposal of sustainability indicators for the waste management from the paper industry within the circular economy model. Water. 10: 1014. https://doi.org/10.3390/w10081014.

  29. Neha, C.R., Pareek, N. and Raverkar, K.P. (2024). Enhancing mungbean (Vigna radiata L.) productivity, soil health and profitability through conjoint use of Rhizobium and PGPR. Legume Research. 47(5): 823-828. doi: 10.18805/LR-4552. 

  30. Narayana, S., Sethiasivan, K., Chauhan, S. and Mathur, R. (2024). Waste liquors generated during handmade paper manufacture from cow dung as a potential source of biofertilizer. Journal of Applied and Natural Science. 16(1): 400-409. https://doi.org/10.31018/jans.v16i1.5209.

  31. Patel, K., Patel, N., Vaghamshi, N., Shah, K., Duggirala, S.M. and Dudhagara, P. (2021). Trends and Strategies in the effluent treatment of pulp and paper industries-A review highlighting reactor options. Current Research in Microbial Sciences. 2: 100077. https://doi.org/10.1016/j.crmicr. 2021.100077.

  32. Pokhrel, D. and Viraraghavan, T. (2004). Treatment of pulp and paper mill wastewater-A review. Sci. Tot. Env. 333: 37- 58. https://doi.org/10.1016/j.scitotenv.2004.05.017.

  33. Reddy, S. (2015). Eco-friendly production of paper products. International Journal of Chemical Concepts. 1: 72-80. https://www.researchgate.net/publication/281854558_ Eco-Friendly_Production_of_Paper_Products.

  34. Renu, S.P., Sahu, U., Bhoyar, M.S., Kumar, M., Singh, U. and Pathak, R. (2016). Ecological success of compatible microbes in consortia isolated from rice rhizosphere for growth and yield of mung bean (Vigna radiata L.). J. Pure Appl Microbiol. 10(4): 3231-3239. doi: 10.22207/JPAM. 10.4.101 https://doi.org/10.22207/JPAM.10.4.101.

  35. Savant, D., Abdulrahman, R. and Ranade, D. (2006). Anaerobic degradation of adsorbable organic halides (AOX) from pulp and paper industry wastewater. Bioresour. Technol. 97: 1092-1104. https://doi.org/10.1016/j.biortech.2004. 12.013.

  36. Sharma, P. and Singh, S.P. (2021). Pollutants characterization and toxicity assessment of pulp and paper industry sludge for safe environmental disposal. Emerging Treatment Technologies for Waste Management. (pp.207-223). 10.1007/978-981-16-2015-7_10.

  37. Singh, U.S. and Tripathi, Y.C. (2020). Characteristics and treatment of pulp and paper mill effluents- A review. International Journal of Engineering and Technical Research (IJETR). 10(11): 19-26.

  38. Sivrikaya, H., Bacak, L., Saracbasi, A., Toroglu, L. and Eroglu, H. (2002). Trace elements in pleurotus sajorcaju cultivated on chemithermomechanical pulp for bioleaching. Food Chemistry. 79: 173-176. https://doi.org/10.1016/S0308- 8146(02)00128-0.

  39. Thompson, G., Swain, J., Kay, M. and Forster, C. (2001). The treatment of pulp and paper mill effluent: A review. Bioresour Technol. 77: 275-286. https://doi.org/10.1016/ S0960-8524(00)00060-2.

  40. Touhari, F., Guetarni, H., Sadaoui, S. and Mehaiguene, M. (2023).  Water quality assessment of the ouled mellouk dam treatment plant, North Western Algeria. Agricultural Science Digest. 43(3): 334-339. doi: 10.18805/ag.DF-497.

  41. Tyagi, V.K., Purushottam and Tomar, A. (2017). Isolation, identification, biochemical and antibiotic sensitivity characterization of rhizobium strains from Vigna mungo (L) Hepper, Cicer aertinum L. and Vigna radiate (L.) R Wilczek in Muzaffarnagar, Uttar Pradesh, India. Int. J. Curr. Microbiol. Appl. Sci. 6(12): 2024-2035. doi. https://doi.org/10.20546/ijcmas. 2017.612.233

  42. United States Environment Protection Act, (US EPA) (1992). https:// www.epa.gov/sites/default/files/201411/documents/ guidelines_exp_assessment.pdf.

  43. United States Environment Protection Act, (US EPA) (2004). https:// www.epa.gov/sites/default/files/2019-08/documents/ 2004-guidelines-water-reuse.pdf.

  44. Yadav, B.K., Mohanty, N., Dash, S., Pradhan, S., Sahoo, B. and Rath, B. (2024). Enhanced yield of mung bean (Vigna radiata L.) using bacterial biofertilizers. Biosciences Biotechnology Research Asia. 21(1): 89-98. https:// dx.doi.org/10.13005/bbra/3205.

  45. Zervakis, G., Yiatras, P. and Balis, C. (1995). Edible mushrooms from olive oil mill wastes. International Biodeterioration and Biodegradation. 38(3-4): 237-243. https://doi.org/ 10.1016/S0964-8305(96)00056-X.
Published In
Indian Journal of Agricultural Research
Article Metrics
Metrics Icon
25
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    Full Research Article

    Utilization of Handmade Paper Industries Effluent Fortified with Growth-promoting Microbes for Enhancing Vigna radiata Growth

    S
    Sunita Chauhan1,*
    P
    Poonam C. Singh2
    R
    Rahul Mishra1,3
    1Kumarappa National Handmade Paper Institute, Sanganer, Jaipur-302 029, Rajasthan, India.
    2CSIR-National Botanical Research Institute, Lucknow-226 001, Uttar Pradesh, India.
    3Khadi and Village Industries Commission, Jaipur-302 004, Rajasthan, India.

    ABSTRACT

    Background: Biofertilizers play an essential role in bringing sustainability to agriculture. They are being focused all over the world to develop and improve technologies for biofertilizers to make them cost-effective and enhance productivity. Handmade papermaking from cow dung results in the generation of effluents, raw liquor (RL) and alkaline black liquor (BL), which are rich in organic carbon and nutrients. The present study was carried out to study the compatibility of RL and BL in agriculture along with microbial biofertilizers.

    Methods: One of the waste effluents (RL) was recently reported to have plant growth-promoting characteristics with the promising results of seed germination and pot studies using green gram seeds at KNHPI. In the present study, the RL and BL efficacy were tested with microbial cultures procured from different sources and isolated at KNHPI for enhancing their biofertilizer potential. The cultures, namely mung Rhizobium (MR), Phosphate Solubilizing Bacteria (PSB-NB), Enterobacter kobei (ECM), Bacillus species isolated from soil B.St (S), Bacillus species isolated from textile industry waste B.St (W) were selected for detailed study in different combinations.

    Result: The combination of RL and BL with different PGPR strains of a compatible nature was observed to show enhanced seed germination and plant growth. The present study provides an insight into the utilization of waste by-products of the handmade paper industry that will have socio-economic implications. This may contribute to bring sustainability to the agriculture sector and help provide employment, income generation and reduce environmental pollution.

    INTRODUCTION

    The pulp and paper sector is recognized as the sixth largest contributor to industrial pollution, following the oil, cement, leather, textile and steel industries, by releasing diverse contaminants into the environment (Savant et al., 2006; Mehmood et al., 2019). Papermaking is highly energy-intensive and water-consuming in terms of freshwater utilization (Pokhrel and Viraraghavan, 2004). Water consumption varies with production methods and may reach up to 60 m³ per ton of paper, generating ~50 m3 of wastewater (Thompson et al., 2001; Ashrafi et al., 2015; Molina-Sanchez et al., 2018). Freshwater is used at every stage, from raw material cultivation to pulping, bleaching and sheet formation, resulting in substantial wastewater generation (Chauhan and Meena, 2021).
           
    Major pollutants of pulp and paper effluents include lignin, fatty acids, dark colour compounds, biochemical oxygen demand (BOD), chemical oxygen demand (COD), adsorbable organic halides (AOX) and volatile organic compounds (VOCs) (Sharma and Singh, 2021). According to the Ministry of Environment and Forest (MOEF), the pulp and paper industry is categorized under the red category due to its high pollution potential (Patel et al., 2021). Wastewater generation ranges from 220-380 m3 per ton of paper produced and is often highly coloured and toxic (Badar and Farooqi, 2012). In India, total water consumption may reach 250-300 m3 per ton of paper (Chaudhary and Paliwal, 2018; Patel et al., 2021). Various treatment approaches, including aerobic, anaerobic, electrochemical, photocatalytic, ozonation, coagulation-flocculation and adsorption techniques have been investigated (Singh and Tripathi, 2020; Touhari et al., 2023). Biological treatment methods rely on microbes to convert organic pollutants into biomass (Sharma and Singh, 2021). However, efficient treatment depends on wastewater characteristics, quantity and reuse requirements, emphasizing the need for well-defined management strategies (Singh and Tripathi, 2020).
           
    Handmade paper refers to sheets produced manually through traditional sheet-forming processes. The Khadi and Village Industries Commission (KVIC) includes paper made using Cylinder Mould Machine/Vat (CMM or CMV) with a deckle width of 102 cm in handmade paper category. Handmade paper is considered socially responsible and sustainable as it is tree-free, eco-friendly and resource-efficient (Chauhan et al., 2022).
           
    It is justified as a cost-effective and energy-saving alternative with strong economic and environmental benefits (Reddy, 2015). Although handmade and mill-made paper share similar principle of production, differences exist in processing, energy use and environmental impact (Chauhan and Hussain, 2009; Chauhan et al., 2022). Both sectors generate coloured effluents that possess environmental and aesthetic concerns, necessitating proper treatment and disposal.
           
    Kulshreshtha et al., (2011) have evaluated the health perspectives of the effluents of the handmade paper industry of the Sanganer region and reported them to be mutagenic with either one strain of Salmonella typhimurium or with both. The presence of dyes and chemicals in the effluents of handmade paper and pulp industries is revealed by its colour, low pH and high COD (Kulshreshtha et al., 2010a). The handmade paper and cardboard industries also produce sludge, at the rate of approximately 10 kgd-1 per industry, in the form of shortened pulp residues which cannot be recycled again. These residues are accumulated in the drain or street and produce enormous odour. Kulshreshtha et al. (2013) have reported the cultivation of Pleurotus citrinopileatus on the sludge of handmade paper and cardboard industrial waste. Agro-industrial and industrial wastes have been exploited for centuries in Asia for the production of oyster mushrooms (Zervakis et al., 1995; Sivrikaya et al., 2002; Kuforiji and Fasidi, 2009; Kulshreshtha et al., 2010b).
           
    Recycled water is defined as wastewater that is treated and reused to supplement water supply (US EPA, 1992). The beneficial utilization of treated wastewater for agriculture is the major water reuse application worldwide (US EPA, 2004). Research shows that wastewater irrigation can result in significant changes to soil’s physical, chemical and biological properties. Kunhikrishnan et al. (2012) have reported the influence of wastewater irrigation on the transformation and bioavailability of heavy metal (loid)s in soil. González et al. (1992) have explored the possibility of utilizing kraft black liquors generated in a paper mill, using Eucalyptus globulus as raw material, for the production of a Nitro-Humic soil conditioner. Liu et al. (2021) have reported a pulping black liquor-based polymer hydrogel as a water retention material and slow-release fertilizer. Similarly, Haile et al., (2021) have reviewed the prospects of making different high-value-added biomaterials from pulp and paper mill wastes.
           
    Therefore, in view of all the above background, the present study was taken up to explore the possibility of improving the use of effluents in the handmade paper industry using cow dung as raw materials. The effluents were augmented with different PGPR strains and evaluated in green gram (Vigna radiata i.e. mung). While the utilization of industrial effluents (Baskar et al., 2026) and PGPRs (Kajić et al., 2025) (in agriculture has been explored independently, this study contributes by systematic evaluation of their combined application, particularly using cow dung-based handmade paper effluents, for improving mung bean growth. Thus the study offers a novel integration of handmade paper industry effluents with plant growth-promoting microbial consortia, providing new insights into their combined potential as eco-friendly biofertilizer formulations for enhancing Vigna radiata growth. 

    MATERIALS AND METHODS

    The present study was carried out in the Biotechnology laboratory of Kumarappa National Handmade Paper Institute (KNHPI), Jaipur, Rajasthan during the Financial year’ 2021-22.
     
    Procurement of standard microbial strains of PGPR
     
    Pseudomonas sps (PSB-NB) and the bacterial strains of rhizobia, namely mung Rhizobium (MR), chickpea Rhizobium (CPR) and pea Rhizobium (PR), were procured from the National Botanical Research Institute (NBRI), Lucknow. Azotobacter sp. (Azo-R) was kindly received from Krishi Vigyan Kendra, Rajsamand. Bacillus megaterium (BM), Bacillus safensis (B.sf) and Enterobacter kobei (ECM) were procured from National Agriculturally Important Microbial Culture Collection (NAIMCC), ICAR-National Bureau of Agriculturally Important Microorganisms (ICAR-NBAIM), Mau. Two Bacillus species were isolated from garden soil (Bst.S) and textile industry effluents (Bst.W) at KNHPI, Jaipur, Rajasthan.
     
    Collection of waste effluents of handmade papermaking
     
    Two types of waste streams/effluents of cow dung based handmade paper industry were collected. Raw liquor (RL) obtained before pulping of the cow dung squeezed in the dewatering machine and after pulping by NaOH (BL) were used in the study (Narayana et al., 2024).
     
    Examining the capability of microbial cultures to utilize RL/BL as the sole source of nutrient
     
    RL and BL plates were prepared separately by adding agar powder (Bacteriological grade, HiMedia, GRM026) at the rate of 3% of the effluents used (RL and BL). The cultures were streaked on RL and BL agar plates. The inoculated plates were incubated at 28°C for 3 to 5 days and the growth of bacterial culture indicated the utilization of RL and BL by the microbes used. Sterilized water agar media supplemented with un-autoclaved RL and BL separately were also included in the study. These plates were inoculated with the available PGPR strains. The plates were then incubated at 28°C and observed for growth, if any.

    Biocompatibility test between microbial strains
     
    Nutrient agar medium was used for checking the compatibility of Rhizobium with different combinations of the available microbial strains. Three standard methods of checking biocompatibility viz. Well Diffusion method, Cross Streaking, Disc and Swab method were used.
     
    Preparation of modified RL and BL solutions
     
    The microbial cultures were grown in their specific media/broth. Rhizobium was grown in Yeast Mannitol Broth (YEMA, Hi Media, M721), AzoR in Jensen’s Broth (Hi Media, GM973) and ECM in King’s B broth (glycerol-10 gpl, K2HPO4-1.5 gpl, MgSO4-1.5 gpl, Peptone-20 gpl). Nutrient broth (HiMedia) was used for growing Bst. (S), Bst.(W), PSB-NB and B.sf All the inoculated plates were incubated for 48 H at 28°C. The 48 H growth cultures were used as primary inoculum (1% of broth) to inoculate the autoclaved and un-autoclaved broth of RL and BL. The biocompatible cultures were inoculated at the rate of 1% each. The inoculated RL and BL solutions were then incubated at 28°C for a period of 48 H. Another set was maintained without incubation- “instant sets”. The treatments of different PGPR strains and RL or BL used are given in Table 1 and 2.

    Table 1: Un-autoclaved and autoclaved set of RL used for in vitro seed germination assay of mung bean.



    Table 2: Un-autoclaved and autoclaved sets of RL and BL with the given consortiums of PGPR strains used.


     
    Surface sterilization of mung seeds       
                    
    Seeds were surface sterilized using 0.01% mercuric chloride solution. For this, Mung seeds were dipped in mercuric chloride solution for 3 minutes and then washed 10 times with sterilized distilled water. The seeds were then allowed to dry inside the Laminar Air Flow chamber.
     
    In vitro seed germination study
     
    In the in vitro seed germination assay, the study aimed to investigate the impact of modified RL/BL solutions on the germination potential of the certified mung bean seeds (Vigna radiata) (Variety bullet) procured from M/S Rajasthan agriculture research institute (RARI), Durgapura, Jaipur. Petri dishes were autoclaved with the circles of Whatman filter paper number 1. The seeds were soaked for four hours in the RL and BL solutions, along with a control set of soaking seeds in tap water.
     
    Estimation of the vigour index
     
    In each of the experiments conducted, the vigour index for each treatment was determined according to the formula from Abdul-Baki and Anderson (1973).
     
    Vigour index = [Mean of root length (cm) + Mean of shoot length (cm)] × Percentage of seed germination

    RESULTS AND DISCUSSION

    Examining the capability of microbial cultures to utilize RL and BL as the sole source of nutrients
     
    All the microbial cultures procured were evaluated for their ability to utilize autoclaved and un-autoclaved RL and BL. All the cultures could grow very well in the autoclaved RL plates. In the case of un-autoclaved RL plates, apart from the inoculated cultures, the indigenous bacteria of the RL also grew in the plates. However, the inoculated bacteria showed growth along the streaks. The culture growth on solidified RL plates is shown in Fig 1A (autoclaved) and 1B (un-autoclaved). Similarly, the cultures grown on solidified BL plates are shown in Fig 2A (autoclaved) and 2B (un-autoclaved).

    Fig 1: Growth of microbial cultures on autoclaved (A) and un-autoclaved (B) RL plates.



    Fig 2: Growth of microbial cultures on autoclaved (A) and un-autoclaved (B) BL plates.


     
    Characterization of Rhizobium strains
     
    The three Rhizobium strains were characterized morphologically and biochemically. The colony characteristics on YMA plates and microscopy showed a typical Rhizobium growth pattern.
     
    Biochemical characterization of Rhizobial strains
     
    All the Rhizobium strains showed typical growth on YMA. The colonies were white to cream with exopolysaccharides secretion. The colonies didn’t take the colour of congo red dye when grown on YMA plates. All the strains were gram-negative and confirmed by Methyl Red (MR test), Catalase and Bromothymol Blue tests. The negative response was recorded for the Voges Proskauer (VP) test, Starch Hydrolysis and Citrate Utilization tests, which showed the absence of Enterobacteriaceae, common contaminants. All the strains endured temperatures up to 45°C. Chowdhury (2015) and Tyagi et al. (2017) reported a similar trend in the biochemical tests.
     
    Biocompatibility studies
     
    In natural conditions, bacteria live in communities and show various kinds of interactions like mutualism, antagonism, synergism, etc. Therefore, their biocompatibility is essential for the use of microbial consortiums. The natural ability or capability of microbes living or existing together in harmony reflects the biocompatibility among them. So, it is always recommended that the biocompatibility of the proposed microbial strains be checked before developing a biofertilizer sample. Kumar and Chandra (2008) have also suggested that the compatibility of Rhizobium sps. should be evaluated before using different consortiums in the field while studying Lentil plants using PSB and PGPRs as biofertilizers. Therefore, the biocompatibility check was performed in the study.  The MR was found to be compatible with bacterial strains viz. BM, ECM, PSB, Bst.(W) and Bst (S) (Table 3). On the other hand, B.sf was found to dominate over Rhizobium sps. Whereas, Rhizobium sps. dominated over all the other Bacillus sps. Overall, MR was found to dominate all the bacterial strains tested except for B.sf Based on phosphate solubilization, IAA production and ARDRA profile, Rhizobium and Bacillus strains have been reported to be compatible with each other (Dhole et al., 2022). 

    Table 3: Compatibility among the microbial cultures.


     
    In vitro seed germination assay
     
    The germination assay was carried out to screen the microbes+ RL or BL combination, which showed effective PGP activity. As per the results shown in Table 4, RL+MR was the best in shoot length for both the autoclaved (39.62% over control) and un-autoclaved (9.83% over control) sets. The un-autoclaved RL+MR was the best combination for root length, resulting in an increase of 32.35% in root length than the control. However, in the autoclaved version, maximum root length could be achieved by using a combination of RL+BM+MR+Gypsum. This showed an increase of 30.31% over control. Maximum fresh weight was achieved in the case of un-autoclaved RL+MR (90.72% over control). In the autoclaved set, RL gave the maximum fresh weight (i.e. 7.58% higher than the control). Dry weight was obtained with the combination of RL+BM+MR+Gypsum for both the autoclaved and un-autoclaved sets, which was 120% and 141.46% higher than the respective control values (Table 4).

    Table 4: In vitro seed germination assay using Un-autoclaved and autoclaved RL added with different microbial cultures.


           
    For the autoclaved sets of instant RL, maximum shoot length and fresh weight could be achieved in the RL+MR+PSB treatment and maximum root length (6.66% over control) was obtained in the RL+MR+BM treatment. Maximum dry weight was obtained in the RL+MR+ECM, which was 28.16% higher than the controls (Table 5).

    Table 5: In vitro seed germination assay (Un-autoclaved and autoclaved set of RL-instant).


           
    For the un-autoclaved set of instant RL, maximum shoot length was obtained in tap water, whereas maximum root length (530%) and fresh weight (55.55%) were obtained in only RL. Dry weight was maximum (18.18% over control) in the case of only RL and RL+MR+BM.
           
    For the autoclaved set of instant BL, maximum shoot length was obtained in the case of BL+MR+ECM (6.35% more than the control) and maximum root length was found equal in the case of water and BL+MR+ECM. Fresh weight was found to be maximum in the case of water, while dry weight was maximum (1.64% increase over control) in the case of BL+MR (Table 6).

    Table 6: In vitro seed germination assay (Un-autoclaved and autoclaved set of BL-Instant).


           
    For the un-autoclaved set of instant BL, shoot length (14.35 % more than the control), fresh weight (57.44% higher than the control) and dry weight (which was 12.08% more than the control) were found to be the best in BL+MR+B.st(W) while maximum root length was achieved in the case of BL+MR+BM which showed an increase of 24.76% over control.
           
    For the autoclaved set of RL-48 hrs, maximum shoot length (i.e. 81.12% more than the control) was achieved in the case of RL+MR+PSB while maximum root length (83.25% over control) was found in RL+MR+B.sf Both the maximum fresh weight (146.26% higher than the control) and dry weight (98% more than the control) were obtained in only RL (Table 7).

    Table 7: In vitro seed germination assay (Un-autoclaved and autoclaved set of RL-48H).


           
    For the un-autoclaved set of RL-48 Hrs, maximum shoot length (8.9% higher than the control) was obtained in the case of RL+MR+BM whereas maximum root length (i.e. 51.81% higher than the control) and fresh weight (108% more than the control) were found in only RL. Dry weight (12.67% more than the control) was maximum in the case of RL+MR.
           
    For the autoclaved set of BL-48 hrs, maximum shoot length (67.72% higher) and fresh weight (6% more than the control) were obtained in the case of BL+MR+ECM, whereas maximum root length and dry weight were found in the case of tap water (Table 8).

    Table 8: In vitro seed germination assay (Un-autoclaved and autoclaved set of BL-48 hours).


           
    For the un-autoclaved set of BL-48 hrs, maximum shoot length, root length and fresh water were obtained in the case of tap water. However, if we compare it with the autoclaved water, then the BL+MR+BM showed the maximum result for root length. Similarly, dry weight (29.19% more than the control) was also the maximum for BL+MR+BM.
           
    Ahmad et al. (2012) have reported that the combined application of Rhizobium and Plant Growth Promoting Rhizobacteria (PGPR) improves the growth and productivity of Mung bean (Vigna radiata L.) under salt-stressed conditions compared with the un-inoculated control or with the individual inoculation of either the Rhizobium or the Phosphate Solubilizing Bacteria (Pseudomonas sps.). In similar lines, mung Rhizobium has shown better effects in combination with the PGPR strains used.
           
    Similarly, Korir et al., (2017) have also shown the synergistic effect of Rhizobium sps. and PGPR, especially the BM, in the bean growth of another leguminous plant, namely Phaseolus vulgaris L., i.e. common bean. This is in concurrence with our results of getting a better effect of mung Rhizobium when combined with the BM.
           
    As per Kaur and Sharma (2016), out of the various bio-fertilizer treatments on Vigna radiata L (Mung bean), the net returns of combined inoculation of Rhizobium and PGPR were found to be maximum. Although they reported that seed yield increased with the inoculation of different biofertilizers, the combined inoculation of Rhizobium and PGPR produced significantly higher seed yield over all the treatments. So, they also reported the synergistic effect of Rhizobium and PGPR sps. on mung bean plants.
           
    In one of the studies reported by Yadav et al. (2024), the plants grown with individual biofertilizers of Rhizobium sps. did not show significant results in the morphological and physical parameters. Still, the consortium of biofertilizers, i.e., Rhizobium sps., Pseudomonas putida and Frateuria aurantia, showed observable yield improvements in mung bean plants.
           
    The PGPR strain named Enterobacter kobei has shown good response while using the BL. Hayat et al., (2010) have also reported that the bacteria belonging to the species Azospirillum, Enterobacter, Klebsiella and  Pseudomonas, have been shown to attach to the root and efficiently colonize root surfaces in which Rhizobium bacteria is already present. Therefore, such PGPRs have the potential to contribute to sustainable plant growth promotion in a synchronized manner if they are compatible with each other.
           
    In the case of microbial consortia, even though they are compatible, their combinations may not always result in higher yields. Many other ecological interactions are going on in the rhizosphere upon consortium application. These interactions may affect the performance of bioinoculants, be it positively or negatively. Inoculants and their consortiums do have an effect on plant growth and yield, but many combinations have to be tried to achieve higher ecological success (Renu et al., 2016).
           
    Khan et al., (2026) reported the role of Plant Growth Promoting Rhizobacteria (PGPR) and plant growth regulators in enhancing the growth of chickpea (Cicer arietinum L.) and minimizing pesticide induced stress in plants.
           
    Neha et al. (2024) demonstrated that co-inoculation of PGPR and Rhizobium with Vigna radiata significantly enhanced root nodulation, biomass accumulation, nutrient uptake and yield attributes. The mung bean was compared with un-inoculated control and single inoculation treatments. Improved soil health and higher N and P uptake indicated the synergistic role of both microbial inoculants in promoting plant growth and soil fertility.
           
    Thus the findings of the present study are in concurrence of the previous reports indicating a positive effect of using Rhizobium in combination with selected PGPR strains. Therefore, an integrated application of potent PGPR strains and Rhizobium with handmade paper industrial effluents may serve as an effective and sustainable approach for improving mung bean productivity. Fig 3 shows the overall effect of biofertilizers tested on vigour index of mung seeds.

    Fig 3: Effect of biofertilizers tested on vigour index of mung seeds.

    CONCLUSION

    From the systematic detailed study, it can be concluded that the effluents of cow dung based handmade papermaking are capable of supporting the growth of bacterial cultures tested. Out of the two types of effluents (RL and BL), RL was found to be a better option for promoting the plant growth parameters. Nevertheless, the quality of these effluents may be improved by inoculating them with the consortium of biocompatible PGPR strains. Both the types of  effluents showed a higher biofertilizer potential in un-autoclaved form than the respective autoclaved forms. Overall, it can be concluded from the germination assay results that RL+ MR is the best combination for both autoclaved and un-autoclaved versions when it was used after 48hours incubation. However, in the case of instant utilization of the modified RL, only RL or water was found to be the best. This might be because the inoculated bacteria could not grow to the sufficient number capable enough to bring out the positive changes. Thus for both RL as well as BL, addition of MR was found to be very effective. Further, the addition of BM with MR showed positive results in both RL as well as BL. This combination was also found best when natural additives like Gypsum was added to modify the effluents.

    ACKNOWLEDGEMENT

    The financial support provided by the Directorate of Science and Technology, Khadi and Village Industries Commission (KVIC), Mumbai is thankfully acknowledged.
           
    The authors are sincerely thankful to the Director, CSIR-National Botanical Research Institute (NBRI), Lucknow and Dr. Puneet Singh Chauhan, Principal Scientist, Division of Plant Microbe Interactions, NBRI, Lucknow for kindly providing cultures and their guidance for the study. 
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

    REFERENCES


    1. Abdul-Baki, A.A. and Anderson, J.D. (1973). Vigour determination in soyabean seed by multiple criteria. Crop Science. 13: 630-633. http://dx.doi.org/10.2135/cropsci1973.00 11183X001300060013x.

    2. Ahmad, M., Zahir, Z.A., Asghar, H.N. and Arshad, M. (2012). The combined application of rhizobial strains and PGPR improves growth and productivity of mung bean (Vigna radiata L.) under salt stressed conditions. Ann. Microbial. 62: 1321-1330. https://doi.10.1007/s13213-011-0380-9.

    3. Ashrafi, O., Yerushalmi, L. and Haghighat, F. (2015). Wastewater treatment in the pulp-and-paper industry: A review of treatment processes and the associated greenhouse gas emission. Journal of Environmental Management. 158: 146-157. https://doi.org/10.1016/j.jenvman.2015.05.010.

    4. Badar, S. and  Farooqi, I. (2012). Pulp and Paper Industry-Manufacturing Process, Waste Water Generation and Treatment. In: Environmental Protection Strategies for Sustainable Development-Strategies for Sustainability. [Malik, A., Grohmann, E. (eds)], Springer, Dordrecht. pp 397-430. https://www.researchgate.net/publication/226218540.

    5. Baskar, M., Kayalvizhi, C. and Bose, C.S.M. (2026). Co-friendly utilisation of distillery effluent in agriculture-A review. Agricultural Reviews. 24(1): 16-30. 

    6. Chaudhary, S. and Paliwal, R. (2018). Techniques for Remediation of Paper and Pulp Mill Effluents: Processes and Constraints. In: Handbook of Environmental Materials Management, 12. Springer International Publishing, Cham. https:// doi.org/10.1016/j.biortech.2011.03.048.

    7. Chauhan, S. and Prasad, S., Meena, S. and Bhatnagar, P. (2022). Exploring the less travelled path of ecofriendly handmade paper production. Innovations in Environmental Biotechnology. (pp.387-414). 10.1007/978-981-16-4445-0_17. https:// www.researchgate.net/publication/360640232.

    8. Chauhan, S. and Meena, B.L. (2021). Introduction to pulp and paper industry: Global scenario. Physical Sciences Reviews. 6(5): 81-109. https://doi.org/10.1515/b_psr- 2020-0014.

    9. Chauhan, S. and Hussain, G. (2009). “Machine made Vs. Handmade Paper” Souvenir of Handmade Paper Industry Published by KVIC During the International Conference on Pulp and Paper Industry (PAPEREX-2009): 75-80.

    10. Chowdhury, D. (2015). Isolation, identification and characterization of bacteria (Rhizobium) from chick pea (Cicer arietinum) and production of biofertilizer. Jr. of Biotechnology and Bioscience. 3(12): 26-29. https://www.researchgate. net/publication/301799385.

    11. Dhole, A.M., Shelat, H.N., Patel, H. and Jhala, Y. (2022). Appraisal of synergistic (co-inoculation) effect of rhizobium and non-rhizobial bacterial endophytes of Vigna radiata. Research Square. https://doi.org/10.21203/rs.3.rs- 1375242/v1.

    12. González, C., Alvarez, R. and Coca, J. (1992). Use of kraft black liquors from a pulp mill for the production of soil conditioners. Waste Management and Research. 10(2): 195-201. https://doi.org/10.1016/0734-242X(92)90072-S.

    13. Haile, A., Galebo, G.G., Tesfaye, T., Mengie, W., Mebrate, M.A., Abuhay, A. and Limeneh, D.Y. (2021). Pulp and paper mill waste-utilization and prospects for high value added biomaterials. Bioresources and Bioprocessing. 8: 35. https://doi.org/10.1186/s40643-021-00385-3.

    14. Hayat, R., Ali, S., Amara, U., Khalid, R. and Ahmed, I. (2010). Soil beneficial bacteria and their role in plant growth promotion:  A review. Annals of Microbiology. 60(4):  579-598. https:// doi.org/10.1007/s13213-010-0117-1.

    15. Kajić, S., Gradinščak, A., Königshofer, D., Fadljević, I., Sikora, S. and Rajnović, I. (2025). Characterization of plant growth promoting rhizobacteria and their benefits on soybean growth. Legume Research. 48(1): 131-136. doi: 10.18805/LRF-819.

    16. Kaur, J. and Sharma, P. (2016). Response of summer mung bean (Vigna radia L.) to rhizobium and PGPR inoculation under different plating methods. Ann. Agric. Res. New series. 37(4): 87-90. https://www.researchgate.net/publication/ 375640816.

    17. Khan, R., Singh, R. and Kumar, R. (2026). Effect of plant growth promoting rhizobacteria (pgpr) and plant growth regulators (PGR) on antioxidant properties of chickpea (Cicer arietinium L.) in presence of toxic effect of thiamethoxam.  Indian Journal of Agricultural Research. 60(1): 115- 122. doi: 10.18805/IJARe.D-6377.

    18. Korir, H., Mungal, N.W., Thuita, M., Hamba, Y. and Masso, C. (2017). Co-inoculation effect of rhizobia and plant growth promoting rhizobacteria on common bean growth in a low phosphorus soil. Frontiers in Plant Science. 8: 141. https://doi: 10.3389/fpls.2017.00141.

    19. Kuforiji, O.O. and Fasidi, I.O. (2009). Biodegradation of agro- industrial wastes by a edible mushroom Pleurotus tuber- regium (Fr.). J. Environ. Biol. 30(3): 355-358. PMID: 20120458. https://pubmed.ncbi.nlm.nih.gov/20120458/.

    20. Kulshreshtha, S., Mathur, N. and Bhatnagar, P. (2011). Handmade paper and cardboard industries: In health perspectives. Toxicology and Industrial Health. 27(6): 515-521. doi: 10.1177/0748233710391992.

    21. Kulshreshtha, S., Mathur, N. and Bhatnagar, P. (2010a). Genotoxic evaluation of handmade paper industrial effluent-A case study. International Journal of Chemical Sciences. 8: 2519-2528.

    22. Kulshreshtha, S., Mathur, N., Bhatnagar, P. and Jain, B. (2010b). Bioremediation of industrial waste through mushroom cultivation. Journal of Environmental Biology/Academy of Environmental Biology, India. 31: 441-444.

    23. Kulshreshtha, S., Mathur, N., Bhatnagar, P. and Kulshreshtha, S. (2013). Cultivation of Pleurotus citrinopileatus on handmade paper and cardboard industrial wastes. Industrial Crops and Products. 41: 340-346. 10.1016/ j.indcrop.2012.04.053.

    24. Kumar, R. and Chandra, R. (2008). Influence of PGPR and PSB on Rhizobium leguminosarum bv. viciae strain competition and symbiotic performance in lentil l. World Journal of Agricultural Sciences. 4(3): 297-301. https://www. researchgate.net/publication/242235746.

    25. Kunhikrishnan, A., Nanthi, S., Bolan, K.M., Seth, L., Ravi, N. and Kim, W. (2012). Chapter Five-the Influence of Wastewater Irrigation on the Transformation and Bioavailability of Heavy Metal (Loid)s in Soil, [Editor(s): Donald, L.] Sparks, Advances in Agronomy, Academic Press. 115: 215- 297. https://doi.org/10.1016/B978-0-12-394276-0.000056.

    26. Liu, X., Li, Y., Meng, Y., Lu, J., Cheng, Y., Tao, Y. and Wang, H. (2021). Pulping black liquor-based polymer hydrogel as water retention material and slow-release fertilizer. Industrial Crops and Products. 165: 113445. https:// doi.org/10.1016/j.indcrop.2021.113445.

    27. Mehmood, K., Rehman, S.K., Wang, F.F., Mahmood, Q., Jadoon, A., Javed, M.F. and Ahmed (2019). Treatment of pulp and paper industrial effluent using physiochemical process for recycling. Water. 11(11): 2393. https://doi:10.3390/ w11112393.

    28. Molina-Sanchez, E., Leyva-Díaz, J.C., Cortes-Garcia, F.J. and Molina-Moreno, V. (2018). Proposal of sustainability indicators for the waste management from the paper industry within the circular economy model. Water. 10: 1014. https://doi.org/10.3390/w10081014.

    29. Neha, C.R., Pareek, N. and Raverkar, K.P. (2024). Enhancing mungbean (Vigna radiata L.) productivity, soil health and profitability through conjoint use of Rhizobium and PGPR. Legume Research. 47(5): 823-828. doi: 10.18805/LR-4552. 

    30. Narayana, S., Sethiasivan, K., Chauhan, S. and Mathur, R. (2024). Waste liquors generated during handmade paper manufacture from cow dung as a potential source of biofertilizer. Journal of Applied and Natural Science. 16(1): 400-409. https://doi.org/10.31018/jans.v16i1.5209.

    31. Patel, K., Patel, N., Vaghamshi, N., Shah, K., Duggirala, S.M. and Dudhagara, P. (2021). Trends and Strategies in the effluent treatment of pulp and paper industries-A review highlighting reactor options. Current Research in Microbial Sciences. 2: 100077. https://doi.org/10.1016/j.crmicr. 2021.100077.

    32. Pokhrel, D. and Viraraghavan, T. (2004). Treatment of pulp and paper mill wastewater-A review. Sci. Tot. Env. 333: 37- 58. https://doi.org/10.1016/j.scitotenv.2004.05.017.

    33. Reddy, S. (2015). Eco-friendly production of paper products. International Journal of Chemical Concepts. 1: 72-80. https://www.researchgate.net/publication/281854558_ Eco-Friendly_Production_of_Paper_Products.

    34. Renu, S.P., Sahu, U., Bhoyar, M.S., Kumar, M., Singh, U. and Pathak, R. (2016). Ecological success of compatible microbes in consortia isolated from rice rhizosphere for growth and yield of mung bean (Vigna radiata L.). J. Pure Appl Microbiol. 10(4): 3231-3239. doi: 10.22207/JPAM. 10.4.101 https://doi.org/10.22207/JPAM.10.4.101.

    35. Savant, D., Abdulrahman, R. and Ranade, D. (2006). Anaerobic degradation of adsorbable organic halides (AOX) from pulp and paper industry wastewater. Bioresour. Technol. 97: 1092-1104. https://doi.org/10.1016/j.biortech.2004. 12.013.

    36. Sharma, P. and Singh, S.P. (2021). Pollutants characterization and toxicity assessment of pulp and paper industry sludge for safe environmental disposal. Emerging Treatment Technologies for Waste Management. (pp.207-223). 10.1007/978-981-16-2015-7_10.

    37. Singh, U.S. and Tripathi, Y.C. (2020). Characteristics and treatment of pulp and paper mill effluents- A review. International Journal of Engineering and Technical Research (IJETR). 10(11): 19-26.

    38. Sivrikaya, H., Bacak, L., Saracbasi, A., Toroglu, L. and Eroglu, H. (2002). Trace elements in pleurotus sajorcaju cultivated on chemithermomechanical pulp for bioleaching. Food Chemistry. 79: 173-176. https://doi.org/10.1016/S0308- 8146(02)00128-0.

    39. Thompson, G., Swain, J., Kay, M. and Forster, C. (2001). The treatment of pulp and paper mill effluent: A review. Bioresour Technol. 77: 275-286. https://doi.org/10.1016/ S0960-8524(00)00060-2.

    40. Touhari, F., Guetarni, H., Sadaoui, S. and Mehaiguene, M. (2023).  Water quality assessment of the ouled mellouk dam treatment plant, North Western Algeria. Agricultural Science Digest. 43(3): 334-339. doi: 10.18805/ag.DF-497.

    41. Tyagi, V.K., Purushottam and Tomar, A. (2017). Isolation, identification, biochemical and antibiotic sensitivity characterization of rhizobium strains from Vigna mungo (L) Hepper, Cicer aertinum L. and Vigna radiate (L.) R Wilczek in Muzaffarnagar, Uttar Pradesh, India. Int. J. Curr. Microbiol. Appl. Sci. 6(12): 2024-2035. doi. https://doi.org/10.20546/ijcmas. 2017.612.233

    42. United States Environment Protection Act, (US EPA) (1992). https:// www.epa.gov/sites/default/files/201411/documents/ guidelines_exp_assessment.pdf.

    43. United States Environment Protection Act, (US EPA) (2004). https:// www.epa.gov/sites/default/files/2019-08/documents/ 2004-guidelines-water-reuse.pdf.

    44. Yadav, B.K., Mohanty, N., Dash, S., Pradhan, S., Sahoo, B. and Rath, B. (2024). Enhanced yield of mung bean (Vigna radiata L.) using bacterial biofertilizers. Biosciences Biotechnology Research Asia. 21(1): 89-98. https:// dx.doi.org/10.13005/bbra/3205.

    45. Zervakis, G., Yiatras, P. and Balis, C. (1995). Edible mushrooms from olive oil mill wastes. International Biodeterioration and Biodegradation. 38(3-4): 237-243. https://doi.org/ 10.1016/S0964-8305(96)00056-X.
    In this Article
      Contact us
      Follow us
      Published In
      Indian Journal of Agricultural Research

      Editorial Board

      View all (0)