ICS 65.080  
 
Reference number  
WDDRS 488 -2: 2022  
© RSB 2022  
  
 RWANDA 
STANDARD  DRS  
488-2  
 
First edition  
2022 -mm-dd 
 
Bio-inoculants  — Specification  — 
Part 2: Phosphate solubilizing bacteria 
(PSB)  
   
DRS 488 -2: 2022  
©RSB 2022  - All rights reserved                  ii 
 In order to match with technological development and to keep continuous progress in industries, 
standards are subject to periodic review. Users shall ascertain that they are in possession of the latest 
edition  
 
 
 
 
 
 
 
 
© RSB 2022 
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ePortal: www.portal.rsb.gov.rw   DRS 488 -2: 2022  
iii                ©RSB 2022  - All rights reserved  
 Contents  Page 
Foreword  ................................ ................................ ................................ ................................ ............................  iv 
1 Scope  ................................ ................................ ................................ ................................ ......................  1 
2 Normative references  ................................ ................................ ................................ ............................  1 
3 Terms and definitions  ................................ ................................ ................................ ...........................  1 
4 Requirements  ................................ ................................ ................................ ................................ .........  1 
4.1 General requirements  ................................ ................................ ................................ ...........................  1 
4.2 Specific requirements  ................................ ................................ ................................ ...........................  2 
5 Packag ing ................................ ................................ ................................ ................................ ...............  2 
6 Labelling  ................................ ................................ ................................ ................................ .................  2 
7 Sampling  ................................ ................................ ................................ ................................ ................  3 
Annex  A (normative)  Determination of number of phosphate solubilizing bacterial cells  ........................  4 
A.1 Preparation of culture medium  ................................ ................................ ................................ ............  4 
A.2 Preparation of serial dilution for cell counts  ................................ ................................ ......................  4 
A.3 Counting  ................................ ................................ ................................ ................................ .................  4 
A.4 Methods for counting solubilisation zones  ................................ ................................ ........................  5 
Annex  B (normative)  Determination of pH and moisture content  ................................ ................................  6 
B.1 Determination of pH  ................................ ................................ ................................ ..............................  6 
B.1.1  Solid bio -fertilizers  ................................ ................................ ................................ ................................  6 
B.1.2  Liquid bio -fertilizers  ................................ ................................ ................................ ..............................  6 
B.2 Determination of moisture content  ................................ ................................ ................................ ...... 6 
B.2.1  Procedure  ................................ ................................ ................................ ................................ ...............  6 
B.2.2  Calculation  ................................ ................................ ................................ ................................ .............  6 
Annex  C (normative)  Determination of soluble phosphorus using ascorbic acid  ................................ ..... 7 
C.1 Principles  ................................ ................................ ................................ ................................ ...............  7 
C.2 Apparatus  ................................ ................................ ................................ ................................ ...............  7 
C.3 Reagents  ................................ ................................ ................................ ................................ ................  7 
C.4 Preparation of reagents  ................................ ................................ ................................ ........................  7 
C.4.1  Sulphomulybdic acid  ................................ ................................ ................................ ............................  7 
C.4.2  Preparation of mixed reagents  ................................ ................................ ................................ .............  8 
C.4.3  Procedure  ................................ ................................ ................................ ................................ ...............  8 
C.5 Calculation  ................................ ................................ ................................ ................................ .............  8 
C.6 Preparation of standard curve  ................................ ................................ ................................ .............  9 
Annex  D (normative)  Guidelines on maintenance and preparation of culture and quality control 
at broth stage  ................................ ................................ ................................ ................................ ....... 10 
D.1 Maintenance of cultures  ................................ ................................ ................................ .....................  10 
D.2 Preparation of inoculum culture and mass culture  ................................ ................................ .........  10 
D.3 Quality control test recommended at broth stage  ................................ ................................ ...........  10 
 DRS 488 -2: 2022  
©RSB 2022  - All rights reserved                  iv 
 Foreword   
Rwanda Standard s are prepared  by Technical Committees and approved by Rwanda Standards Board (RSB) 
Board  of Directors in  accordance with the procedures of RSB, in compliance with Annex 3 of the WTO/TBT 
agreement on the preparation, adoption and application of standards.  
The main task of technical committees is to prepare national standards.  Final Draft Rwanda S tandards 
adopted by Technical committ ees are ratifie d by me mbers of RSB Board of Director s for publication and 
gazettment as Rwanda Standards.  
DRS 488 -2 was prepared by Technical Committee RSB/TC 007, Agrochemicals . 
In the preparation of this standard, reference was made to the following standard  (s): 
WD-ARS 1452 -2:20217,  Bio-inoculants — Part 2: Specifications for phosphate solubilizing  bacteria  
The assistance de rived from the above source is hereby acknowledged with thanks.  
DRS  488 consists of the following parts, under the general title Bio-inoculant s — Specification : 
⎯ Part 1: Nitrogen fixing bacteria  
⎯ Part 2: Phosphate solubilizing bacteria  
⎯ Part 3: Potash solubilizing bacteria  
Committee membership  
The following organizations were represented on the Technical Committee on Agrochemicals  (RSB/TC 007) in 
the preparation of this standard.  
AGROPY Ltd  
University of Rwanda/College of Education (UR/CE)  
Ministry of Environment (MoE)  
University of Rwanda/Colle ge of Sciences and Technology (UR/CST)  
Rwanda Inspectorate, Competition and Consumer Protection Authority (RICA)  
Rwanda Forensic Laboratory (RFL)  
Rwanda Agriculture and Animal Resources Development Board (RAB)  DRS 488 -2: 2022  
v                ©RSB 2022  - All rights reserved  
 National Agricultural Export Development Board (NAEB)  
Rwanda Investigation Bureau (RIB)  
Rwanda  Standards Board  (RSB) – Secretariat  DRS 488 -2: 2022  
©RSB 2022  - All rights reserved                  vi 
 Introduction   
Commercial farming to meet food security involving intensive land use relies heavily on agricultural  inputs 
such as chemical fertilizers and synthetic pesticides. However, increasing continuous use of  chemical 
fertilizers over years has caused environmental damage such as ground water  contamination, eutrophication 
of water bodies. Long term sole reliance on excessive use of chem ical fertilizers and synthetic pesticides has 
also adversely impacted on soil quality, fertility and soil  microbial biodiversity.  
With the increasing demand for food to meet the growing population, food production can be  enhanced by 
increasing the producti vity of existing agricultural land sustainably and/or bring  additional land under 
cultivation. Ensuring sustainable long term land productivity can be achieved  through an integrated nutrient 
management system involving a combination of environmentally  friendly sources of plant nutrients including 
organic microbial fertilizers and chemical fertilizers.  
Bio-inoculant s are defined as preparations containing living cells or latent cells of efficient strains of  
microorganisms that help crop plants’ uptake of nut rients by their interactions in the rhizosphere  when applied 
through seed or soil. They accelerate certain microbial processes in the soil which  augment the extent of 
availability of nutrients in a form easily assimilated by plants. Bio -fertilizers  generat e plant nutrients like 
nitrogen and phosphorus through their activities in the soil or  rhizosphere and make available in a gradual 
manner to plants.  
Bio-inoculant s are preparations containing live microbes which help in enhancing soil  fertility by fixing 
atmospheric nitrogen, solubili zation of phosphorous, mobilization of nutrients or  decomposing organic wastes 
or by augmenting plant growth by producing growth hormones with  their biological activities.  
Potential benefits of using Bio-inoculant s are: 
a) To prov ide an eco ‐friendly alternate renewable source of nutrients.  
b) Sustain soil health  
c) Decompose plant residues, and stabilize C:N ratio of soil  
d) To enhance soil biological activity and improve availability of plant nutrients.  
e) To improve chemical, physical and biological properties of soil.  
f) To promote plant growth and improve crop yield.  
g) Solubilize and mobilize nutrients  
Bio-inoculant s are supplement to chemical fertilizers but not substitute to it. They usually cause  increase in 
crop yield. However , they do not cause marked increase in productivity like chemicals.  Efficiency of microbial 
fertilizer is markedly dependent on soil character, for example moisture  content, pH, temperature organic 
matter and types of resident microorganism. When these fac tors are unfavourable microbial fertilizers may not 
be effective in increasing the soil fertility.  
 DRS 488 -2: 2022  
vii                ©RSB 2022  - All rights reserved  
 Phosphate solubili zing bacteria (PSB)  
Phosphate solubilizing bacteria (PSB) are beneficial bacteria capable of solubilizing inorganic  phosphorus 
from insolu ble compounds. Many phosphate solubilizing bacteria (PSB) belong to the  Pseudomonas, Bacillus, 
Rhizobium, Agrobacterium, Burkholderia, Achromobacter, Micrococcus,  Aerobacter, Enterobacter, 
Flavobacterium, and Erwinia genera.  
Most soils are deficient in sol uble forms of phosphorus (P), one of the major essential macronutrients  required 
for plant growth. Phosphorus makes up to about 0.2% of plant dry weight and has a defined  role in plant 
metabolism such as cell division, development, photosynthesis, breakdow n of sugar,  nuclear transport within 
the plant, transfer of genetic characteristics from one generation to another  and regulation of metabolic 
pathways.  
The plants obtain their phosphate requirements from the soil pool as inorganic phosphate, produced  by 
weathering by parent rock or as organic phosphate derived from decayed plant, animal or  microorganisms.  
The phosphate available for plant growth depends not only on the total amount of phosphorous in  the 
environment but also on its solubility, which in turn is dictated by chemical reactions and  biological interaction 
in the soil. The makeup of a soil (soil texture) and its acidity (pH) determine the  extent to which nutrients are 
available to plants. The diverse soil phosphate forms can be genera lly categorized as soil solution phosphate, 
insoluble organic and insoluble inorganic phosphate.  
Uses of PSB  
PSB can be used for all crops including paddy, millets, oilseeds, pulses and vegetables. Methods  
recommended for application are:  
⎯ Seed treatment  
⎯ Seedling dipping  
⎯ Soil application  
 
 DRS 488 -2: 2022  
1                ©RSB 2022  - All rights reserved  
 Bio-inoculants  — Specification  — Part 2: Phosphate solubilizing bacteria 
(PCB)  
1 Scope   
This Draft Rwanda Standard  specifies the requirements , sampling  and test methods for phosphate solubili zing 
Bacterial inoculant (PSBI) for use in laboratories.  It is applicable to both so lid and liquid microbial bio ‐fertilizers.  
It does not  cover field efficacy test.  
2 Normative references   
The following documents are referred to in the text in such a way that some or all of  their content constitutes 
requirements of this document.  For dated references, only the edition  cited applies. For undated references, 
the latest edition of the referenced document (including any amendments) applies.  
RS ISO 8157 , Fertilizers and Soil conditioners — Vocabulary  
DRS  488-1: 2021, Bio-fertilizer — Specifications — Part 1: Nitrogen fixing bacteria  
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and performance 
testing of culture media  
3 Terms and definitions   
For the purposes of this standard, the terms and def initions given in RS ISO 8157, DRS 488 -1 and the 
following apply.  
3.1 
phosphate solubilizing bacterial inoculants (PSBI)  
a product having high population of a strain(s) of bacteria intended to solubili ze insoluble phosphate  of the soil.  
4 Requirements  
4.1 General requirements  
4.1.1  For solid bio ‐inoculant s, the carrier used shall be of a nature that is not harmful to the  environment 
and the colour of the inoculants would be dependent on the colour of the carrier.  
4.1.2  Carrier material such as peat, lignite, peat, charcoal or other similar material may be  used. It shall be 
neutralized with calcium carbonate and then sterilized. The carrier  material shall be in the form of powder 
capable of passing through 150 to 212 micron  (72 to 100 mesh) sieve .  
©RSB 2022  - All rights reserved                  2 
 4.1.5  Specified mother culture shall be obtained from any recognized institution maintaining  the mother 
culture. The manufacturer may control the quality of broth as given in  Annex D.  
4.2 Specific requirements  
The bio‐inoculant s shall comply with the requirements of Table 1.  
Table 1  — Requirements for phosphate solubilizing bio‐inoculant s 
 Parameter  Requirement  Methods of test  
(a) Viable cell count, CFU, 
min. 5.0  107 cell/g of [powder, or granule or 
carrier material (dry mass basis)  
or 
1.0 x108 cell/ml of liquid  Annex A  
(b) Contamination level  No contamination with other  
micro‐organisms at 10-5 dilution  Annex A  
(c) pH 6.5 ‐ 7.5 for moist/ dry powder  
5.0 – 7.5 for liquid based  Annex B  
(d) Moisture content, %, max 
(in case of carrier based)  30‐40 Annex B  
(e) Efficiency character  The strain shall have phosphate  
solubili zing capacity in range of  30%, 
when tested  spectrophotome trically. In 
terms  of zone formation, minimum 5 mm  
solubilization zone in prescribed  media 
having at least 3 mm  thickness.  Annex C  
 
5 Packaging  
The bio‐inoculant s shall be pack aged in polyethylene packs, thickness of which shall not be less  than 100 
micron s. 
6 Labelling  
Each pack shall be labeled legibly and indelibly with the following information:  
a) Name of product;  
b) Name, address and phone number of the manufacturer;  
c) Intended crop;  
d) Type of carrier used;  
e) Batch number;  
f) Production date and expiry date;  DRS 488 -2: 2022  
3                ©RSB 2022  - All rights reserved  
 g) Net quantity in kg or ml;  
h) Rate of application per ha or m2; 
i) Storage instruction  
j) Microbial count (cfu/ml or cfu/g);  
k) Direction for use of PSBI shall be printed briefly on the packets. A separate pamphlet may  preferably be 
given with it.  
7 Sampling  
The representative samples of the PSBI shall be drawn as prescribed for Rhizobium inoculant in  Annex A of 
DRS 488 -1. 
  
©RSB 2022  - All rights reserved                  4 
 Annex  A 
(normative)  
 
Determination of number of phosphate solubilizing bacterial cells  
A.1 Preparation of culture medium  
A.1.1  Use a medium of the following composition: 
Glucose  10.0 g 
Tri‐calcium phosphate 5.0 g 
Ammoniu m sulphate 0.5 g 
Magnesium sulphate  0.1 g 
Sodium chloride  0.2 g 
Yeast extract 0.5 g 
Mang anese sulphate  Trace 
Ferrous suIphate Trace 
Distilled water  1000 ml 
Agar  15.0 g 
pH adjusted  to 7 ± 0.2 
A.1.2  The culture medium shall be prepared in accordance with ISO 11133 or using any other accepta ble 
validated test meth od. 
A.2 Preparation of serial dilution for cell counts  
A.2.1  Dispense 30 g of PSBI  in 270 ml of sterile water  and shake  for 10 minutes on  a reciprocal 
shaker. 
A.2.2  Make  serial dilutions up to 10-7 level. 
A.2.3  Pipette out 0.2 ml aliquots of 10-5 to 10-7 dilution and deliver it on the Petri dishes containing set 
medium as described in A.1. 
A.2.4  Spread the aliquots ov er the  plate. Invert the plates and place them in the incubator at 28 ± 2 C 
for 3 days. 
A.2.5  Use 3 replicates of 10-5, 10-6 and 10-7 dilution. 
 
A.3 Counting  
Count the total number of colonies  on the plates  including colonies with solubiliza tion zone  with the help of 
a colony counter.  DRS 488 -2: 2022  
5                ©RSB 2022  - All rights reserved  
 A.4 Methods for counting solubilisation zones  
A.4.1  Take 10 g of PSBI  (BF) in 90 ml in water.  
A.4.2  Make  a tenfold dilution series up to 10-7 
A.4.3  Take 0.2 ml aliquots of 10-5 to 10-7 dilution using sterile pipettes and delivered  to Petri dishes 
containing Pikovskayei’s media. 
A.4.4  Spread  it unifor mly. Invert the plate  and incubate them up to 2 weeks at 28 ± 2 C. 
A.4.5  Count  the colonies showing hallow cones and measure their diameter. Minimum  accep table zone 
is 10 mm in diameter.  
 
   
©RSB 2022  - All rights reserved                  6 
 Annex  B 
(normative)  
 
Determination of pH and moisture content  
B.1 Determination of pH  
B.1.1  Solid bio -fertilizers  
Make suspension of 20g of the PSB into 50 ml of distilled water and shake on a rotary  shaker for 2h. 
Filter this suspension and determine  the pH of filtrate with the help of pH meter.  
B.1.2  Liquid bio -fertilizers  
Take 100 ml of inoculants into a 250 ml clean and dry conical flask and shake on a rotary shaker for 2h. 
Determine pH using a pH meter.  
B.2 Determination of moisture content  
The moisture content test is applicable only for powder granules or carrier material. 
B.2.1  Procedure  
B.2.1.1  Measure 10 g of sample in a pre ‐weighed container.  
B.2.1.2  Heat the sample for 24 hours in an air oven at 80 C.  
B.2.1.3  Allow to cool in a desiccator and take the weight.  
B.2.2  Calculation  
The loss in weight represents  the moisture. 
Moisture content, % = 
100sampledry  oven the of Weightdrying on  weightin Loss  DRS 488 -2: 2022  
7                ©RSB 2022  - All rights reserved  
 Annex  C 
(normative)  
 
Determination of soluble phosphorus using ascorbic acid  
C.1 Principles  
C.1.1  Soluble phosphorus forms hetropoly molybdo ‐phosphate complex with molybdate ions which on 
reduction produces a characteristic blue colour measured at 840 to 880 nm.  
C.1.2  Considering the higher stabil ity of the ascorbic acid, easiness to handle, higher tolerance to the 
concentration of interfering ions, possibilities to use it with all types of acids and higher stability the developed 
colour (from 10 to 60 min), ascorbic acid instead of stanous chlorid e is nowadays use as the reducing agent 
for the hetropoly molybdo ‐phosphate complex formed by the soluble phosphate ions on addition of ammonium 
molybdate solution.  
C.2 Apparatus  
Spectrophotometer capable of transmission measurem ents at 840 to 880 nm. Extractant: It is olsen extract. 
C.3 Reagents  
C.3.1  Ammonium molybdate [(NH 4)6M07024.4H20] 
C.3.2  L‐Ascorbic  Acid  
C.3.3  p‐Nitrophenol  
C.3.4  10 N H2S04 
C.3.5  4 N H2S04 
C.4 Preparation of reagents  
C.4.1  Sulphomulybdic acid  
C.4.1.1  Take 20 g of ammonium molybdate and dissolve in 300 ml of distilled water.  
C.4.1.2  Add slowly 450 ml of 10 N H2SO4. 
C.4.1.3  Cool the above mixture  and add 100 ml of 0.5 perce nt solution  of antimony potassium  tartrate. 
C.4.1.4  Cool and make the volume to one litre.  
©RSB 2022  - All rights reserved                  8 
 C.4.1.5  Store  in glass bottle  away from direct sunligh t. 
C.4.2  Preparation of mixed reagents  
C.4.2.1  Add 1.5 g of L‐ascorbic acid in 100 ml of the above  stock solution  and mix. Add 5 ml of this 
solution to develop colour.  
C.4.2.2  Mixed reagent is to be prepared fresh as it does not keep for more than 24 h. 
C.4.3  Procedure  
C.4.3.1  Weigh the required material in a 100 ml conical flask.  
C.4.3.2  Add 50 ml of extractant and shake it of 30 min on a rotary shaker.  
C.4.3.3  Filter the suspension through Whatman filter paper No. 40. If the filtrate is coloured then add a tea 
spoon of Darco ‐60 (activated phosphorus free carbon), shake and fiIter.  
C.4.3.4  Take a known aliquot (5 to 25 ml) of the extract in a 50 ml volumetric flask.  
C.4.3.5  Add 5 drops of p ‐nitrophenol indicator (1.5 percent so lution in water) and adjust the pH of the 
extract between 2 and 3 with the help of 4NH 2SO 4. The yellow colour will disappear when the pH of the 
solution becomes 3. Swirl gently to avoid loss of the solution along with the evolution of CO4.(CO 2.) 
C.4.3.6  When the CO 2 evolution has subsided, wash down the neck of the flask and dilute the solution to 
about 40 ml.  
C.4.3.7  Add 5 ml of the sulphomolybdic acid mixed reagent containing ascorbic acid. Swirl the content 
and make up the volume.  
C.4.3.8  Measure the tra nsmission after 30 min at 880 nm using red filter. The blue colour developed 
remains stable up to 60 minutes.  
C.4.3.9  Record the concentration of phosphorus (P) in the extract from the standard curve.  
C.5 Calculation  
Calculate the concentration  of soluble Phosphor us using the following formula: 
P (%) = 
10050 1050z-6

xy  
where,  
x Weight of the substance taken (g) DRS 488 -2: 2022  
9                ©RSB 2022  - All rights reserved  
 y Volume of the extract  taken for P determ ination (ml) 
z Reading from the standard curve (ppm)  against percent transmission recorded 
Volume of the extractant  added = 50 ml 
Volume made after colour developed = 50 ml 
C.6 Preparation of standard curve  
Prepare standard curve  using 0.1 to 0.6 ppm P in 50 ml volumetric flask. Plot the standard curve  by 
taking  concentration  of soluble P on x‐axis and percent T on y‐axis using a semi‐log graph paper. It  is a 
straight line relationship between the soluble P and percent T when plotted  on a semi‐log graph pa per.  
©RSB 2022  - All rights reserved                  10 
 Annex  D 
(normative)  
 
Guidelines on maintenance and preparation of culture and quality control at broth 
stage  
D.1 Maintenance of cultures  
D.1.1  Maintain pure cultu re of PSBI on the medium  as described  in A.1 in the form of slants. 
D.1.2  Transfer a loopful  of pure culture to each  of the agar slants aseptically  in an inoculation  room  
and incub ate at 28 ± 2°C for three  days. Al ways keep pure culture below  5°C. 
D.2 Preparation of inoculum culture and mass culture  
Inoculum culture  and mass  cultu re shall be prepared  as described  in G.1.2  (a), G.1.2  (b) and G.1.3  of the 
DRS 488 -1 changi ng the media composition  as mentioned  in A.1 of this standard.  
D.3 Quality control test recommended at broth stage  
D.3.1  Qualitative test  
D.3.1.1  Check for free from visible  conta minants  by microsco pe and observing  solubilization  zones. 
D.3.1.2  The pH of bacterial broth  shall normally be between  6.5 to 7. 
D.3.1.3  Gram  stain ing test shall be carried out as described in G.1.4.1.3 of DRS 488 -1. 
D.3.2  Quantitative test  
Viable cell count  DRS 488 -2: 2022  
11                ©RSB 2022  - All rights reserved  
 Bibliography  
[1] IS 8268:2001, Rhizobium inoculants — Specification 
[2] IS 14807:2000, Phosphate solubilising bacter ial inoculant (PSBI) — Speci fication 
[3] KS 2356:2011, Bio fertilizer — Specificat ion 
[4] Biofertilizers  and Organic Ferti lizers  in Fertilizer (Control) ord er, 1985, published by National 
Centre of Organic Fa rming, Depa rtment  of Agriculture and Cooperation,  Ministry of Agricu lture, Govt of 
India 
 
 DRS 488 -2: 2022  
 
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